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Posts Tagged ‘Oxidative phosphorylation’


Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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New Insights on the Warburg Effect [2.2]

Larry H. Bernstein, MD, FCAP, Curator, Writer

https://pharmaceuticalintelligence.com/8/05/15/lhbern/New_Insights_on_the_Warburg_Effect_%5B2.2%5D

 

New Insights on the Warburg Effect [2.2]

Defective Mitochondria Transform Normal Cells into Tumors

GEN News Jul 9, 2015

Ninety-one years ago Otto Warburg demonstrated that cancer cells have impaired respiration, which became known as the Warburg Effect. The interest in this and related work was superceded in the last quarter of the twentieth century by work on the genetic code. Now there is renewed interest.

An international research team reports that a specific defect in mitochondria plays a key role in the transition from normal cells to cancerous ones. The scientists disrupted a key component of mitochondria of otherwise normal cells and the cells took on characteristics of malignant cells.

Their study (“Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming”) is published Oncogene and was led by members of the lab of Narayan G. Avadhani, Ph.D., the Harriet Ellison Woodward Professor of Biochemistry in the department of biomedical sciences in the school of veterinary medicine at the University of Pennsylvania. Satish Srinivasan, Ph.D., a research investigator in Dr. Avadhani’s lab, was the lead author.

This is consistent with the 1924 observation by Warburg that cancerous cells consumed glucose at a higher rate than normal cells (Meyerhof ratio) and had defects in their grana, the organelles that are now known as mitochondria. He postulated that these defects led to problems in the process by which the cell produces energy. But the process called oxidative phosphorylation was not yet known. Further work in his laboratory was carried out by Hans Krebs and by Albert Szent Gyorgyi elucidating the tricarboxylic acid cycle.  The discovery of the importance of cytochrome c and adenosine triphosphate in oxidative phosphorylation was made in the post World War II period by Fritz Lippman, with an important contribution by Nathan Kaplan. All of the name scientists, except Kaplan, received Nobel Prizes. The last piece of the puzzle became the demonstation of a sequence of hydrogen transfers on the electron transport chain. The researchers above have now shown that mitochondrial defects indeed contributed to the cells becoming cancerous.

“The first part of the Warburg hypothesis has held up solidly in that most proliferating tumors show high dependence on glucose as an energy source and they release large amounts of lactic acid,” said Dr. Avadhani. “But the second part, about the defective mitochondrial function causing cells to be tumorigenic, has been highly contentious.”

To see whether the second part of Warburg’s postulation was correct, the researchers took cell lines from the skeleton, kidney, breast, and esophagus and used RNA molecules to silence the expression of select components of mitochondrial cytochrome oxidase C, or CcO, a critical enzyme involved in oxidative phosphorylation. CcO uses oxygen to make water and set up a transmembrane potential that is used to synthesize ATP, the molecule used for energy by the body’s cells.

The biologists observed that disrupting only a single protein subunit of cytochrome oxidase C led to major changes in the mitochondria and in the cells themselves. “These cells showed all the characteristics of cancer cells,” noted Dr. Avadhani.

The normal cells that converted to cancerous cells displayed changes in their metabolism, becoming more reliant on glucose by utilization of the glycolytic pathway. They reduced their synthesis of ATP.  Oxidative phosphorylation was reduced in concert with the ATP reduction. The large switch to glycolysis as primary energy source is a less efficient means of making ATP that is common in cancer cells.

The cells lost contact inhibition and gained an increased ability to invade distant tissues, both hallmarks of cancer cells. When they were grown in a 3D medium, which closely mimics the natural environment in which tumors grow in the body, the cells with disrupted mitochondria formed large, long-lived colonies, akin to tumors.

The researchers also silenced cytochrome oxidase C subunits in breast and esophageal cancer cell lines. They found that the cells became even more invasive, according to Dr. Srinivasan. The team then looked at actual tumors from human patients and found that the most oxygen-starved regions, which are common in tumors, contained defective versions of CcO.

“That result alone couldn’t tell us whether that was the cause or effect of tumors, but our cell system clearly says that mitochondrial dysfunction is a driving force in tumorigenesis,” explained Dr. Avadhani.

The researchers observed that disrupting CcO triggered the mitochondria to activate a stress signal to the nucleus, akin to an SOS alerting the cell that something was wrong. Dr. Avadhani and his colleagues had previously seen a similar pathway activated in cells with depleted mitochondrial DNA, which is also linked to cancer.

Building on these findings, Dr. Avadhani and members of his lab will examine whether inhibiting components of this mitochondrial stress signaling pathway might be a strategy for preventing cancer progression.

“We are targeting the signaling pathway, developing a lot of small molecules and antibodies,” said Dr. Avadhani. “Hopefully if you block the signaling the cells will not go into the so called oncogenic mode and instead would simply die.”

In addition, they noted that looking for defects in CcO could be a biomarker for cancer screening.

 

Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism

Rafael Moreno-Sánchez, Alvaro Marín-Hernández, Emma Saavedra, Juan P. Pardo, Stephen J. Ralph, Sara Rodríguez-Enríquez
Intl J Biochem Cell Biol 7 Feb 2014; 50:10-23
http://dx.doi.org/10.1016/j.biocel.2014.01.025

The supply of ATP in mammalian and human cells is provided by glycolysis and oxidative phosphorylation (OxPhos). There are no other pathways or processes able to synthesize ATP at sufficient rates to meet the energy demands of cells. Acetate thiokinase or acetyl-CoA synthetase, a ubiquitous enzyme catalyzing the synthesis of ATP and acetate from acetyl-CoA, PPi and AMP, might represent an exception under hypoxia in cancer cells, although the flux through this branch is negligible (≤10%) when compared to the glycolytic flux (Yoshii et al., 2009).

Glycolysis in human cells can be defined as the metabolic process that transforms 1 mol of glucose (or other hexoses) into 2 moles of lactate plus 2 moles of ATP. These stoichiometric values represent a maximum and due to the several reactions branching off glycolysis, they will be usually lower under physiological conditions, closer to 1.3–1.9 for the lactate/glucose ratio (Travis et al., 1971; Jablonska and Bishop, 1975; Suter and Weidemann, 1975; Hanson and Parsons, 1976; Wu and Davis, 1981; Pick-Kober and Schneider, 1984; Sun et al., 2012). OxPhos is the metabolic process that oxidizes several substrates through the Krebs cycle to produce reducing equivalents (NADH, FADH2), which feed the respiratory chain to generate an H+.

Applying basic biochemical principles, this review analyzes data that contrasts with the Warburg hypothesis that glycolysis is the exclusive ATP provider in cancer cells. Although disregarded for many years, there is increasing experimental evidence demonstrating that oxidative phosphorylation (OxPhos) makes a significant contribution to ATP supply in many cancer cell types and under a variety of conditions.

Substrates oxidized by normal mitochondria such as amino acids and fatty acids are also avidly consumed by cancer cells. In this regard, the proposal that cancer cells metabolize glutamine for anabolic purposes without the need for a functional respiratory chain and OxPhos is analyzed considering thermodynamic and kinetic aspects for the reductive carboxylation of 2-oxoglutarate catalyzed by isocitrate dehydrogenase.

In addition, metabolic control analysis (MCA) studies applied to energy metabolism of cancer cells are reevaluated. Regardless of the experimental/environmental conditions and the rate of lactate production, the flux-control of cancer glycolysis is robust in the sense that it involves the same steps:

  • glucose transport,
  • hexokinase,
  • hexosephosphate isomerase, and
  • glycogen degradation,

all at the beginning of the pathway; these steps together with phosphofructokinase 1 also control glycolysis in normal cells.

The respiratory chain complexes exert significantly higher flux-control on OxPhos in cancer cells than in normal cells. Thus, determination of the contribution of each pathway to ATP supply and/or the flux-control distribution of both pathways in cancer cells is necessary in order to identify differences from normal cells which may lead to the design of rational alternative therapies that selectively target cancer energy metabolism.

Fig. 1. Labeling patterns of 13C-glutamate or 13C-glutamine mitochondrial metabolism in cancer cells.

Fig. 2. Survey in PubMed of papers published in the field of tumor mitochondrial metabolism from 1951 to September 2013.

 

Emerging concepts in bioenergetics and cancer research: Metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy

Emilie Obre, Rodrigue Rossignol
Intl J Biochem Cell Biol 2015; 59:167-181
http://dx.doi.org/10.1016/j.biocel.2014.12.008

The field of energy metabolism dramatically progressed in the last decade, owing to a large number of cancer studies, as well as fundamental investigations on related transcriptional networks and cellular interactions with the microenvironment. The concept of metabolic flexibility was clarified in studies showing the ability of cancer cells to remodel the biochemical pathways of energy transduction and linked anabolism in response to glucose, glutamine or oxygen deprivation.

A clearer understanding of the large scale bioenergetic impact of C-MYC, MYCN, KRAS and P53 was obtained, along with its modification during the course of tumor development. The metabolic dialog between different types of cancer cells, but also with the stroma, also complexified the understanding of bioenergetics and raised the concepts of metabolic symbiosis and reverse Warburg effect.

Signaling studies revealed the role of respiratory chain derived reactive oxygen species for metabolic remodeling and metastasis development. The discovery of oxidative tumors in human and mice models related to chemoresistance also changed the prevalent view of dysfunctional mitochondria in cancer cells. Likewise, the influence of energy metabolism-derived oncometabolites emerged as a new means of tumor genetic regulation. The knowledge obtained on the multi-site regulation of energy metabolism in tumors was translated to cancer preclinical studies, supported by genetic proof of concept studies targeting LDHA, HK2, PGAM1, or ACLY.

Here, we review those different facets of metabolic remodeling in cancer, from its diversity in physiology and pathology, to the search of the genetic determinants, the microenvironmental regulators and pharmacological modulators.

 

Pyruvate kinase M2: A key enzyme of the tumor metabolome and its medical relevance

Mazurek, S.
Biomedical Research 2012; 23(SPEC. ISSUE): Pages 133-142

Tumor cells are characterized by an over expression of the glycolytic pyruvate kinase isoenzyme
type M2 (abbreviations: M2-PK or PKM2). In tumor metabolism the quaternary structure of M2-PK (tetramer/dimer ratio) determines whether glucose is used for glycolytic energy regeneration (highly active tetrameric form, Warburg effect) or synthesis of cell building blocks (nearly inactive dimeric form) which are both prerequisites for cells with a high proliferation rate. In tumor cells the nearly inactive dimeric form of M2- PK is predominant due to direct interactions with different oncoproteins. Besides its key functions in tumor metabolism recent studies revealed that M2-PK may also react as protein kinase as well as co activator of transcription factors. Of medical relevance is the quantification of the dimeric form of M2-PK with either an ELISA or point of care rapid test in plasma and stool that is used for follow-up studies during therapy (plasma M2-PK) and colorectal cancer (CRC) screening (fecal M2-PK; mean sensitivity for CRC in 12 independent studies with altogether 704 samples: 80% ± 7%). An intervention in the regulation mechanisms of the expression, activity and tetramer: dimer ratio of M2-PK has significant consequences for the proliferation rate and tumorigenic capacity of the tumor cells, making this enzyme an intensively

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Oxidation and Synthesis of Fatty Acids

Author and Curator: Larry H. Bernstein, MD, FCAP 

 

Lipid Metabolism

http://www.elmhurst.edu/~chm/vchembook/622overview.html

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidation to produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.

metabolism of fats

metabolism of fats

http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

fattyacidspiral

fattyacidspiral

http://www.elmhurst.edu/~chm/vchembook/images/620fattyacidspiral.gif

 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.

 

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2
 NET 12 ATP
 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP
GRAND TOTAL  130 ATP

Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.

This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.

  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.

.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.

My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.

The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1964/lynen-bio.html

http://onlinelibrary.wiley.com/store/10.1002/anie.201106003/asset/image_m/mcontent.gif?v=1&s=1e6dc789dfa585fe48947e92cc5dfdcabd8e2677

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

http://science.howstuffworks.com/dictionary/famous-scientists/biologists/feodor-lynen-info.htm

Fyodor Lynen

Fyodor Lynen

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Summary, Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP 

 

This portion of a series of chapters on metabolism, proteomics and metabolomics dealt mainly with carbohydrate metabolism. Amino acids and lipids are presented more fully in the chapters that follow. There are features on the

  • functioning of enzymes and proteins,
  • on sequential changes in a chain reaction, and
  • on conformational changes that we shall also cover.

These are critical to developing a more complete understanding of life processes.

I needed to lay out the scope of metabolic reactions and pathways, and their complementary changes. These may not appear to be adaptive, if the circumstances and the duration is not clear. The metabolic pathways map in total
is in interaction with environmental conditions – light, heat, external nutrients and minerals, and toxins – all of which give direction and strength to these reactions. A developing goal is to discover how views introduced by molecular biology and genomics don’t clarify functional cellular dynamics that are not related to the classical view.  The work is vast.

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from carbon dioxide and water by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are consumed by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts.

Carbohydrates are used for short-term fuel, and even though they are simpler to metabolize than fats, they don’t produce as equivalent energy yield measured by ATP.  In animals, the concentration of glucose in the blood is linked to the pancreatic endocrine hormone, insulin. . In most organisms, excess carbohydrates are regularly catabolized to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates.

Glucose is metabolized obtaining ATP and pyruvate by way of first splitting a six-carbon into two three carbon chains, which are converted to lactic acid from pyruvate in the lactic dehydrogenase reaction. The reverse conversion is by a separate unidirectional reaction back to pyruvate after moving through pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that convert pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the citric acid cycle. This multi-enzyme complex is related structurally and functionally to the oxoglutarate dehydrogenase and branched-chain oxo-acid dehydrogenase multi-enzyme complexes. In eukaryotic cells the reaction occurs inside the mitochondria, after transport of the substrate, pyruvate, from the cytosol. The transport of pyruvate into the mitochondria is via a transport protein and is active, consuming energy. On entry to the mitochondria pyruvate decarboxylation occurs, producing acetyl CoA. This irreversible reaction traps the acetyl CoA within the mitochondria. Pyruvate dehydrogenase deficiency from mutations in any of the enzymes or cofactors results in lactic acidosis.

PDH-rxns The acetyl group is transferred to coenzyme A

PDH-rxns The acetyl group is transferred to coenzyme A

http://guweb2.gonzaga.edu/faculty/cronk/biochem/images/PDH-rxns.gif

Typically, a breakdown of one molecule of glucose by aerobic respiration (i.e. involving both glycolysis and Kreb’s cycle) is about 33-35 ATP. This is categorized as:

Glycogenolysis – the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues.

Glycogenolysis in liver provides circulating glucose short term.

Glycogenolysis in muscle is obligatory for muscle contraction.

Pyruvate from glycolysis enters the Krebs cycle, also known as the citric acid cycle, in aerobic organisms.

Anaerobic breakdown by glycolysis – yielding 8-10 ATP

Aerobic respiration by Kreb’s cycle – yielding 25 ATP

The pentose phosphate pathway (shunt) converts hexoses into pentoses and regenerates NADPH. NADPH is an essential antioxidant in cells which prevents oxidative damage and acts as precursor for production of many biomolecules.

Glycogenesis – the conversion of excess glucose into glycogen as a cellular storage mechanism; achieving low osmotic pressure.

Gluconeogenesis – de novo synthesis of glucose molecules from simple organic compounds. An example in humans is the conversion of a few amino acids in cellular protein to glucose.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

The hormone insulin is the primary glucose regulatory signal in animals. It mainly promotes glucose uptake by the cells, and it causes the liver to store excess glucose as glycogen. Its absence

  1. turns off glucose uptake,
  2. reverses electrolyte adjustments,
  3. begins glycogen breakdown and glucose release into the circulation by some cells,
  4. begins lipid release from lipid storage cells, etc.

The level of circulatory glucose (known informally as “blood sugar”) is the most important signal to the insulin-producing cells.

  • insulin is made by beta cells in the pancreas,
  • fat is stored n adipose tissue cells, and
  • glycogen is both stored and released as needed by liver cells.
  • no glucose is released to the blood from internal glycogen stores from muscle cells.

The hormone glucagon, on the other hand, opposes that of insulin, forcing the conversion of glycogen in liver cells to glucose, and then release into the blood. Growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon.  These hormones are referred to as stress hormones because they are released under the influence of catabolic proinflammatory (stress) cytokines – interleukin-1 (IL1) and tumor necrosis factor α (TNFα).

Net Yield of GlycolysisThe preparatory phase consumes 2 ATP

The pay-off phase produces 4 ATP.

The gross yield of glycolysis is therefore

4 ATP – 2 ATP = 2 ATP

The pay-off phase also produces 2 molecules of NADH + H+ which can be further converted to a total of 5 molecules of ATP* by the electron transport chain (ETC) during oxidative phosphorylation.

Thus the net yield during glycolysis is 7 molecules of ATP*
This is calculated assuming one NADH molecule gives 2.5 molecules of ATP during oxidative phosphorylation.

Cellular respiration involves 3 stages for the breakdown of glucose – glycolysis, Kreb’s cycle and the electron transport system. Kreb’s cycle produces about 60-70% of ATP for release of energy in the body. It directly or indirectly connects with all the other individual pathways in the body.

The Kreb’s Cycle occurs in two stages:

  1. Conversion of Pyruvate to Acetyl CoA
  2. Acetyl CoA Enters the Kreb’s Cycle

Each pyruvate in the presence of pyruvate dehydrogenase (PDH) complex in the mitochondria gets converted to acetyl CoA which in turn enters the Kreb’s cycle. This reaction is called as oxidative  decarboxylation as the carboxyl group is removed from the pyruvate molecule in the form of CO2 thus yielding 2-carbon acetyl group which along with the coenzyme A forms acetyl CoA.

The PDH requires the sequential action of five co-factors or co-enzymes for the combined action of dehydrogenation and decarboxylation to take place. These five are TPP (thiamine phosphate), FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide), coenzyme A (denoted as CoA-SH at times to depict role of -SH group) and lipoamide.

Acetyl CoA condenses with oxaloacetate (4C) to form a citrate (6C) by transferring its acetyl group in the presence of enzyme citrate synthase. The CoA liberated in this reaction is ready to participate in the oxidative decarboxylation of another molecule of pyruvate by PDH complex.

Isocitrate undergoes oxidative decarboxylation by the enzyme isocitrate dehydrogenase to form oxalosuccinate (intermediate- not shown) which in turn forms α-ketoglutarate (also known as oxoglutarate) which is a five carbon compound. CO2 and NADH are released in this step. α-ketoglutarate (5C) undergoes oxidative decarboxylation once again to form succinyl CoA (4C) catalysed by the enzyme α-ketoglutarate dehydrogenase complex.

Succinyl CoA is then converted to succinate by succinate thiokinase or succinyl coA synthetase in a reversible manner. This reaction involves an intermediate step in which the enzyme gets phosphorylated and then the phosphoryl group which has a high group transfer potential is transferred to GDP to form GTP.

Succinate then gets oxidised reversibly to fumarate by succinate dehydrogenase. The enzyme contains iron-sulfur clusters and covalently bound FAD which when undergoes electron exchange in the mitochondria causes the production of FADH2.

Fumarate is then by the enzyme fumarase converted to malate by hydration(addition of H2O) in a reversible manner.

Malate is then reversibly converted to oxaloacetate by malate dehydrogenase which is NAD linked and thus produces NADH.

The oxaloacetate produced is now ready to be utilized in the next cycle by the citrate synthase reaction and thus the equilibrium of the cycle shifts to the right.

The NADH formed in the cytosol can yield variable amounts of ATP depending on the shuttle system utilized to transport them into the mitochondrial matrix. This NADH, formed in the cytosol, is impermeable to the mitochondrial inner-membrane where oxidative phosphorylation takes place. Thus to carry this NADH to the mitochondrial matrix there are special shuttle systems in the body. The most active shuttle is the malate-aspartate shuttle via which 2.5 molecules of ATP are generated for 1 NADH molecule. This shuttle is mainly used by the heart, liver and kidneys. The brain and skeletal muscles use the other shuttle known as glycerol 3-phosphate shuttle which synthesizes 1.5 molecules of ATP for 1 NADH.

Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH. The importance of this pathway can easily be underestimated.  The main source for energy in respiration was considered to be tied to the high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+. The pentose phosphate shunt is essential for the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

The pyridine nucleotide transhydrogenase reaction concerns the energy-dependent reduction of TPN by DPNH. In 1959, Klingenberg and Slenczka made the important observation that incubation of isolated liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate acceptor resulted in a rapid and almost complete reduction of  the intramitochondrial TPN. These and related findings led Klingenberg and co-workers (1-3) to postulate the occurrence of a ATP-controlled transhydrogenase reaction catalyzing the reduction of TPN by DPNH.  (The role of transhydrogenase in the energy-linked reduction of TPN.  Fritz Hommes, Ronald W. Estabrook, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6. http://dx.doi.org:/10.1016/0006-291X(63)90017-2/).

Further studies observed the coupling of TPN-specific dehydrogenases with the transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine nucleotide (TPN). The studies showed the direct interaction between TPNHz and DPN, in the presence of transhydrogenase to yield products having the properties of TPN and DPNHZ. The reaction involves a transfer of electrons (or hydrogen) rather than a phosphate. (Pyridine Nucleotide Transhydrogenase  II. Direct Evidence for and Mechanism of the Transhydrogenase Reaction* by  Nathan 0. Kaplan, Sidney P. Colowick, And Elizabeth F. Neufeld. (From The Mccollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland) J. Biol. Chem. 1952, 195:107-119.) http://www.JBC.org/Content/195/1/107.Citation
Notation: TPN, NADP; DPN, NAD+; reduced pyridine nucleotides: TPNH (NADPH2), DPNH (NADH).

Note: In this discussion there is a detailed presentation of the activity of lactic acid conversion in the mitochondria by way of PDH. In a later section there is mention of the bidirectional reaction of lactate dehydrogenase.  However, the forward reaction is dominant (pyruvate to lactate) and is described. This is not related to the kinetics of the LD reaction with respect to the defining characteristic – Km.

Biochemical Education Jan 1977; 5(1):15. Kinetics of Lactate Dehydrogenase: A Textbook Problem.
K.L. MANCHESTER. Department of Biochemistry, University of Witwatersrand, Johannesburg South Africa.

One presupposes that determined Km values are meaningful under intracellular conditions. In relation to teaching it is a simple experiment for students to determine for themselves the Km towards pyruvate of LDH in a post-mitochondrial supernatant of rat heart and thigh muscle. The difference in Km may be a factor of 3 or 4-fold.It is pertinent then to ask what is the range of suhstrate concentrations over which a difference in Km may be expected to lead to significant differences in activity and how these concentrations compare with pyruvate concentrations in the cell. The evidence of Vesell and co-workers that inhibition by pyruvate is more readily seen at low than at high enzyme concentration is important in emphasizing that under intracellular conditions enzyme concentrations may be relatively large in relation to the substrate available. This will be particularly so in relation to [NADH] which in the cytoplasm is likely to be in the ~M range.

A final point concerns the kinetic parameters for LDH quoted by Bergmeyer for lactate estimations a pH of 9 is recommended and the Km towards lactate at that pH is likely to be appreciably different from the quoted values at pH 7 — Though still at pH 9 showing a substantially lower value for lactate with the heart preparationhttp://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2877%2990013-9/pdf

Several investigators have established that epidermis converts most of the glucose it uses to lactic acid even in the presence of oxygen. This is in contrast to most tissues where lactic acid production is used for energy production only when oxygen is not available. This large amount of lactic acid being continually produced within the epidermal cell must be excreted by the cell and then carried away by the blood stream to other tissues where the lactate can be utilized. The LDH reaction with pyruvate and NADH is reversible although at physiological pH the equilibrium position for the reaction lies very far to the right, i.e., in favor of lactate production. The speed of this reaction depends not only on the amount of enzyme present but also on the concentrations of the substances involved on both sides of the equation. The net direction in which the reaction will proceed depends solely on the relative concentrations of the substances on each side of the equation.
In vivo there is net conversion of pyruvate (formed from glucose) to lactate. Measurements of the speed of lactate production by sheets of epidermis floating on a medium containing glucose indicate a rate of lactate production of approximately 0.7 rn/sm/
mm/mg of fresh epidermis.Slice incubation experiments are presumably much closer to the actual in vivo conditions than
the homogenate experiments. The discrepancy between the
two indicates that in vivo conditions are far from optimal for the conversion of pyruvate to lactate. Only 1/100th of the maximal activity of the enzyme present is being achieved. The concentrations of the various substances involved are not
optimal in vivo since pyruvate and NADH concentrations are
lower than lactate and NAD concentrations and this might explain the in vivo inhibition of LDH activity. (Lactate Production And Lactate Dehydrogenase In The Human Epidermis*. KM. Halprin, A Ohkawara. J Invest Dermat 1966; 47(3): 222-6.)
http://www.nature.com/jid/journal/v47/n3/pdf/jid1966133a.pdf

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Introduction to Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP

 

Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence.  It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.

There are essentially two major sources of fuel, mainly, carbohydrate and fat.  A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.

Carbohydrates are a major fuel utilized by way of either of two pathways.  They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle.  Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.

Carbohydrate metabolism has either of two routes of utilization.  This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle.  In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate.  In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital.  Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld,  who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.

This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.

The subchapters are as follows:

1.1      Carbohydrate Metabolism

1.2      Studies of Respiration Lead to Acetyl CoA

1.3      Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

1.4      The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy

Complex I or NADH-Q oxidoreductase

Complex I or NADH-Q oxidoreductase

Fatty acid oxidation and ETC

Fatty acid oxidation and ETC

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The multi-step transfer of phosphate bond and hydrogen exchange energy

Curator: Larry H. Bernstein, MD, FCAP, Leaders in Pharmaceutical Intelligence

In this subtext of the series we expand on a tie between respiration and glycolysis, and the functioning of the mitochondrion to discover the key role played by oxidative phosphorylation, “acetyl coenzyme A, and electron transport.  This was crucial to understanding cellular energetics, which explains the high energy of fatty acid catabolism from stored adipose tissue, and the criticality of the multi-step sequence of reactions in energy transfer.

This portion considerably provides a response to the TWO points made by Jose EDS Rosallis:

  1. Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. This poiny needs further clarification, but he makes important observations here.
  • Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it had changed from active form of the previous day to a non-active form.

The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity.

  • This assay was repeated with glycogen phosphorylase and the result was the opposite it increases its activity.

This led to the discovery of cAMP activated protein kinase and the assembly of a very complex system in the glycogen granule that is not a simple carbohydrate polymer. Instead

  • it has several proteins assembled and preserves the capacity to receive from a single event (rise in cAMP) two opposing signals with maximal efficiency,
  • stops glycogen synthesis, as long as levels of glucose 6 phosphate are low and
  • increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat these assays with

  • PK I, PKII and PKIII of M. Rouxii and Sutherland route to cAMP failed in this case.

I ask Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer)
(Nathan O. Kaplan discovery) indicating this as his idea. The reason was my “chief” (SP) more than once,  said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things. ” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.

[This reminds me of when I was studying the emergence of lactic dehysrogenase isoenzyme patterns in the developing eye lens of cattle, I raised reservations about Elliott Vessells challenge to Nathan Kaplan, but that not being my primary problem, my brilliant mentor (H.M.), a very young full professor of anatomy said – leave that to NOK.}

Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is

  • glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved.

This dialogue explains why during the Schroedinger’s “What is life?” reading with him he asked me if from biochemist in exile, to biochemist I expressed all of my thoughts to him. Since I had considered that Schrödinger did not confront Darlington & Haldane for being in exile. This may explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes in a way that suggests the the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of
that year(1971).

  1. show in detail with different colors what carbons belongs to CoA a huge molecule, in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield in comparison with anaerobic glycolysis. The idea is how much must have being spent in DNA sequences to build that molecule in order to use only two atoms of carbon. Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy). Latter, millions of years, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/
  1. Signaling transduction tutorial.
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-transduction-tutorial/
  1. Carbohydrate metabolism
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy

  1. Protein synthesis and degradation
  2. Subcellular structure
  3. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Oxidation-Reduction Reactions

Rachel Casiday, Carolyn Herman, and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/cytochromes.html

 

OX-Phos steps

OX-Phos steps

http://s1.hubimg.com/u/6583902_f496.jpg

 

Key Concepts:

  • ATP as Free-Energy Currency in the Body
  • Coupled Reactions
    • Standard Free-Energy Change for Coupled Reactions
    • ATP Dephosphorylation Coupled to Nonspontaneous Reactions
    • Coupled Reactions to Generate ATP
  • Structure and Function of the Mitochondria
  • Oxidation-Reduction Reactions in the Electron-Transport Chain
    • Electron-Carrier Proteins (NOTE: This section includes a separate link and an animation.)
    • Relationship Between Reduction Potentials and Free Energy
  • Proton Gradient as Means of Coupling Oxidative and Phosphorylation Components of Oxidative Phosphorylation
  • ATP Synthetase Uses Energy From Proton Gradient to Generate ATP

Every day, we build bones, move muscles, eat food, think, and perform many other activities with our bodies. All of these activities are based upon chemical reactions. However, most of these reactions are not spontaneous (i.e., they are accompanied by a positive change in free energy, DG>0) and do not occur without some other source of free energy. Hence, the body needs some sort of “free-energy currency,” (Figure 1) a molecule that can store and release free energy when it is needed to power a given biochemical reaction.

The four questions:

  1. How does the body “spend” free-energy currency to make a nonspontaneous reaction spontaneous? The answer, which is based on thermodynamics, is to use coupled reactions.
  2. How is food used to produce the reducing agents (NADH and FADH2) that can regenerate the free-energy currency? The answer, from biology, is found in glycolysis and the citric-acid cycle.
  3. How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)? Once again, coupled reactions are key.
  4. What mechanism does the body use to couple the reducing agent reactions and the generation of ATP? ATP is synthesized primarily by a two-step process consisting of an electron-transport chain and a proton gradient.  This process is based on electrochemistry and equilibrium, as well as thermodynamics.

The free-energy change (DG) for the net reaction is given by the sum of the free-energy changes for the individual reactions.  The phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.This step actually consists of two reactions:

(1) the phosphorylation of glycerol, and

(2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

      Glycerol + HPO42- –>  (Glycerol-3-Phosphate)2- + H2O DGo= +9.2 kJ
(nonspontaneous)
(2)
+      ATP4- + H2O –>       ADP3- + HPO42- + H+ DGo30.5 kJ
(spontaneous)
(3)
     Glycerol + ATP4- –> (Glycerol-3-Phosphate)2- +ADP3- + H+ DGo21.3 kJ
(spontaneous)
(4)
   

ATP is the most important “free-energy-currency” molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous; i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body’s use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

structure of ATP

structure of ATP

This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP.jpg

flowchart of food energy

flowchart of food energy

This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy, so that it is available to the body in a readily accessible form.

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart1.jpg

Glycolysis   Glucose + 2 HPO42- + 2 ADP3- + 2 NAD+ –>
2 Pyruvate + 2 ATP4- + 2 NADH + 2 H+ + 2 H2O
(5)
Intermediate Step   2(Pyruvate + Coenzyme A + NAD+ –>
Acetyl CoA + CO2 + NADH)
(6)
Citric-Acid Cycle 2(Acetyl CoA + 3 NAD++ FAD + GDP3-
+ HPO42- + 2H2O –> 2 CO2 + 3 NADH + FADH2
+ GTP4- + 2H+ + Coenzyme A)
(7)

The structures of the important molecules in Equations 5-7 are shown in Table 1, below.

How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.

pyruvate

pyruvate

  Pyruvate

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/pyruvate.jpg

acetylCoA

acetylCoA

Acetyl CoA

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

NADH

NADH

NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/acetylCoA.jpg

 

FADH2

FADH2

FADH2

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/FADH2.jpg

two-dimensional representations of several important molecules in Equations 5-7.

As seen in Equations 5-7 in the blue box, glycolysis and the citric-acid cycle produce a net total of only four ATP or GTP molecules (GTP is an energy-currency molecule similar to ATP) per glucose molecule. This yield isfar below the amount needed by the body for normal functioning, and in fact is far below the actual ATP yield for glucose in aerobic organisms (organisms that use molecular oxygen). For each glucose molecule the body processes, the body actually gains approximately 30 ATP molecules! (See Figure 4, below.)  So, how does the body generate ATP?

The process that accounts for the high ATP yield is known as oxidative phosphorylation. A quick examination of Equations 5-7 shows that glycolysis and the citric-acid cycle generate other products besides ATP and GTP, namely NADH and FADH2 (blue). These products are molecules that are oxidized (i.e., give up electrons) spontaneously. The body uses these reducing agents (NADH and FADH2) in an oxidation-reduction reaction .  As you will see later in this tutorial, it is the free energy from these redox reactions that is used to drive the production of ATP.

flowchart - making of ATP

flowchart – making of ATP

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/flowchart2.jpg

This flowchart shows the major steps involved in breaking down glucose from the diet and converting its chemical energy to the chemical energy in the phosphate bonds of ATP, in aerobic (oxygen-using) organisms. Note: In this flowchart, red denotes a source of carbon atoms (originally from glucose),green denotes energy-currency molecules, and blue denotes the reducing agents that can be oxidized spontaneously.

In the discussion above, we see that glucose by itself generates only a tiny amount of ATP. However, during the breakdown of glucose, a large amount of NADH and FADHis produced; it is these reducing agents that dramatically increase the amount of ATP produced. How does this work?

How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)?

As discussed in an earlier section about coupling reactions, ATP is used as free-energy currency by coupling its (spontaneous) dephosphorylation (Equation 3) with a (nonspontaneous) biochemical reaction to give a net release of free energy (i.e., a net spontaneous reaction). Coupled reactions are also used to generate ATP by phosphorylating ADP. The nonspontaneous reaction of joining ADP to inorganic phosphate to make ATP (Equation 8, below, and Figure 2, above) is coupled to the oxidation reaction of NADH or FADH(Equation 9, below). (Recall, NADH and FADH2 are produced in glycolysis and the citric-acid cycle as described in the blue box). For simplicity, we shall henceforth discuss only the oxidation of NADH; FADH2 follows a very similar oxidation pathway.

The oxidation reaction for NADH has a larger, but negative, DG than the positive DG required for the formation of ATP from ADP and phosphate. This set of coupled reactions is so important that it has been given a special name: oxidative phosphorylation. This name emphasizes the fact that an oxidation (of NADH) reaction (Equation 9 and Figure 5, below) is being coupled to a phosphorylation (of ADP) reaction (Equation 8, below, and Figure 2, above). In addition, we must consider the reduction reaction (gaining of electrons) that accompanies the oxidation of NADH. (Oxidation reactions are always accompanied by reduction reactions, because an electron given up by one group must be accepted by another group.) In this case, molecular oxygen (O2) is the electron acceptor, and the oxygen is reduced to water (Equation 10, below) .

The individual reactions of interest for oxidative phosphorylation are:

Phosphorylation

ADP3- + HPO42- + H+ –>
ATP4- + H2O

DGo= +30.5 kJ
(nonspontaneous)
(8)
oxidation

NADH –> NAD+ + H+ +  2e

DGo158.2 Kj
(spontaneous)
(9)
reduction

1/2 O2 + 2H+ + 2e –> H2O

DGo61.9 kJ
(spontaneous)

                                                                       (10)                                    

The net reaction is obtained by summing the coupled reactions, as shown in Equation 11, below.

ADP3- + HPO42- + NADH + 1/2 O2 + 2H+ –>
ATP4- + NAD+ + 2 H2O
DGo= -189.6 kJ
(spontaneous)
(11)

The molecular changes that occur upon oxidation of NADH are shown:

NAD+_NADH

NAD+_NADH

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/NAD+_NADH.jpg

This is a two-dimensional (ChemDraw) representation showing the change that occurs when NADH is oxidized to NAD+. “R” represents the part of the structure that is shown in black in the drawing of NADH in Table 1, and does not change during the oxidation half-reaction. The molecular changes that occur upon oxidation are shown in red.

In this tutorial, we have seen that nonspontaneous reactions in the body occur by coupling them with a very spontaneous reaction (usually the ATP reaction shown in Equation 3). We have just seen that ATP is produced by coupling the phosphorylation reaction with NADH oxidation (a very spontaneous reaction). But we have not yet answered the question: by what mechanism are these reactions coupled?

Coupling Reactions in Biological Systems

Every day your body carries out many nonspontaneous reactions. As discussed earlier, if a nonspontaneous reaction is coupled to a spontaneous reaction, as long as the sum of the free energies for the two reactions is negative, the coupled reactions will occur spontaneously. How is this coupling achieved in the body? Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme. Consider again the phosphorylation of glycerol (Equations 2-4). Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver. The product of glycerol phosporylation, glycerol-3-phosphate (Equation 2), is used in the synthesis of phospholipids.

Glycerol kinase is a large protein comprised of about 500 amino acids. X-ray crystallography of the protein shows us that there is a deep groove or cleft in the protein where glycerol and ATP attach (see Figure 6, below). Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol. Instead of two separate reactions where ATP loses a phosphate (Equation 3) and glycerol picks up a phosphate (Equation 2), the enzyme allows the phosphate to move directly from ATP to glycerol (Equation 4).

The coupling in oxidative phosphorylation uses a more complicated (and amazing!) mechanism, but the end result is the same: the reactions are linked together, the net free energy for the linked reactions is negative, and, therefore, the linked reactions are spontaneous.

glyckin

glyckin

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/glyckin.jpg

This is a schematic representation of ATP and glycerol bound (attached) to glycerol kinase. The enzyme glycerol kinase is a dimer (consists of two identical subuits). There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate (spontaneous) and glycerol gaining a phosphate (nonspontaneous) are linked together as one spontaneous process

Questions on ATP: The Body’s Free-Energy Currency (How Free-Energy Currency Works)

  • Biological systems involve many molecules containing phosphate groups, such as ATP. Although ATP is the most commonly used free-energy currency, any of these phosphorylated molecules could, in theory, be used as free-energy currency. The standard free-energy change (DGo) for the dephosphorylation (removal of a phosphate group) of several biological compounds is given below:
Acetyl phosphate DGo = -47.3 kJ/mol
Adenosine triphosphate (ATP) DGo = -30.5 kJ/mol
Glucose-6-phosphate DGo = -13.8 kJ/mol
Phosphoenolpyruvate (PEP) DGo = -61.9 kJ/mol
Phosphocreatine DGo = -43.1 kJ/mol

Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency (i.e., the amount of nonspontaneous reactions enabled per phosphate removed from a molecule of free-energy currency) from the most efficient to the least efficient.

  • What, if any, changes are there in the shape of the ring as NADH is oxidized to NAD+(see Figure 5)? (Hint: Consider which atoms lie in the same plane in each structure.)

Mechanism of Coupling the Oxidative-Phosphorylation Reactions

In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles (specialized cellular components) known as mitochondria. A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions.

Synthesis of ATP (Equation 8) is coupled with the oxidation of NADH (Equation 9) and the reduction of O2 (Equation 10). There are three key steps in this process:

  1. Electrons are transferred from NADH, through a series of electron carriers, to O2. The electron carriers are proteins embedded in the inner mitochondrial membrane. (More detail about the structure of the mitochondria is presented in the next section.) (See Figure 7a.)
  2. Transfer of electrons by these carriers generates a proton (H+) gradient across the inner mitochondrial membrane. (See Figure 7b.)
  3. When Hspontaneously diffuses back across the inner mitochondrial membrane, ATP is synthesized. The large positive free energy of ATP synthesis is overcome by the even larger negative free energy associated with proton flow down the concentration gradient. (See Figure 7c.)

These steps are outlined below.

  1. Electron Transport (Oxidation-Reduction Reactions) Through a Series of Proteins in the Inner Membrane of the Mitochondria
e_transfer

e_transfer

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/e_transfer.jpg

Generation of H+(Proton) Concentration Gradient Across the Inner Mitochondrial Membrane During the Electron-Transport Process (via a Proton Pump)

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/gradient.jpg

Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+Back to the Matrix Inside the Inner Mitochondrial Membrane

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/ATP_produced.jpg

The three major steps in oxidative phosphorylation are

(a) oxidation-reduction reactions involving electron transfers between specialized proteins embedded in the inner mitochondrial membrane; 

(b) the generation of a proton (H+) gradient across the inner mitochondrial membrane (which occurs simultaneously with step (a)); and 

(c) the synthesis of ATP using energy from the spontaneous diffusion of electrons down the proton gradient generated in step (b).

Note: Steps (a) and (b) show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron (green) from another protein in the electron-transport chain, an Fe(III) ion in the center of a heme group (purple) embedded in the protein is reduced to Fe(II). The coordinates for the protein were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

Cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP. Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs.

Structure and Function of the Mitochondria

mitochondria

mitochondria

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/mitochondria.jpg

This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below. An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure.

The mitochondrial membranes are crucial for this organelle’s role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane ispermeable to most small molecules and ions, because it contains large protein channels called porins. The inner membrane is impermeable to most ions and polar molecules. The inner membrane is the site of oxidative phosphorylation. Although the membrane is mostly impermeable, it contains special H+ (proton) channels and pumps that enable the coupling of the redox reaction involving NADH and O2 (Equations 9-10) to the phosphorylation reaction of ADP (Equation 8), as described below (“Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation”). (Recall the discussion of protein channels in the “Maintaining the Body’s Chemistry: Dialysis in the Kidneys” Tutorial .)

As shown in Figure 8, inside the inner membrane is a space known as the matrix; the space between the two membranes is known as the intermembrane space. The matrix side of the inner membrane has a negative electrical charge relative to the intermembrane space due to an H+ gradient set up by the redox reaction (Equations 9 and 10). This charge difference is used to provide free energy (G) for the phosphorylation reaction (Equation 8).

Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation

Phosphorylation of ADP (Equation 8) is coupled to the oxidation-reduction reaction of NADH and O2 (Equations 9 and 10). Electrons are not transferred directly from NADH to O2, but rather pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. Why? This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion. As we shall see, it is this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis.

Two major types of mitochondrial proteins (see Figure 9, below) are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.

  1. The electron carriers (NADH-Q reductase, ubiquinone (Q), cytochrome reductase, cytochrome c, and cytochrome oxidase shown in shades of purple in Figure 9 below) transport electrons in a stepwise fashion from NADH to O2.  Three of these carriers (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) are also proton pumps, and simultaneously pump H+ ions (protons) from the matrix to the intermembrane space. (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.) The protons that are pumped across the membrane complete the redox reaction (Equations 9 and 10). The creation of a proton gradient across the membrane is one way of storing free energy.
  2. ATP synthetase (shown in red in Figure 9 below) allows H+ ions to diffuse back into the matrix and uses the free energy released to synthesize ATP from ADP and HPO42-. The ATP synthetase is essential for the phosphorylation to occur (Equation 8). (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.)

The electron carriers can be divided into three protein complexes (NADH-Q reductase (1), cytochrome reductase (3), and cytochrome oxidase (5)) that pump protons from the matrix to the intermembrane space, and two mobile carriers (ubiquinone (2) and cytochrome c (4)) that transfer electrons between the three proton-pumping complexes. (Gold numbers refer to the labels on each protein in Figure 9, below.) Because electrons move from one carrier to another until they are finally transferred to O2, the electron carriers (shown in Figure 9,below) are said to form an electron-transport chain.

Figure  below, is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on “View the Movie.”

Proteins of inner space

Proteins of inner space

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/Proteins.jpg

This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation. Click the blue button below to download QuickTime 4.0 to view the movie.

NADH-Q reductase (1), cytochrome reductase (3) , and cytochrome oxidase (5) are electron carriers as well as proton pumps, using the energy gained from each electron-transfer step to move protons (H+) against a concentration gradient, from the matrix to the intermembrane space.Ubiquinone (Q) (2) and cytochrome c (Cyt C) (4) are mobile electron carriers. (Ubiquinone is not actually a protein.) All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials. Together, these electron carriers form a “chain” to transport electrons from NADH to O2. The path of the electrons is shown with the green dotted line.

ATP synthetase (red) has two components: a proton channel (allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix), and a catalytic component to catalyze the formation of ATP.

For a more complete description of each step in oxidative phosphorylation (indicated by the gold numbers), click here.

view movie

view movie

http://www.apple.com/quicktime/

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/movie.jpg

http://www.chemistry.wustl.edu/~edudev/LabTutorials/Cytochromes/images/QuickTime.jpg

Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that, although NADH donates two electrons and O2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time. Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial.

In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron tranfers from one electron carrier to the next spontaneous?

What causes electrons to be transferred down the electron-transport chain?

As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in a iron-sulfur (Fe-S) group or in a heme group. (Recall the heme group from the Chem 151 tutorial “Hemoglobin and the Heme Group: Metal Complexes in the Blood“.) The iron in these groups is alternately oxidized and reduced between Fe(II) (reduced) or Fe(III) (oxidized) states.

Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential (e) of a reduction half reaction is, the greater the tendency is for the species to accept an electron. Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change.

Reduction Potentials and Relationship to Free Energy

An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential (e). By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction (i.e., the opposite reaction). The electrical potential for an oxidation-reduction reaction is calculated by

erxn = eoxidation + ereduction (12)

For example, for the overall reaction of the oxidation of NADH paired with the reduction of O2, the potential can be calculated as shown below.

Reduction Potentials ereduction
NAD+ + 2H+ + 2e –> NADH + H+ -0.32 V
(1/2) O2 + 2H+ + 2e –> H2O +0.82 V

The overall reaction is

NADH + H–> NAD+ + 2H+ + 2e eoxidation = 0.32 V
(1/2) O2 + 2H+ + 2e –> H2O ereduction = 0.82 V
net: NADH + (1/2)O2 + H+ –>
H2O + NAD+
erxn = 1.14 V

The electrical potential (erxn) is related to the free energy (DG) by the following equation:

DG= -nFerxn (13)

where n is the number of electrons transferred (in moles, from the balanced equation), and F is the Faraday constant (96,485 Coulombs/mole). (Using this equation, DG is given in Joules; one Joule = 1 Volt x 1 Coulomb.)

Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy (DG =-220 kJ); i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16, below.

reduced cytochrome c–> oxidized cytochrome c+ e eoxidation = – .220 V (14)
oxidized cytochrome c + e –> reduced cytochrome c ereduction = .250 V (15)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = 0.030 V (16) Spontaneous

We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

reduced cyt c–> oxidized cyt c+ e eoxidation = – .220 V (17)
oxidized cyt b + e –> reduced cyt b ereduction = – 0.34 V (18)
NET: reduced cyt c1 + oxidized cyt c –>
oxidized cyt c+ reduced cyt c
erxn = – 0.56 V (19) NOT Spontaneous

Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in DG (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

Complex Name Half Reaction Reduction Potential
Cytochrome reductase

(also known as cytochrome b-c1 complex)

(3 in Figure 9)

Cytochrome b (Fe(III) center)
+ e –>
Cytochrome b (Fe(II) center)
-0.34 V
(at pH 7, T=30oC)
Cytochrome c1 (Fe(III) center)
+ e– –>
Cytochrome c1 (Fe(II) center)
+0.220 V
(at pH 7, T=30oC)
Cytochrome c

(4 in Figure 9)

Cytochrome c (Fe(III) center)
+ e– –>
Cytochrome c (Fe(II) center)
+0.250 V
(at pH 7, T=30oC)
Cytochrome oxidase

(5 in Figure 9)

Cytochrome oxidase
( Fe(III) center) + e– –>
Cytochrome oxidase
(Fe(II) center)
+0.285 V
(at pH 7.4, T=25oC)
Table 2

To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

Hence, the electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H+. As we shall see below, this huge concentration gradient leads to the production of ATP.

Questions on Electron Carriers: Steps in the Electron-Transport Chain; Reduction Potentials and Relationship to Free Energy

  • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
  • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H+ ions) in the intermembrane space is increased.  Could cells move protons (H+ ions) from the matrix to the intermembrane space without transporting electrons?  Why or why not?

 ATP Synthetase: Production of ATP

We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily-available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

ATP synthetase is a protein consisting of two important segments: a transmembrane proton channel, and a catalytic component located inside the matrix. The proton-channel segment allows H+ ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Thus, since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.

Questions on ATP Synthetase: Production of ATP

  • A scientist has created a phospholipid-bilayer membrane containing ATP-synthetase proteins. Instead of a proton gradient, this scientist has created a large Cs+ gradient (many Cs+ ions on the side of the membrane without the catalytic unit, and few Cs+ ions on the side of the membrane with the catalytic unit). Would you expect the ATP-synthetase proteins in this membrane to be able to generate ATP, given an abundant supply of ADP and phosphate? Briefly, explain your answer. (HINT: Draw on your knowledge of the structure of protein channels to predict what effect replacing H+ ions with Cs+ ions would have.)
  • Certain toxins allow H+ ions to move freely across the inner mitochondrial membrane (i.e., without needing to pass through the channel in ATP synthetase). What effect do you expect these toxins to have on the production of ATP? Briefly, explain your answer.

Summary

In this tutorial, we have learned that the ability of the body to perform daily activities is dependent on thermodynamic, equilibrium, and electrochemical concepts.   These activities, which are typically based on nonspontaneous chemical reactions, are performed by using free-energy currency. The common free-energy currency is ATP, which is a molecule that easily dephosphorylates (loses a phosphate group) and releases a large amount of free energy. In the body, the nonspontaneous reactions are coupled to this very spontaneous dephosphorylation reaction, thereby making the overall reaction spontaneous (DG < 0). As the coupled reactions occur (i.e., as the body performs daily activities), ATP is consumed and the body regenerates ATP by using energy from the food we eat (Figure 3). As seen in Figure 4, the breakdown of glucose (glycolysis) obtained from the food we eat cannot by itself generate the large amount of ATP that is needed for metabolic energy by the body. However, glycolysis and the subsequent step, the citric-acid cycle, produce two easily oxidized molecules: NADH and FADH2. These redox molecules are used in an oxidative-phosphorylation process to produce the majority of the ATP that the body uses. This oxidative-phosphorylation process consists of two steps: the oxidation of NADH (or FADH2) and the phosphorylation reaction which regenerates ATP. Oxidative phosphorylation occurs in the mitochondria, and the two reactions (oxidation of NADH or FADHand phosphorylation to generate ATP) are coupled by a proton gradient across the inner membrane of the mitochondria (Figure 9). As seen in Figures 7 and 9, the oxidation of NADH occurs by electron transport through a series of protein complexes located in the inner membrane of the mitochondria. This electron transport is very spontaneous and creates the proton gradient that is necessary to then drive the phosphorylation reaction that generates the ATP. Hence, oxidative-phosphorylation demonstrates that free energy can be easily transferred by proton gradients. Oxidative-phosphorylation is the primary means of generating free-energy currency for aerobic organisms, and as such is one of the most important subjects in the study of bioenergetics (the study of energy and its chemical changes in the biological world).

Additional Link:

  • This fun description of oxidative phosphorylation by Dr. E.J.Oakeley contains step-by-step animated illustrations of the redox reactions involved, as well as a quiz to test your understanding of the material.

References:

Alberts, B. et al. In Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.: New York, 1994, pp. 653-684.

Becker, W.M. and Deamer, D.W. In The World of the Cell, 2nd ed., The Benjamin/Cummings Publishing Co., Inc.: Redwood City, CA, 1991, pp. 291-307.

Fasman, G.D. In Handbook of Biochemistry and Molecular Biology, 3rd ed., CRC Press, Inc.: Cleveland, OH, 1976, Vol. I (Physical and Chemical Data), pp. 132-137.

Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.

Moa, C., Ozer, Z., Zhou, M. and Uckun, F. X-Ray Structure of Glycerol Kinase Complexed with an ATP Analog Implies a Novel Mechanism for the ATP-Dependent Gylcerol Phosphorylation by Glycerol Kinase.Biochemical and Biophysical Reaearch Communications. 1999, 259, 640-644.

Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.

Stryer, L. In Biochemistry, 4th. ed., W.H. Freeman and Co.: New York, 1995, pp. 490, 509, 513, 529-557.

Zubay, G. Biochemistry, 3rd. ed., Wm. C. Brown Publishers: Dubuque, IA, 1983, p. 42.

Acknowledgements:

The authors thank Dewey Holten (Washington University in St. Louis) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.

Copyright 1999, Washington University, All Rights Reserved.

 

 

 

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