Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deletion mutations in the NH2- and COOH-terminal regions of the epsilon subunit of Escherichia coli ATP synthase were constructed making use of the AatII and HincII restriction enzyme sites. The resultant mutated epsilon species were analyzed for in vivo functionality and for recognition by anti-epsilon monoclonal antibodies. Deletion of residues Asp-7 through Gln-14 (epsilon delta D7-Q14) resulted in reduced ability to complement uncC mutants as determined by growth yields on limiting glucose medium and by formation of small colonies on plates with succinate as the source of carbon and energy. None of the other mutants was notably impaired. Upon induction to obtain overexpression, the NH2-terminal deletion mutants were expressed at levels comparable to the wild-type epsilon subunit, but the COOH-terminal deletion mutants were expressed less strongly, suggesting that residues in the latter region are important for protein stability. Monoclonal antibody epsilon-1, which cannot bind to epsilon when it is part of F1-ATPase, recognized the COOH-terminal deletions well, but the NH2-terminal deletions poorly. Additional epitope mapping using epsilon fusion proteins revealed that residues required for the epsilon-1 epitope extend to between Thr-77 and Arg-85. Monoclonal antibody epsilon-4, which can bind to epsilon when it is part of F1-ATPase, recognized the NH2-terminal deletions well, but hardly recognized the COOH-terminal deletions, indicating a role of residues located COOH-terminal to Ile-131 in recognition by this antibody. Epitope mapping using the fusion proteins revealed that the residues required by epsilon-4 begin in the region between Val-78 and Met-95. These results imply a two-domain structure of epsilon and orient the subunit within the enzyme.
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PMID:Orientation of the epsilon subunit in Escherichia coli ATP synthase. 768 93

An F1-ATPase-defective mutant, TBLA-1, was constructed by the transduction of a defective gene for the alpha subunit of F1-ATPase, atpA401, into Escherichia coli W1485lip2, a lipoic acid-requiring pyruvic acid producer. The pyruvic acid production of the strain TBLA-1 was found to be improved markedly compared with that of strain W1485lip2. In cultures using a jar fermentor, the strain W1485lip2 consumed 50 g/liter of glucose and produced 25 g/liter of pyruvic acid after culture for 32h, while strain TBLA-1 consumed the same amount of glucose, and produced more than 30 g/liter of pyruvic acid in a 24-h culture. A revertant, No. 63-1, derived from the strain TBLA-1, had a normal level of F1-ATPase activity, and showed a similar pattern of pyruvic acid production to that of strain W1485lip2.
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PMID:Pyruvic acid production by an F1-ATPase-defective mutant of Escherichia coli W1485lip2. 776 10

The highly conserved beta subunit of the Escherichia coli F1F0 ATPase was divided into three sections, each of which was exchanged with the homologous section of the beta subunit of the obligate aerobe Bacillus megaterium. Plasmids coding for the resultant six chimeric beta subunits varied in their abilities to complement two E. coli beta mutants as measured by testing transformed cells for aerobic growth on a nonfermentable carbon source or anaerobic growth on rich medium containing glucose. Two chimeras were able to restore both growth on succinate and anaerobic growth on rich medium. The genetic results corresponded to increased levels of membrane-bound ATPase and ATP synthase activities. These chimeric subunits were therefore capable of being assembled into functional E. coli ATPase complexes. The results indicate that chimeric beta subunits can be used to analyze assembly of the beta subunit and that the final 181 amino acids of the beta subunit might contain a region involved in functional energy coupling.
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PMID:Construction and function of chimeric beta subunits containing regions from the beta subunits of the F1F0 ATPases of Escherichia coli and Bacillus megaterium. 782 74

Two strains of Escherichia coli that lack the epsilon subunit of the F1F0 ATP synthase have been constructed. They are shown to be viable but with very low growth yields (28%). These strains can be complemented by plasmids carrying wild-type uncC, but not when epsilon is overproduced. These results indicate that epsilon is not essential for growth on minimal glucose medium and that the level of its expression affects the assembly of the ATP synthase.
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PMID:Construction and plasmid-borne complementation of strains lacking the epsilon subunit of the Escherichia coli F1F0 ATP synthase. 783 27

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic beta-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802-5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose-->malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling. 801 51

When Ehrlich ascites carcinoma (EAC) cells and thymocytes were treated with uncoupler or rotenone in glucose-free medium, rapid ATP depletion was observed in both types of the cells. Oligomycin slowed down ATP loss in thymocytes, but not in EAC cells. Thus, mitochondrial ATP hydrolysis appears to be significant in deenergized thymocytes in contrast to EAC cells, in which other ATP consuming reactions were prevailing. Complete deenergization of mitochondria by uncoupler or rotenone in these cells resulted in inactivation of mitochondrial ATPase by 65-75%. The effect was observed after complete and rapid (20-30 s) disruption of the cells with detergent, Lubrol WX. ATPase was blocked by the specific inhibitor protein (IF1) as it was shown by the studies on reactivation of this enzyme. When respiration is blocked but ATP content is supported by glycolysis, mitochondrial ATPase is not suppressed by IF1, and maintains the energization of mitochondria. It is concluded that under complete de-energization of mitochondria IF1 significantly inhibits mitochondrial ATP hydrolysis and may slow down ATP loss in thymocytes and EAC cells.
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PMID:Mitochondrial ATP hydrolysis and ATP depletion in thymocytes and Ehrlich ascites carcinoma cells. 827 14

The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.
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PMID:Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts. 836 48

Residue beta Y331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and lin-benzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized beta Y331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kappa cat/Km and lin-benzo-ADP binding characteristics similar to wild type. Fluorescence from beta W331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites. ATP, ADP, or AMPPNP caused virtually complete quenching of beta W331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme. Therefore the beta W331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions. We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates. Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site. We also measured AMPPNP and ADP binding parameters. For ADP, the "on" rate at the first catalytic site was much faster (> or = 5 x 10(5) M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed. For AMPPNP, the "on" rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.
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PMID:Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied. 837 71

A metabolic model of fuel sensing has been proposed in which malonyl-CoA and long-chain acyl-CoA esters may act as coupling factors in nutrient-induced insulin release (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney J, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802-5810, 1992). To gain further insight into the control of malonyl-CoA content in islet tissue, we have studied the short- and long-term regulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the beta-cell. These enzymes catalyze the formation of malonyl-CoA and its usage for de novo fatty acid biogenesis. ACC mRNA, protein, and enzymatic activity are present at appreciable levels in rat pancreatic islets and clonal beta-cells (HIT cells). Glucose addition to HIT cells results in a marked increase in ACC activity that precedes the initiation of insulin release. Fasting does not modify the ACC content of islets, whereas it markedly downregulates that of lipogenic tissues. This indicates differential regulation of the ACC gene in lipogenic tissues and the islets of Langerhans. FAS is very poorly expressed in islet tissue, yet ACC is abundant. This demonstrates that the primary function of malonyl-CoA in the beta-cells is to regulate fatty acid oxidation, not to serve as a substrate for fatty acid biosynthesis. The anaplerotic enzyme pyruvate carboxylase, which allows the replenishment of citric acid cycle intermediates needed for malonyl-CoA production via citrate, is abundant in islet tissue. Glucose causes an elevation in beta (HIT)-cell citrate that precedes secretion, and only those nutrients that can elevate citrate induce effective insulin release. The results provide new evidence in support of the model and explain why malonyl-CoA rises markedly and rapidly in islets upon glucose stimulation: 1) glucose elevates citrate, the precursor of malonyl-CoA; 2) glucose enhances ACC enzymatic activity; and 3) malonyl-CoA is not diverted to lipids. The data suggest that ACC is a key enzyme in metabolic signal transduction of the beta-cell and provide evidence for the concept that an anaplerotic/malonyl-CoA pathway is implicated in insulin secretion.
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PMID:Evidence for an anaplerotic/malonyl-CoA pathway in pancreatic beta-cell nutrient signaling. 854 64

Special features of glucose metabolism in pancreatic beta-cells are central to an understanding of the physiological role of these cells in glucose homeostasis. Several of these characteristics are emphasized: a high-capacity system for glucose transport; glucose phosphorylation by the high-Km glucokinase (GK), which is rate-limiting for glucose metabolism and determines physiologically the glucose dependency curves of many processes in beta-cell intermediary and energy metabolism and of insulin release and is therefore viewed as glucose sensor; remarkably low activity of lactate dehydrogenase and the presence of effective hydrogen shuttles to allow virtually quantitative oxidation of glycolytic NADH; the near absence of glycogen and fatty acid synthesis and of gluconeogenesis, such that intermediary metabolism is primarily catabolic; a crucial role of mitochondrial processes, including the citric acid cycle, electron transport, and oxidative phosphorylation with FoF1 ATPase governing the glucose-dependent increase of the ATP mass-action ratio; a Ca(2+)-independent glucose-induced respiratory burst and increased ATP production in beta-cells as striking manifestations of crucial mitochondrial reactions; control of the membrane potential by the mass-action ratio of ATP and voltage-dependent Ca2+ influx as signal for insulin release; accumulation of malonyl-CoA, acyl-CoA, and diacylglycerol as essential or auxiliary metabolic coupling factors; and amplification of the adenine nucleotide, lipid-related, and Ca2+ signals to recruit many auxiliary processes to maximize insulin biosynthesis and release. The biochemical design also suggests certain candidate diabetes genes related to fuel metabolism: low-activity and low-stability GK mutants that explain in part the maturity-onset diabetes of the young (MODY) phenotype in humans and mitochondrial DNA mutations of FoF1 ATPase components thought to cause late-onset diabetes in BHEcdb rats. These two examples are chosen to illustrate that metabolic reactions with high control strength participating in beta-cell energy metabolism and generating coupling factors and intracellular signals are steps with great susceptibility to genetic, environmental, and pharmacological influences. Glucose metabolism of beta-cells also controls, in addition to insulin secretion and insulin biosynthesis, an adaptive response to excessive fuel loads and may increase the beta-cell mass by hypertrophy, hyperplasia, and neogenesis. It is probable that this adaptive response is compromised in diabetes because of the GK or ATPase mutants that are highlighted here. A comprehensive knowledge of beta-cell intermediary and energy metabolism is therefore the foundation for understanding the role of these cells in fuel homeostasis and in the pathogenesis of the most prevalent metabolic disease, diabetes.
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PMID:Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. 854 69


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