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)

The immediate and direct regulation of insulin release by circulating nutrients, especially glucose, is thought to be mediated in the pancreatic B-cell by a sequence of metabolic, ionic, and motile events. On the basis of previous work, it is assumed that the process by which glucose is recognized as an insulinotropic agent entirely depends on the metabolic changes evoked by the sugar in the islet cells. Several factors are considered as possible candidates for the coupling between these metabolic changes and subsequent ionic events such as altered phosphate, chloride, sodium, potassium, and calcium handling. It is acknowledged that changes in the concentrations of glycolytic intermediates and cyclic nucleotides (adenosine- or guanosine-3', 5'-cyclic monophosphate), or both, could play a modulatory role upon stimulated insulin release. However, the initiation of insulin release seems to depend on the generation of two essential coupling factors: H+ and reduced pyridine nucleotides. The changes in H+ fluxes may account for the glucose-induced decrease in K+ and Ca2+ fractional outflow rate, all three parameters displaying hyperbolic-like dose-response curves with half-maximal values at noninsulinotropic glucose concentrations. The changes in NAD(P)H concentration may account for a glucose-induced Ca2+--Ca2+ exchange process due to a change in affinity of a native ionophoretic system. The dose-response curves for these parameters yield a sigmoidal pattern analogous to that which depicts the rate of insulin release at increasing glucose concentrations. It is proposed that such a coupling between metabolic and cationic events is operative in response to other insulinotropic nutrients and that its time course may be relevant to the phasic aspect of insulin release. Thus, the nutrient-induced release of insulin (and possibly other pancreatic hormones), which is essential for the regulation of fuel homeostasis, would depend on the capacity of circulating nutrients to act as a fuel in the islet cells. This concept raises a question as to the existence and nature of feedback mechanisms regulating the metabolic fluxes in the islet cells as a function of their energy expenditure.
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PMID:Insulin release: the fuel hypothesis. 3 43

Mammalian and avian muscles were examined histochemically and biochemically to determine the relative contribution of membrane bound (mitochondrial and sarcotubular) ATPases under the same conditions employed for myofibrillar ATPase. For histochemically investigated Ca+(+)-ATPase activity following incubation at pH 9.4 according to the calcium-citro-phosphate technique, avian muscle displayed distinct mitochondrial localization in both dark and light staining fibres. However, mitochondrial localization did not occur in mammalian muscle fibres. Pretreatment of unfixed frozen sections with ouabain, cyanide and acetone did not prevent the reticular distribution in avian muscle fibres. The present study demonstrates that "myofibrillar" localization is achieved by the Ca+(+)-precipitation technique: provided frozen sections are pretreated with cold acetone, fixed in a fixative containing oligomycin or azide and then incubated in a medium containing glycine-NaO H as buffer. Mitochondria prepared by successive mechanical homogenization or by Nagarse treatment plus 2 min homogenization develop different ATPase activities at pH 9.4 7.4 6.0 and 4.35 as well as stimulation by 70 mM Ca++ at these pHs compared to those ATPase activities in the homogenate of mixed hamster hind leg muscles. Glycerol-3-phosphate dehydrogenase and creatine kinase (both located at the outer surface of the inner mitochondrial membrane) and succinate dehydrogenase and glutamate dehydrogenase (localized at the inner mitochondrial membrane and in the matrix resp.) also show different activities in both mitochondria preparations indicating different membrane properties of both mitochondria. Evidence is obtained that using the calcium-citro-phosphate technique at pH 9.4 oligomycin-sensitive and -insensitive ATPases are activated by Ca++ in both mitochondria preparations. Since in muscle homogenate less than 10% of Ca+(+)-stimulated ATPase activity is oligomycin-sensitive, mitochondrial ATPase exhibit only a small portion of total ATPase from mixed hamster hind leg muscles.
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PMID:Histochemical and biochemical investigations of adenosine triphosphatase in vertebrate mixed muscles. 4 33

1. The effects of phosphate and electron transport on the ATPase induced in rat-liver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin. 2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase. 3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (greater than 0.2 muM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin. 4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations. 5. It is proposed that the steady-state concentration of endogenous P-i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P-i.
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PMID:The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria. 12 70

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.
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PMID:Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate. 12 85

The homogeneous rat liver F1-ATPase preparation of Catterall and Pedersen (Catterall, W.A., and Pedersen, P.L. (1971) J. Biol. Chem. 246, 4987-4994) has been crystallized from a solution containing phosphate and ATP by precipitation with ammonium sulfate. Most of the resultant crystals are cubes of approximately 0.3 to 0.6 mm per side. X-ray precession photographs show that the crystals are rhombohedral, space group R32 (D37 NO155) with hexagonal cell dimensions a = 148 A, c = 368 A. The molecular weight of the asymmetric unit of the crystals is 190,000 or about half the molecular weight (384,000) of the rat liver enzyme indicating that the crystallographic 2-fold axes of symmetry coincide with a molecular symmetry axis. The crystals diffract to at least 3.5 A and therefore this is the first report of an ATPase preparation in which crystals suitable for x-ray analysis have been obtained.
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PMID:Adenosine triphosphatase from rat liver mitochondria. Crystallization and x-ray diffraction studies of the F1-component of the enzyme. 14 72

1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.
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PMID:The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration. 16 20

To study ESR absorption of mitochondrial ATPase a special flow system was developed allowing to maintain surviving conditions for mitochondria. It has been shown that ESR free radical signal of mitochondria observed at room temperature (g-factor 2.00 and halfwidth of about 15 Gs) depends on their metabolic state. An increase of free radical content during mitochondrial energization can be associated with operation of ATPsynthetase. It is supposed that free radicals take part in the reaction of ADP phosphorylation and that a molecule of ADP itself bound in the active centre of ATPase can become a free radical to facilitate the process of inorganic phosphate incorporation.
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PMID:[Participation of free radicals in ATP synthesis]. 21 18

Laser Raman spectroscopy has been used to study a phosphate transfer reaction from ATP to Pi or arsenate in dimethyl sulfoxide. The spectra support a mechanism involving Mg-2+ binding to the alpha or beta phosphates of ATP leaving the third phosphate free for the transfer reaction. The data also indicate the formation of a relatively stable intermediate which is facilitated by the presence of dimethyl sulfoxide and a dicarboxylic acid (maleate). The intermediate has a Raman spectrum with a band at 1090.5 cm- minus 1 similar to the end product ADP, but is formed much more rapidly. Since the model reaction has many features in common (e.g., activation by maleate) with the transfer reactions catalyzed by coupling factors from spinach chloroplast, Raman spectroscopy may also prove to be a useful tool in the elucidation of biological energy transfer reactions.
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PMID:Laser Raman spectroscopy as a mechanistic probe of the phosphate transfer from adenosine triphosphate in a model system. 112 86

The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase nor its ability to be activated by the proton gradient. It seems therefore to block only the proton flow required by the catalytic function of the enzymes. Venturicidin, however, also slows down the deactivation of the thiol-reduced ATPases during uncoupled ATP hydrolysis, following a delta mu H+ activation, but phloridzin, a reversible F1 inhibitor, has the same effect. Tentoxin, an irreversible F1 inhibitor, decreases the rate of ATP hydrolysis but does not affect the rate of deactivation. These findings suggest that catalytic and regulatory H(+)-binding sites are different. No distinction can be made, if any, between protons involved in unmasking the thiol-sensitive groups of F1 and in activating the enzyme. The effect of venturicidin and phloridzin on the deactivation is consistent with an inhibitory effect of newly formed--by ATP hydrolysis--ADP molecules, which might affect the enzyme without passing through the medium. Phosphate at millimolar concentration has an effect similar to low concentrations of phloridzin and venturicidin, probably by a simple back-reaction effect.
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PMID:An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase. 131 60

The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme. A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins. The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop. The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding. Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches. A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands. Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution. Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included. Magnesium is not required for the interaction with the purine nucleotides. Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E. coli. While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain. 133 55


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