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)

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.
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PMID:Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria. 217 20

Ion-selective electrodes recorded the pH (7.49 +/- 0.05, n = 8) and pCa (6.72 +/- 0.03, n = 40) in samples (approximately 1 microliter) of isolated Myxicola axoplasm mounted within 760-micron diameter plastic tubes. We determined the interactions between Ca2+ and H+ on axoplasmic buffers by microinjecting CaCl2 or HCl into the axoplasmic samples at a distance 75-125 micron from the tips of the electrodes (distance = r). When axoplasmic pH was lowered 0.97 +/- 0.095 from its resting value (measured at r = 125 micron) by injecting 4 nmol HCl, pCa dropped 0.30 +/- 0.05 (n = 6). When expressed in units of concentration, these data show that a HCl injection of approximately 4 mmol/l axoplasm increased H+ and Ca2+ activity by approximately 0.3 microM. Lowering axoplasmic pCa 2.20 +/- 0.43 (r = 75 micron) (n = 3) by injecting 40 pmol CaCl2 had only a small effect on pH. In other experiments, two Ca2+ electrodes measured the Ca2+ activity 125 and 375 micron from the site of CaCl2 injection. Evidence of Ca2+ buffering was obtained when the Ca2+ activity at these two locations was below that expected for simple Ca2+ diffusion away from the injection site. Centrifuged axoplasm (100,000 g) taken from the bottom of the centrifuge tube had a somewhat greater Ca2+ buffering capacity than that taken from the top of the tube. Electron microscopic studies of the centrifuged axoplasm showed a greater concentration of mitochondria and other axoplasmic vesicles in the bottom of the centrifuge tube. Ruthenium red (20-40 micrograms/ml) greatly reduced Ca2+ buffering. The mitochondrial inhibitors CN (2 mM) and oligomycin (a mixture of oligomycin A, B, and C, 5 micrograms/ml) also reduced Ca2+ buffering but were not as effective as ruthenium red. Axoplasm in which ATP and mitochondrial substrates were removed by dialysis was unable to lower free Ca2+ when the concentration of this ion was elevated to approximately 10 microM. In the presence of oligomycin to block mitochondrial ATPase, and with Mg2+ -ATP as the only source of energy, axoplasm lowered Ca2+ activity slowly; with succinate as the only metabolic substrate, axoplasm rapidly lowered the Ca2+ activity from approximately 10 microM to below 1 microM.
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PMID:Calcium and proton buffering and diffusion in isolated cytoplasm from Myxicola axons. 242 Jan 93

The effects of gramicidin S (GS), an antibiotic, on the rat heart membrane ATPases and contractile activity of the right ventricle strips were investigated. GS inhibited sarcolemmal Ca2+-stimulated ATPase (IC50 = 3 microM), Ca2+/Mg2+ ATPase which is activated by millimolar Ca2+ or Mg2+ (IC50 = 3.4 microM), and sarcoplasmic reticulum Ca2+-stimulated ATPase (IC50 = 6 microM). The type of inhibition for the sarcolemmal Ca2+/Mg2+ ATPase by GS was apparently uncompetitive, while that for Ca2+-stimulated ATPases in sarcolemma or sarcoplasmic reticulum was of mixed type. Other ATPases, including mitochondrial ATPase, sarcolemmal Na+-K+ ATPase, and myofibrillar ATPase, were not inhibited by this agent. GS also decreased the rat right ventricle maximum force development (half-maximal inhibitory concentration was 2-4 microM), maximum velocity of contraction, and maximum velocity of relaxation. The resting tension was increased by GS to over 200%. The contractile actions of GS were mostly irreversible upon washing the muscle 3 times over a 10-min period. Decreased Ca2+, Mg2+, Na+, K+ concentrations in the perfusate increased the effects of GS. These findings showed that GS was a potent inhibitor of divalent cation ATPases of heart sarcolemma and sarcoplasmic reticulum and it is suggested that these membrane effects may explain the cardiodepressant action of this agent.
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PMID:Influence of gramicidin S on cardiac membrane Ca2+/Mg2+ ATPase activities and contractile force development. 247 7

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force. 248 79

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.
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PMID:Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex. 252 40

Although the binding of nucleotides at the noncatalytic sites of F1-ATPase has been regarded as probably having some type of regulatory function, only limited observations have been reported that support such a role. We present here results showing that the presence of ATP at noncatalytic sites can give a fivefold enhancement of the rate of GTP hydrolysis by the chloroplast F1-ATPase. Heat-activation of the chloroplast F1-ATPase in the presence of ATP, followed by column separation from the medium nucleotides gives an enzyme with two of the three noncatalytic sites filled with ATP. In contrast, heat-activation in the presence of ADP gives an enzyme with only one noncatalytic site filled with ADP. Such an enzyme with two noncatalytic sites empty catalyzes MgGTP hydrolysis only very slowly. The filling of a second noncatalytic site with ATP by exposure of the enzyme to ATP without Mg2+ present, followed by column separation, markedly increases the rate of GTP hydrolysis. A further increase occurs when a third noncatalytic site is filled by exposure to Mg2+ and ATP. The rate of MgATP hydrolysis is the same for the enzyme heat-activated in the presence of ATP or ADP, probably because MgATP, unlike MgGTP, rapidly binds to both catalytic and noncatalytic sites.
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PMID:Modulation of the GTPase activity of the chloroplast F1-ATPase by ATP binding at noncatalytic sites. 252 43

The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons. (a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added. (b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid. (c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.
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PMID:Effect of polyamines on mitochondrial F1-ATPase catalyzed reactions. 253 Feb 10

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.
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PMID:Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. 253 Feb 15

The amount of F1-ATPase in chromatophores from Rhodospirillum rubrum was determined by Western blotting using anti-RrF1 rabbit antibodies. 9.1 mmol F1 (mol bacteriochlorophyll)-1 was obtained or 14% of the total protein content of the chromatophores. The turnover rate of the F0F1-ATPase was 17 molecules ATP s-1 during synthesis, 2 molecules ATP s-1 during hydrolysis under coupled conditions with Mg2+ as the divalent cation, and 7 molecules ATP s-1 during hydrolysis in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Binding of 1 mol oligomycin/mol F0F1-ATPase was found to inhibit the activities of the enzyme completely. A single binding site was found with a Kd of approximately 2 microM.
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PMID:Amount and turnover rate of the F0F1-ATPase and the stoichiometry of its inhibition by oligomycin in Rhodospirillum rubrum chromatophores. 253 30

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.
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PMID:The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase. 253 13


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