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 affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].
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PMID:Determination of the roles of active sites in F1-ATPase by controlled affinity labeling. 253 46

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase. 255 70

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.
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PMID:Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles. 282 18

Monoatomic alcohols at definite concentration stimulated or inhibited activities of liver mitochondrial ATPase, anion-sensitive ATPase in erythrocyte membranes and Na, K-ATPase in rat brain depending on the ratio of concentrations of the natural substrate components--Mg2+ complex and ATP. Interrelationships between the enzymes and alcohols hydrophobic properties were found.
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PMID:[Hydrophobic properties of various phosphohydrolases]. 284 17

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.
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PMID:AlF4- reversibly inhibits 'P'-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase. 284 38

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.
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PMID:Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. 285 69

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at 10(-9) and 10(-6) M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.
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PMID:Regulation of oxidative phosphorylation in mitochondria by external free Ca2+ concentrations. 285 85

The rate of mitochondrial ATPase inactivation by the naturally occurring inhibitor protein in the presence of saturating ATP and Mg2+ at pH 8.0 depends hyperbolically on the amount of inhibitor added; the upper limit of an apparent first-order constant for the inactivation process is 1.0(-1) at 25 degrees C. A dramatic difference in the inactivation rate is observed when the protein inhibitor is added to the same assay system from either acidic (pH 4.8) or alkaline (pH 8.2) solutions. The slow reversible transition of the inhibitor from its rapidly reacting 'acidic' form to the slow reacting 'alkaline' form occurs when the solution of the protein inhibitor is subjected to a pH-jump from 4.8 to 8.2 (t1/2 approximately 30s at 25 degrees C). The pH-profile of the inhibitor active/inactive equilibrium suggests that a group with pKa approximately 6.5 is involved in the transition. Treatment of the inhibitor protein with a histidine-specific reagent (e.g. diethyl pyrocarbonate) abolishes its inactivating effect on the ATPase activity. It is concluded that the protonation/deprotonation of the inhibitor protein followed by its slow conformational changes is the rate-limiting step in the inhibitor-ATP synthetase interaction.
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PMID:Interaction between the mitochondrial ATP synthetase and ATPase inhibitor protein. Active/inactive slow pH-dependent transitions of the inhibitor protein. 286 10

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.
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PMID:Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1. 286 Jan 7

The F1-ATPase from the uncD11 mutant of E. coli (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., & Futai, M. (1980) J. Biochem. 88, 695-703), showed different enzymological properties from the wild-type enzyme. The mutant F1-ATPase had biphasic kinetics and essentially the same Km values as the wild-type enzyme, although its Vmax values were lower. The mutant enzyme showed altered sensitivities to dicyclohexylcarbodiimide (DCCD), azide and quercetin; it was less sensitive than the wild-type to quercetin and DCCD, and its Mg2+-dependent ATPase activity was slightly more resistant to azide than that of the wild-type, whereas its Ca2+-dependent activity was more sensitive. On the other hand, the mutant and wild-type F1 were inhibited equally by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). The fact that the Mg2+- and Ca2+-dependent F1-ATPase activities of the wild-type and mutant responded differently to quercetin and azide suggested that their mechanisms of action were different. Previous studies (Noumi, T., Mosher, M.E., Natori, S., Futai, M., & Kanazawa, H. (1984) J. Biol. Chem. 259, 10071-10075) indicated that Ser is replaced by Phe at residue 174 of the beta subunit of the mutant. Thus the Ser residue or its neighboring area(s) may constitute the binding site of DCCD, quercetin and azide.
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PMID:Change of inhibitor sensitivities of Escherichia coli F1-ATPase due to a mutational substitution of Phe for Ser at residue 174 of the beta subunit. 286 63


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