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 inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.
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PMID:The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. 286 44

The purified, soluble F1-ATPase was modified by several covalently reacting inhibitors, either known or considered to bind to the active site bearing beta-subunit, to cause partial inhibition up to 99%. The modified enzyme was then reconstituted in the presence of OSCP (oligomycin sensitivity conferring protein) with submitochondrial particles (SMP) almost completely (greater than 99%) denuded of active F1-ATPase and was assayed for oligomycin-sensitive ATPase and oxidative phosphorylation activities. The inhibitors used were 1-fluoro-2,4-dinitrobenzene (FDNB), N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCD), quinacrine mustard (QM), 5-(dimethylamino)-naphthalene-1-sulfonyl chloride (dansyl-Cl), 5'-[p-(fluoro-sulfonyl)benzoyl]adenosine (FSBA), and N,N'-dicyclohexylcarbodiimide (DCCD). The SMP reconstituted with unmodified F1 exhibited oxidative phosphorylation and oligomycin-sensitive ATPase (in the presence of uncouplers) activities as high as 500 nmol min-1 mg-1 and 8 mumol min-1 mg-1, respectively. The systems reconstituted with F1 modified to cause various degrees of inhibition with FDNB, EEDQ, CMCD, QM, and dansyl-Cl exhibited the same degree of inhibition of oxidative phosphorylation and oligomycin-sensitive ATPase activities as the inhibition of the ATPase activity of the modified F1 before reconstitution. The systems reconstituted with FSBA-modified F1 showed the following relative degrees of inhibition: oxidative phosphorylation greater than oligomycin-sensitive ATPase of particles greater than ATPase of soluble F1. In contrast, the systems reconstituted with DCCD-modified F1 showed much greater inhibition of oligomycin-sensitive ATPase than of oxidative phosphorylation activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibitory chemical modifications of F1-ATPase: effects on the kinetics of adenosine 5'-triphosphate synthesis and hydrolysis in reconstituted systems. 623 51

The natural ATPase inhibitor (IF1) from beef heart mitochondria was labeled with phenyl (14C)isothiocyanate [(14C)PITC]. Chemical labeling by (14C)PITC does not modify the inhibitory properties of IF1, provided the number of residues of (14C)PITC bound per molecule of IF1 is lower than five to six, which corresponds to the average labeling of roughly half of the available lysine residues in IF1. This partially labeled, fully active, IF1 was used to determine the binding stoichiometry of IF1 with respect to F1 and to localize the inhibitor binding sites in F1-ATPase. The pattern of loss of ATPase activity of F1 with increasing amounts ot (14C)PITC-IF1 indicated that the ATPase activity is fully inhibited when 1 mol of IF1 is bound to 1 mol of F1. As F1 contains at least 2 beta subunits, this points to a half-site reactivity of F1 with respect to IF1. Sites of interaction between (14C)PITC-IF1 and F1 subunits were investigated by the use of two cross-linking reagents which act as "zero length" cross-linkers, 1-ethyl-3-[(dimethylamino)propyl]carbodiimide (EDAC) and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline (EEDQ); the products of cross-linking were analyzed by NaDodSO4-polyacrylamide gel electrophoresis. IF1 was found to bind preferentially to the beta subunit of F1. Among the cross-linked products formed by reaction of EDAC or EEDQ with subunits of F1, one of them, the beta gamma dimer, did not accumulate when IF1 was added to F1 prior to cross-linking.
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PMID:Radiolabeling of natural adenosine triphosphatase inhibitor with phenyl (14C)isothiocyanate and study of its interaction with mitochondrial adenosine triphosphatase. Localization of inhibitor binding sites and stoichiometry of binding. 739 10