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)

When the bovine mitochondrial F1-ATPase is inactivated with dicyclohexyl[14C]carbodiimide and then gel-filtered, from 2 to 3 g atoms of 14C are incorporated/mol of enzyme. Prior inactivation of the enzyme by the modification of an essential tyrosine residue with 4-chloro-7-nitrobenzofurazan, a reaction that can be reversed by thiols, does not affect the irreversible inactivation of the ATPase by dicyclohexyl[14C]carbodiimide. During the large scale modification of the F1-ATPase by dicyclohexyl[14C]carbodiimide which led to 70% inactivation, 1.9 g atoms of 14C were incorporated/mol of enzyme. Isolation of the alpha, beta, and gamma subunits from this large scale inactivation revealed that the gram atoms of 14C bound per mol of each of the subunits was: alpha, 0.04; beta, 0.56; and gamma, 0.04. The majority of the radioactivity in a cyanogen bromide digest of the 14C-labeled beta subunit was isolated in a fragment that has the following amino acid sequence: Glu-Leu-Ile-Asn-Asn-Val-Ala-Lys-Ala-His-Gly-Gly-Tyr-Ser-Val-Phe-Ala-Gly-Val-Gly -Glu-Arg-Thr-Arg-Glu-Gly-Asn-Asp-Leu-Tyr-Glu*-His-Met; where Glu* represents the N gamma-glutamyl derivative of dicyclohexyl[14C]urea.
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PMID:Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit. 611 57

Reaction of the photoaffinity label 8-azido-[2-3H] ATP with bovine heart mitochondrial F1-ATPase abolishes its enzyme activity; inhibition is prevented by ATP (Wagenvoord, R.J., van der Kraan, I., and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). More than 65% of the radioactivity is associated with the beta-subunit and about 25% with the alpha-chains. Radioactivity in the beta-subunit is localized in two specific regions. One corresponds to residues 1-12 (Runswick, M.J., and Walker, J.E. (1983) J. Biol. Chem. 258, 3081-3089), a region which is nonessential for catalysis. Radioactivity in the second region is localized predominantly on three amino acids, lysine 301, isoleucine 304, and tyrosine 311. It seems likely that these residues are found in the vicinity of the ATP binding site of F1-ATPase.
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PMID:The sites of labeling of the beta-subunit of bovine mitochondrial F1-ATPase with 8-azido-ATP. 622 27

Inactivation of the bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.5, led to the incorporation of 1.42 g atoms of 14C/mol. Treatment of the inactivated enzyme with dithiothreitol removed 0.99 g atom of 14C/mol of enzyme which was accompanied by reactivation of the ATPase. Therefore, of the 1.42 mol of 7-chloro-4-nitro-[14C]benzofurazan incorporated per mol of bovine heart mitochondrial F1-ATPase, 0.43 mol was present on lysine residues and 0.99 mol was present on tyrosine residues. When the inactivated enzyme was treated with 10 mM sodium dithionite at pH 6.0, 10% of the activity was recovered which was accompanied by a 10% loss in covalently bound 14C. Following dithionite treatment, that part of the 14C which remained covalently bound could not be removed by subsequent treatment of the labeled enzyme with dithiothreitol. It is presumed that dithionite reduces the 4-nitro group of the covalently bound reagent, converting it to 4-amino[14C]benzofurazan derivatives at lysine and tyrosine residues. The moles of 4-amino[14C]benzofurazan incorporated per mol of the isolated subunits were: alpha, 0.18; beta, 0.30; gamma, 0.03; and delta plus epsilon, less than 0.01. Gel filtration of a cyanogen bromide digest of the labeled beta subunit on Sephadex G-75 resolved a major 14C peak which contained 83% of the 14C recovered. The major, radioactive tryptic fragment derived from this peak was purified by gel filtration on Sephadex G-75 followed by reversed phase high performance liquid chromatography. Automatic Edman degradation of this peptide showed that the 14C was released at the position occupied by beta-Tyr-311.
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PMID:Identification of the essential tyrosine residue in the beta subunit of bovine heart mitochondrial F1-ATPase that is modified by 7-chloro-4-nitro[14C]benzofurazan. 623 10

Bovine heart mitochondrial ATPase is inhibited after covalent modification with 4-chloro-7-nitrobenzofuroxan. The kinetics of the reaction are indistinguishable from those for the reaction of an essential tyrosine residue of the ATPase with 4-chloro-7-nitrobenzofurazan that have been described previously [Ferguson et al. (1975) Eur. J. Biochem. 54, 117-126]. 4-Fluoro-7-nitrobenzofurazan inhibits the ATPase with a pseudo-first-order rate constant that is tenfold greater than that for 4-chloro-7-nitrobenzofurazan. These data indicate that the rate-limiting step for reaction of the enzyme with these reagents is formation of a Meisenheimer complex at the C-4 position and that the modified tyrosine is probably on the surface of the protein. No evidence was found for more complex patterns of reactivity of 4-chloro-7-nitrobenzofurazan and its analogues. Both ammonium 4-chloro-7-sulphobenzofurazan and ammonium 4-fluoro-7-sulphobenzofurazan fail to react with the ATPase. The utility of these reagents as alternatives to the nitro derivatives may be limited owing to their slow reaction rates. After modification on tyrosine by 4-chloro-7-nitrobenzofurazan, the nitrobenzofurazan group can be transferred by an intramolecular process to lysine [Ferguson et al. (1975) Eur. J. Biochem. 54, 127-133]. ATPase with the lysine thus modified is shown to be reactive towards 4-chloro-7-nitrobenzofurazan in a manner indistinguishable from the native enzyme. This indicates that the intramolecular transfer occurs at sufficient distance to avoid steric hindrance to the second reaction, and that the lysine does not participate in a neighbouring group effect to enhance the reactivity of the tyrosine.
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PMID:The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4-chloro-7-nitrobenzofurazan and related compounds. 623 12

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .
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PMID:Identification of an essential lysine residue in the beta subunit of the F1-ATPase from the thermophilic bacterium, PS3, using 7-chloro-4-nitro[14C]benzofurazan. 623 50

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.
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PMID:Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan. 623 61

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.
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PMID:Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding. 631 39

The natural mitochondrial ATPase inhibitor (IF1) was modified with a radioactivity labeled heterobifunctional and photosensitive reagent, methyl 4-azido(14C)benzimidate ((14C)MABI). Titration experiments of IF1 by (14C)MABI and tryptic maps of (14C)MABI-IF1 indicated that specific lysine residues in IF1 are preferentially labeled by (14C)MABI. Under appropriate conditions of labeling (1 to 2 lysine residues modified per IF1), MABI-IF1 exhibited the same inhibitory potency as native IF1 on the hydrolytic activity of the coupling factor 1 of mitochondrial ATPase (F1). The same conditions were required for inhibition of F1 by MABI-IF1 and IF1 (slightly acidic pH and presence of ATP and MgCl2). In photolabeling experiments, (14C)MABI-IF1 was used to investigate the localization of IF1 binding sites on F1. Upon photoirradiation, MABI-IF1 bound selectively to the beta subunit of soluble or membrane-bound F1. Adenylyl imidodiphosphate and quercetin, two compounds which partially mimic the inhibitory effect of IF1 on ATPase activity of F1, markedly prevented the binding of (14C)MABI-IF1 to F1; on the other hand, aurovertin, a specific ligand of the beta subunit of F1, did not affect the interaction between (14C)MABI-IF1 and F1. In the absence of light, (14C)MABI-IF1 was used as a reversible radiolabeled ligand with respect to membrane bound F1 to investigate F1-IF1 interactions to inside-out submitochondrial particles as a function of the energy state of the particles. Oxidation of NADH by submitochondrial particles resulted in a decrease of bound (14C)MABI-IF1; the effect was counteracted by antimycin. The data suggested that added (14C)MABI-IF1 is capable of exchanging with IF1 bound to F1 in submitochondrial particles and that the rate and extent of (14C)MABI-IF1 release are triggered by the proton-motive force developed by the particles.
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PMID:Photoaffinity labeling of mitochondrial adenosine triphosphatase by an azido derivative of the natural adenosine triphosphate inhibitor. 645 97

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

The subunit c protein of mitochondrial ATP synthase accumulates in lysosomal storage bodies of numerous tissues in human subjects with certain forms of ceroid-lipofuscinosis, a degenerative hereditary disease. Subunit c appears to constitute a major fraction of the total storage-body protein. Lysosomal accumulation of subunit c has also been reported in putative animal models (dogs, sheep and mice) for ceroid-lipofuscinosis. In humans with the juvenile form of the disease, hydrolysates of total storage-body protein have been found to contain significant amounts of epsilon-N-trimethyl-lysine (TML). TML is also abundant in storage-body protein hydrolysates from affected dogs and sheep. These findings suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. In a putative canine model for human juvenile ceroid-lipofuscinosis, residue 43 of the storage-body subunit c was previously found to be TML. In the present study, subunit c was isolated from the storage bodies of humans with juvenile ceroid-lipofuscinosis, and from sheep and mice with apparently analogous diseases. In all three species, partial amino acid sequence analysis of the stored subunit c indicated that the protein contained TML at residue 43. These findings strongly suggest that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.
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PMID:Mitochondrial ATP synthase subunit c stored in hereditary ceroid-lipofuscinosis contains trimethyl-lysine. 757 23


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