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)

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1.
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PMID:A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae. 13 3

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.
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PMID:Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site. 14 73

The mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analogue, p-fluorosulfonylbenzoyl-5'-adenosine. This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer containing 20% glycerol. Under these conditions, 4.2 g atoms of 14C are incorporated per 350,000 g of enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM p-fluorosulfonyl[14C]benzoyl-5'-adenosine. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was associated with the two largest subunits. The isolated alpha subunit contained 0.45 g atom of 14C/mol and the isolated beta subunit contained 0.88 g atom of 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the beta subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled beta subunit is contained ina tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-Tyr-Asp-Val-Ala-Arg, where Tyr is the radioactive derivative of the tyrosine residue that was sulfonylated during the inactivation.
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PMID:Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine. 15 Apr 16

The emission maximum of the fluorescence spectrum of mitochondrial F1-ATPase is shifted from 305 to 334 nm when the excitation wavelength is altered from 270 to 300 nm. This indicates that both tyrosine and tryptophan contribute to the intrinsic fluorescence of the F1-ATPase.
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PMID:Intrinsic fluorescence of mitochondrial F1-ATPase. 15 35

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.
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PMID:The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation. 15 5

The dicyclohexylcarbodiimide-binding protein of Aspergillus nidulans has been identified as the smallest subunit of the mitochondrial ATPase complex, and has a molecular weight of approximately 8000. It is extractable from whole mitochondria and from the purified enzyme in neutral chloroform/methanol, contains 30% polar amino acids, and the N-terminal amino acid has been identified as tyrosine. Using a double-labelling technique in the absence and presence of cycloheximide, followed by immunoprecipitation of the enzyme complex with antiserum against Neuospora crassa F1 ATPase, it has been shown that this subunit is synthesized on cytoplasmic ribosomes.
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PMID:Mitochondrial ATPase complex of Aspergillus nidulans and the dicyclohexylcarbodiimide-binding protein. 15 78

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.
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PMID:On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine. 15 96

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.
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PMID:The mitochondrial ATPase. Evidence for a single essential tyrosine residue. 23 39

1. When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4-chloro-7-nitrobenzofurazan, is incubated at pH 9.0, the 7-nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N-nitrobenzofurazan ATPase has little or no activity. 2. The fluorescence of the N-nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP. 3. Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N-nitrobenzofurazan chromophore is exclusively in the beta subunit. 4. The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds. 5. The interaction of the N-nitrobenzofurazan ATPase with aurovertin is reported.
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PMID:The mitochondrial ATPase. Selective modification of a nitrogen residue in the beta subunit. 23 40

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).
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PMID:Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation. 131 7


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