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)

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.
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PMID:Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump. 290 67

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.
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PMID:In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri. 292 75

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.
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PMID:Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa. 621 16

Active transport of catecholamines into chromaffin granules is driven by the transmembrane pH gradient and membrane potential, created by an electrogenic proton-translocating ATPase in the granule membrane. The ATPase activity of highly purified chromaffin granule membranes is inhibited by a number of agents in common with mitochondrial ATPase, and also by antibodies raised against mitochondrial F1. Dichloromethane treatment of these membranes solubilizes an enzyme that is closely similar to mitochondrial F1, but distinguishable from it by its interaction with specific antisera and the inhibitor aurovertin. Chromaffin granule membranes contain a low-molecular-weight protein that reacts with dicyclohexylcarbodiimide; it can be extracted into chloroform-methanol, and is of higher electrophoretic mobility than the corresponding mitochondrial protein. Evidence is presented that this is a component of the proton-translocating ATPase complex.
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PMID:Proton-translocating ATPase of chromaffin granule membranes. 628 79

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.
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PMID:Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae. 632 65

1. The isolation of the mitochondrial ATPase F1 and its beta-subunit from commercial baker's yeast (Saccharomyces cerevisiae) is described. 2. The molecular weight determined by ultracentrifugation is 340000 +/- 30000. Gel chromatography indicates a molecular weight of 300000 +/- 20000. 3. Fluorimetric titration of the isolated enzyme with aurovertin reveals two binding sites per molecule. The isolated beta-subunit binds aurovertin in a 1 : 1 stoicheiometry. It is concluded that the ATPase molecule contains two aurovertin-binding beta-subunits. 4. The stabilizing agent methanol influences both the measured Kd and the concentration of binding sites for aurovertin. These results fit a model in which both F1 and aurovertin are distributed between aqueous and methanol phases. 5. The effect of methanol on the ATPase activity can be described in terms of the model proposed by Recktenwald and Hess (Recktenwald, D. and Hess, B. (1977) FEBS Lett. 76, 25-28). It is proposed that methanol enhances the affinity of the regulatory site for ATP, but at higher concentrations prevents the interaction between the regulatory and catalytic sites. 6. Since HSO(-3), a typical effector of the assumed regulatory site of F1, has no effect on the binding of aurovertin, it is concluded that the binding site of aurovertin is not correlated with the regulatory site. 7. The inhibition of ATPase activity by aurovertin is slowly (t 1/2 = 70 s) induced during turnover conditions. 8. From the effect of methanol on the inhibition of ATPase activity by aurovertin it is concluded that under turnover conditions the conformation is such that the aurovertin-binding sites have a 6-fold higher affinity for methanol than under resting conditions.
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PMID:Studies on the structure and conformation of yeast mitochondrial ATPase using aurovertin and methanol as probes. 644 70

1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.
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PMID:Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria. 645 Dec 41

The effects of glycerol and methanol upon beef heart mitochondrial ATPase (F1) were studied. Glycerol was found to be a potent reversible inhibitor of the F1-catalyzed hydrolysis of ATP and ITP. The inhibition of ATP hydrolysis was linear with respect to glycerol concentrations, while that of ITP was not. From the temperature dependence of Vmax for F1-catalyzed ATP and ITP hydrolysis in glycerol or methanol solutions, the energy of activation and the enthalpy of activation were calculated. The inhibitory effect of ADP on F1 hydrolytic activity was studied in three solvent systems (totally aqueous, 20% methanol, and 20% glycerol). Compared to the aqueous system, methanol decreased the potency of ADP as an inhibitor, and glycerol enhanced the potency.
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PMID:Kinetic and thermodynamic properties of beef heart mitochondrial ATPase: effect of co-solvent systems. 645 17

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In soluble F1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP in ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.
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PMID:Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol. 646 56

Subunit c of the Escherichia coli F1F0-ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent-solubilized F0 subunits a and b to form a functionally active H+ channel. The rates of H+ uptake by the proteoliposomes containing the reconstituted F0 complex were comparable to those observed with native F0 reconstituted without subunit dissociation. The F0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of the F1-ATPase sector of the complex. Reconstitution of D61N and D61G mutant c subunits with wild-type subunits a and b produced an inactive F0. Hybrid F0 complexes, formed with mixtures of wild-type and D61N or D61G mutant c subunits, were also prepared. Formation of an active F0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e. active F0 formation was gradually disrupted as the mutant/wild-type ratio was increased from 0.05 to 0.2. The hybrid reconstitution studies support a model where inactivation of one of the 9-12 c subunits found in F0 is sufficient to abolish activity.
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PMID:Reconstitution of the Fo complex of Escherichia coli ATP synthase from isolated subunits. Varying the number of essential carboxylates by co-incorporation of wild-type and mutant subunit c after purification in organic solvent. 758 91


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