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)

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.
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PMID:The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa. 294 21

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.
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PMID:Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9. 295 97

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

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.
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PMID:Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide. 622 13

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 effect of inorganic phosphate, ADP, ATP, and their analogues on the rate of labeling of F1-ATPase by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and phenylglyoxal have been investigated. Analysis of the kinetic data indicate that the labeled functional groups of the essential tyrosine and arginine residues respectively are both located at the catalytic site of F1. The active phenolic group of tyrosine is located closer to the bound inorganic phosphate or the gamma-phosphate group than the alpha- and beta-phosphate groups of the bound ATP at the catalytic site, whereas the guanidinium group of arginine is located closer to the alpha- and beta-phosphate groups of the bound ATP than to its gamma-phosphate group or the bound inorganic phosphate. The kinetically deduced dissociation constants are 1.3 mM and 210 microM for the inorganic phosphate and aDP respectively bound to this catalytic site. Labeling the essential tyrosine residue by NBD-Cl has been found to facilitate subsequent labeling of the essential arginine residue by phenylglyoxal.
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PMID:Effect of phosphate and adenine nucleotides on the rate of labeling of functional groups at the catalytic site of F1-ATPase. 645 51

The role of tyrosine in the catalytic mechanism of nucleoside triphosphate hydrolysis by beef heart mitochondrial ATPase is explored. We compare the rates of the ATPase reaction by both nitrated and native F1 at both pH 8 and pH 6. The pH-activity profile of nitrated F1 is compared to the pH-activity profile of the unmodified enzyme. These data indicate that the phenolic group of an active-site tyrosine must be protonated during the hydrolysis reaction. Deuterium oxide is used in the reaction buffer to explore the role of protons in the ATPase reaction. Kinetic constants of the nucleoside triphosphates are obtained at various levels of D2O using both the nitrated and native forms of F1. Several nucleoside diphosphates are used as inhibitors of F1-catalyzed ITP hydrolysis. Dissociation constants of these inhibitors are obtained at both low and high concentrations of D2O for both the nitrated and native F1. We explore the possibility that a tyrosine and an arginine lie in close proximity in the F1 active site by studying the effects of sequential modification of arginine and tyrosine. These results are interpreted in terms of possible ATP hydrolysis mechanisms. Two possible roles for tyrosine in the hydrolysis of nucleoside triphosphates by F1 are suggested.
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PMID:The effect of nitration and D2O on the kinetics of beef heart mitochondrial adenosine triphosphatase. 645 57

An energy-transducing pathway in halobacteria is described. Arginine mediates substrate level phosphorylation and allows the cells to grow anaerobically. Bacteriorhodopsin plus light can function as an alternative energy source under these conditions, provided the cells contain the pigment when transferred to the anaerobic environment. Therefore the selection of mutants functionally defective in ATP synthase or bacteriorhodopsin becomes possible.
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PMID:Anaerobic growth of halobacteria. 693 39

The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.
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PMID:Beta-gamma subunit interaction is required for catalysis by H(+)-ATPase (ATP synthase). Beta subunit amino acid replacements suppress a gamma subunit mutation having a long unrelated carboxyl terminus. 755 18

Deletion mutations in the NH2- and COOH-terminal regions of the epsilon subunit of Escherichia coli ATP synthase were constructed making use of the AatII and HincII restriction enzyme sites. The resultant mutated epsilon species were analyzed for in vivo functionality and for recognition by anti-epsilon monoclonal antibodies. Deletion of residues Asp-7 through Gln-14 (epsilon delta D7-Q14) resulted in reduced ability to complement uncC mutants as determined by growth yields on limiting glucose medium and by formation of small colonies on plates with succinate as the source of carbon and energy. None of the other mutants was notably impaired. Upon induction to obtain overexpression, the NH2-terminal deletion mutants were expressed at levels comparable to the wild-type epsilon subunit, but the COOH-terminal deletion mutants were expressed less strongly, suggesting that residues in the latter region are important for protein stability. Monoclonal antibody epsilon-1, which cannot bind to epsilon when it is part of F1-ATPase, recognized the COOH-terminal deletions well, but the NH2-terminal deletions poorly. Additional epitope mapping using epsilon fusion proteins revealed that residues required for the epsilon-1 epitope extend to between Thr-77 and Arg-85. Monoclonal antibody epsilon-4, which can bind to epsilon when it is part of F1-ATPase, recognized the NH2-terminal deletions well, but hardly recognized the COOH-terminal deletions, indicating a role of residues located COOH-terminal to Ile-131 in recognition by this antibody. Epitope mapping using the fusion proteins revealed that the residues required by epsilon-4 begin in the region between Val-78 and Met-95. These results imply a two-domain structure of epsilon and orient the subunit within the enzyme.
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PMID:Orientation of the epsilon subunit in Escherichia coli ATP synthase. 768 93


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