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)

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.
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PMID:Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. 353 63

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.
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PMID:ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP. 613 89

A soluble Mg-dependent ATPase, similar to the mitochondrial ATPase from beef heart, has been isolated from heart mitochondria of salmon (Salmo salar). The salmon heart ATPase has 5 subunits with molecular weights similar to the beef heart enzyme, but the Stoke's radius of the intact salmon enzyme is larger. The salmon heart ATPase is less temperature labile than the beef heart enzyme. The salmon heart ATPase is strongly inhibited by ADP, and the inhibition is highly temperature dependent. The ITPase activity is also inhibited by IDP (Ki = 180 micron). 2,4-Dinitrophenol in small concentrations stimulates the ITPase activity as well as the ATPase activity of the "washed" salmon heart enzyme. However, in an enzyme preparation which had been freed of most of the bound nucleotides by dialysis in the presence of glycerol (Roveri et al., 1980) the ITPase activity is not stimulated by 2,4-dinitrophenol.
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PMID:Some properties of isolated mitochondrial ATPase from salmon heart. 615 Aug 4

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.
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PMID:Alterations of the alpha or beta subunits of the mitochondrial ATPase in yeast mutants. 621 96

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two-dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min-1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.
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PMID:Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria. 622 Oct 23

The mitochondrial F1-ATPase from bean (Vicia faba L.) was solubilized by a chloroform treatment of mitochondrial membranes and purified by centrifugation on a glycerol gradient. The active fraction contained 5 subunits: alpha (Mr = 52,000), beta (Mr = 51,000), gamma (Mr = 34,000), delta (Mr = 23,800), and epsilon (Mr = 22,900). Purified coupled mitochondria were incubated in the presence of [ 35S ]methionine and malate to allow mitochondrial translation to occur. The largest labeled polypeptide (Mr = 52,000) was present in the chloroform extract, co-sedimented with the F1-ATPase on glycerol gradient and co-migrated with the alpha subunit upon two-dimensional electrophoresis. The results indicate that the alpha subunit of bean mitochondrial ATPase is translated on mitoribosomes, in contrast to the situation in other organisms.
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PMID:The alpha subunit of a plant mitochondrial F1-ATPase is translated in mitochondria. 622 32

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.
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PMID:Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae. 622 52

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.
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PMID:Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe. 623 Mar 52

In the yeast Schizosaccharomyces pombe, the structural gene mutations A23-13 (alpha-) and B59-1 (beta-) which totally prevent the expression of either the alpha or the beta subunits of the mitochondrial ATPase, were shown by classical genetic mapping studies to be both located on chromosome I but genetically unlinked. It is concluded that the structural genes ATP1 and ATP2 for the alpha and beta subunits of the mitochondrial ATPase are not organized in a cluster. By both meiotic recombination frequency analysis and gene transfer studies, three single nuclear mutations affecting to different extents the electrophoretic mobility of the beta polypeptide were located on the chromosome I very close to the mutation B59-1 (beta-). Two mutations involved a defective ATPase activity and the inability to grow on glycerol (gly). One of these mutants E5-23 (beta") exhibited a beta subunit of slightly reduced electrophoretic mobility. The other mutation F1-10 (beta) was associated with a beta subunit of normal electrophoretic mobility. The plasmid pMa2 (Boutry, M., Vassarotti, A., Ghislain, M., Douglas, M., Goffeau, A. (1984) J. Biol. Chem. 259, 2840-2844) containing the structural gene for the beta subunit complemented the mutants E5-23 (beta") and F1-10 (beta) as well as B59-1 (beta-). These three mutations are therefore likely to affect the beta structural gene itself or a very contiguous gene contained in the 5.4-kilobase genomic insert of pMa2. The mutation F1-10 (beta) was mapped between E5-23 (beta") and B59-1 (beta-) by analysis of the meiotic recombination frequencies. Another mutation F25-28-11 (beta') was responsible for an appreciable decrease of electrophoretic mobility of the beta subunit which, however, did not affect either the ATPase activity or the ability to grow on glycerol (GLY). This mutant transformed by pMa2 was able to express the structural gene for the wild type beta subunit and the resulting transformants synthesized and assembled both the beta and beta' subunits. It is concluded that the mutation F25-28-11 (beta') also affects the structural gene for the beta subunit and does not affect genes controlling the processing machinery.
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PMID:Independent loci for the structural genes of the yeast mitochondrial alpha and beta ATPase subunits. 623 Mar 53

Miconazole [( 1-[2-(2,4 dichlorophenyl)-2-(2,4 dichlorophenyl)methoxy]ethyl]-1 H-imidazole) completely inhibited growth of Saccharomyces cerevisiae and Candida albicans on glycerol at 10 microM . 50 microM was needed to achieve the same effect during growth on glucose. Miconazole inhibited competitively the mitochondrial ATPase of S. cerevisiae with a Ki of 1 microM. F1 activity of the enzyme was not affected. Mutants resistant to miconazole were isolated. The ATPase of these mutants was resistant to 10 microM miconazole. Higher concentrations of miconazole inhibited the ATPase of the plasma membrane. The inhibition of the S. cerevisiae enzyme was competitive with a Ki of 50 microM. The results point to the mitochondrial ATPase as the primary target of miconazole action at least during growth on non-fermentable carbon sources.
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PMID:Mode of action of miconazole on yeasts: inhibition of the mitochondrial ATPase. 623 81


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