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)

Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase. The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself. The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial F0 is not known. The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria. 285 48

An azido derivative of the oligomycin sensitivity conferring protein (OSCP) was prepared by alkylation with the bifunctional reagent p-azido phenacyl bromide. Azido-OSCP was fully biologically active in the dark. Upon photoirradiation of a mixture of beef heart mitochondrial F1-ATPase and azido-OSCP, the resulting covalent photoproducts were separated by polyacrylamide gel electrophoresis in the presence of Na dodecyl sulfate and characterized by an immunochemical procedure. OSCP was found to react with the alpha and the beta subunits of F1 with strong preference for the alpha subunit.
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PMID:Photolabeling of mitochondrial F1-ATPase by an azido derivative of the oligomycin-sensitivity conferring protein. 286 14

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.
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PMID:Coupling factor activity of the purified pea mitochondrial F1-ATPase. 286 69

The nearest neighbor relationships between the Fo subunits of bovine mitochondrial H+-ATPase were studied by using copper-o-phenanthroline, an SH-oxidizing cross-linking reagent. The cross-linked samples of purified H+-ATPase, F1-ATPase or Fo were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and the disulfide cross-linked polypeptides were identified by enzyme-linked immunosorbent assay and immunoblot transfer using subunit specific antisera. SDS-PAGE of H+-ATPase showed several cross-links, although none involved subunits of Fo sector linked to those of F1. Both H+-ATPase and Fo showed formation of a 45-kDa product. Upon reduction, the 45-kDa component gave rise to a 21-kDa band, identified as oligomycin-sensitivity-conferring protein (OSCP), and a 24-kDa band. These two proteins thus appear to be near neighbors with their cysteine residues in close proximity with each other. Under the conditions of cross-linking, there was a concentration-dependent decrease in the Pi-ATP exchange activity of the intact H+-ATPase as well as of H+-ATPase reconstituted with copper-o-phenanthroline-treated Fo and untreated F1. The site of inhibition appeared to residue in the Fo sector. Loss of Pi-ATP exchange occurred at the same time as formation of the 45-kDa product. Our present data showing copper-o-phenanthroline-induced interactions of the 24-kDa protein with the OSCP and simultaneous inactivation of Pi-ATP exchange activity of the complex strengthen earlier suggestions [Hadikusumo, R.G., Hertzog, P.J. & Marzuki, S. (1984) Biochim. Biophys. Acta 765,258-267] that the 24-kDa protein may be a bona fide subunit of Fo.
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PMID:Cross-linking of bovine mitochondrial H+-ATPase by copper--o-phenanthroline. Interaction of the oligomycin-sensitivity-conferring protein with a 24-kDa protein. 286 96

A simple analytical procedure for comparing the rates of inactivation of an enzyme in the presence and absence of its substrate is proposed. The rapid inactivation of yeast F1-ATPase during the catalytic reaction was found to be due to certain anions rather than due to ATP or ADP. MgATP failed to protect the enzyme but substituting sulfate, acetate, bicarbonate, or N-tris(hydroxymethyl)methyl-2-aminoethane sulfonate anions and preincubation with ADP prevented the inactivation.
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PMID:Use of Swinbourne plots to study potential suicide substrates: effects of ATP and ADP on yeast mitochondrial F1-ATPase. 286 16

Two proteinaceous factors, 15K and 9K proteins, which acted together to stabilize the inactivated yeast F1F0-ATPase-inhibitor complex [Hashimoto, T., et al. (1984) J. Biochem. 95, 131-136] were hardly distinguishable from the sigma and epsilon subunits, respectively, of yeast F1-ATPase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. However, they were clearly distinguishable from these subunits by analyses of the sequences at their amino terminals and by immunoblotting combined with SDS polyacrylamide gel electrophoresis. The two stabilizing factors and an ATPase inhibitor existed in mitochondria in equimolar ratios to F1-ATPase. These three protein factors were not present in purified F1-ATPase or in F1F0-ATPase preparations, but remained in the mitochondrial membranes after extraction of F1F0-ATPase with Triton X-100. These observations strongly suggest that the two stabilizing factors and the ATPase inhibitor form a regulatory substructure of mitochondrial ATP synthase, in addition to the F1 and F0 subunits.
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PMID:Existence of stoichiometric amounts of an intrinsic ATPase inhibitor and two stabilizing factors with mitochondrial ATP synthase in yeast. 287 60

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.
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PMID:Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies. 287 75

ATPase/ATP synthase preparations originally contain protein bands in the 28-31-kDa region. The present study demonstrates separation of the band at 29 kDa (adenine nucleotide translocator) from a band at approximately 31 kDa. In cholate/ammonium sulfate-extracted ATP synthases removal of the 31-kDa band results in decrease of ATP-Pi exchange and oligomycin sensitivity of the ATPase activity. It is suggested that the protein band at 31 kDa is heterogeneous, containing diverse activities, the identities of which are yet to be determined.
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PMID:ATP synthase complex from beef heart mitochondria. Separation of protein bands in the region of 28-31 kDa. 287 90

Subunit c of the membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli has been isolated under nondenaturing conditions (Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515-518) and antibodies have been raised in rabbits. The primary antisera did not recognize the antigen when present in the same buffer as used for the immunization. Surprisingly, in one of the three antisera a strong antibody binding was observed when intact F0, a.c complex or reconstituted subunit c was provided as the antigen. Incorporation of subunit c into liposomes together with subunits a and b forming an active, H+-translocating complex was not required for the recognition by the antiserum. Subunit c prepared by chloroform/methanol extraction or by chromatography in the presence of sodium dodecyl sulfate was not recognized by the anti-c antiserum when incorporated into liposomes.
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PMID:Conformation-specific antiserum raised against subunit c of ATP synthase (F1F0) from Escherichia coli. 287 91

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.
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PMID:Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase. 287 81


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