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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)

Repeated extraction of bovine heart submitochondrial particles with ammonia and EDTA (AE) yields a preparation that is highly deficient in coupling factor B (FB). The activity of the thrice extracted particle (AE-P3) in ATP-driven NAD+ reduction by succinate and the 32Pi-ATP exchange activity were substantially stimulated, 8-fold and 5-fold, respectively, by purified FB. To decrease the basal activity of the particle further, the residual FB in AE-P3 was inactivated by treatment with the -SH reagent, 4-vinylpyridine. The resulting particle was depleted of F1 by treatment with 3.5 M NaBr. This particle was incorporated into asolectin liposomes alone and in the presence of added FB. Passive proton conduction in the FB-deficient proteoliposomes was negligible and restored in the presence of FB. The H+ conductance was inhibited extensively by oligomycin and partially by F1-ATPase. The results show absolute dependence on FB for functioning of the FO proton channel.
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PMID:Coupling factor B is a component of the Fo proton channel of mitochondrial H+-ATPase. 243 94

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

Membrane energization by ATP has been measured in vesicles containing purified bovine heart mitochondrial H+-ATPase (ATP synthase) with the voltage-sensitive dye oxonol VI. The dithiol chelator, Cd2+, and the thiol oxidant, copper o-phenanthroline, produced discharge of the membrane potential when added at the steady state and inhibited its establishment when added prior to energization by ATP. These effects, which were reversed by dithiothreitol, were not accompanied by an increase in the nonspecific H+ permeability of the membrane. Passive H+ conduction in proteoliposomes containing F0 (hydrophobic segment of ATP synthase) was assayed by the quenching of 9-aminoacridine fluorescence after establishing a K+ diffusion potential. This conductance was blocked by Cd2+, an inhibitor of coupling factor B (FB). Labeling of F0 with 115Cd2+ at the concentrations that inhibited the F0 conductance followed by gel electrophoresis yielded a single radioactive band with a molecular weight corresponding to FB, the presence of which in the F0 preparation was confirmed by immunoblot staining. The data offer strong evidence that FB is an essential component of the H+ channel of F0, because H+ conduction through the channel is inhibited by chemical modification of FB.
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PMID:Evidence for the involvement of coupling factor B in the H+ channel of the mitochondrial H+-ATPase. 614 19

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers. 647 76

Factor B is a subunit of the mammalian ATP synthase complex, whose existence has been controversial. This paper describes the molecular and functional properties of a recombinant human factor B, which when added to bovine submitochondrial particles depleted of their factor B restores the energy coupling activity of the ATP synthase complexes. The mature human factor B has 175 amino acids and a molecular mass of 20,341 Da. The preparation is water-soluble, monomeric, and is inactivated by monothiol- and especially dithiol-modifying reagents, probably reacting at its cysteine residues Cys-92 and Cys-94. A likely factor B gene composed of 5 exons has been identified on chromosome 14q21.3, and the functional role of factor B in the mammalian ATP synthase complex has been discussed.
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PMID:Factor B and the mitochondrial ATP synthase complex. 1174 38

Factor B is a water-soluble protein, which is required for the coupled activity of the mitochondrial ATP synthase complex. Specific removal of factor B from well-coupled bovine heart submitochondrial particles (SMP) results in uncoupling and the loss of ATP-driven membrane potential formation and reverse electron transfer from succinate to NAD. Addition of recombinant human factor B (molecular mass 20,341 Da) to factor B-depleted SMP (AE-SMP) restores these properties [G.I. Belogrudov, and Y. Hatefi, (2002) J. Biol. Chem. 277, 6097-6103]. This paper shows that extraction and purification of ATP synthase complex (complex V) from bovine heart mitochondria results in extensive loss of factor B. Addition of recombinant human factor B to AE-SMP completely restores the lost oxidative phosphorylation and ATP-32P(i) exchange activities of the particles and increases the ATP-32P(i) exchange activity of complex V by 2.5-fold. These results further indicate that factor B is an essential component of the mammalian ATP synthase complex.
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PMID:Factor B is essential for ATP synthesis by mitochondria. 1236 15

Bovine factor B, a polypeptide required for the coupled activity of the mitochondrial ATP synthase complex, was cloned. A novel expression system for overproducing the recombinant bovine factor B was developed, which yielded the recombinant polypeptide at a level of 12-15 mg of protein per liter of bacterial culture. Reconstitution of the recombinant polypeptide with factor B-depleted ammonia, EDTA-treated submitochondrial particles (AE-SMP) restored the formation of substrate-driven DeltapH gradient across vesicular membranes, presumably by blocking a proton leak. The proton leak in the AE-SMP could also be blocked by the F0 inhibitors oligomycin and dicyclohexylcarbodiimide, but not the F1-ATPase inhibitors efrapeptin and aurovertin B. The six factor B thiols titrated rapidly with Ellman's reagent, and two of these, presumably Cys92 and Cys94, gained protection following treatment of factor B with a vicinal dithiol-specific reagent phenylarsine oxide (PAO). Similarly, Cd2+, whose binding to factor B is believed to also involve a vicinal dithiol, and PAO, protected approximately 2 Cys residues against labeling with sulfhydryl-specific fluorescent reagent fluorescein-5'-maleimide. The circular dichroism spectra showed that binding of Cd2+ and Zn2+, but not Ca2+ to bovine factor B caused small but reproducible changes in the secondary structure elements of the polypeptide.
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PMID:Bovine factor B: cloning, expression, and characterization. 1657 55

Since the early studies on the resolution and reconstitution of the oxidative phosphorylation system from animal mitochondria, coupling factor B was recognized as an essential component of the machinery responsible for energy-driven ATP synthesis. At the phenomenological level, factor B was agreed to lie at the interface of energy transfer between the respiratory chain and the ATP synthase complex. However, biochemical characterization of the factor B polypeptide has proved difficult. It was not until 1990 that the N-terminal amino acid sequence of bovine mitochondrial factor B was reported, which followed, a decade later, by the report describing the amino acid sequence of full-length human factor B and its functional characterization. The present review summarizes the recent advances in structure-functional studies of factor B, including its recently determined crystal structure at 0.96 A resolution. Ectopic expression of human factor B in cultured animal cells has unexpectedly revealed its role in shaping mitochondrial morphology. The supramolecular assembly of ATP synthase as dimer ribbons at highly curved apices of the mitochondrial cristae was recently suggested to optimize ATP synthesis under proton-limited conditions. We propose that the binding of the ATP synthase dimers with factor B tetramers could be a means to enhance the efficiency of the terminal step of oxidative phosphorylation in animal mitochondria.
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PMID:Recent advances in structure-functional studies of mitochondrial factor B. 1937 34