Cross-linking of bovine mitochondrial H+-ATPase by copper--o-phenanthroline. Interaction of the oligomycin-sensitivity-conferring protein with a 24-kDa protein.
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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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D010084:Oxidation-Reduction / Q000187:drug effects; D010455:Peptides / Q000302:isolation & purification; D010618:Phenanthrolines; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D008565:Membrane Proteins; D010455:Peptides; D010618:Phenanthrolines; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; C025205:1,10-phenanthroline