Cross‐linking of bovine mitochondrial H+‐ATPase by copper–o‐phenanthroline

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Abstract
The nearest neighbor relationships between the F0 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 F0 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 F0 sector linked to those of F1. Both H+-ATPase and F0 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 F0 and untreated F1. The site of inhibition appeared to residue in the F0 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 F0.

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