Interactions between the oligomycin sensitivity conferring protein and beef heart mitochondrial F1-ATPase. 2. Identification of the interacting F1 subunits by crosslinking

Abstract

Interactions between oligomycin sensitivity conferring protein (OSCP) and subunits of beef heart mitochondrial F1-ATPase were explored by cross-linking at an OSCP/F1 molar ratio close to 1 to ensure specific high-affinity binding of OSCP to F1. Cross-links between F1 subunits and OSCP were established by means of 2 zero length cross-linkers, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline. The cross-linked products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Coomassie blue staining revealed 2 cross-linked products of MW 75,000 and 80,000, which could result from the binding of OSCP to the .alpha. and .beta. subunits of F1. Definite identification of the cross-linked products was achieved by chemical labeling with specific radiolabeled reagents and by blotting on nitrocellulose filters followed by immunocharacterization with anti-.alpha., anti-.beta., and anti-OSCP antibodies. OSCP was found to cross-link with the .alpha. and .beta. subunits of F1.