The F 0 subunits of bovine mitochondrial ATP synthase complex: Purification, antibody production, and interspecies cross-immunoreactivity

Abstract

The known subunits of the membrane sector F 0 of the bovine mitochondrial ATP synthase complex are subunits b, d, 6, F 6, OSCP (oligomycin sensitivity-conferring protein), the DCCD (dicyclohexylcarbodiimide) binding proteolipid, and A6L. The first six subunits were purified from SMP or preparations of the ATP synthase complex, and monospecific antibodies were raised against each. The antisera were shown to be competent for immunoblotting, and each antiserum recognized a single polypeptide of the expected M r in preparations of the ATP synthase complex. Immunoblots utilizing antibodies to OSCP and subunits d and 6, which exhibit the same M r on dodecyl sulfate—polyacrylamide gels, showed clearly that these polypeptides are immunologically distinct. Immunological cross-reactivity was demonstrated between bovine, human, rat, Saccharomyces cerevisiae, Paracoccus denitrificans, and Escherichia coli for subunit 6; between bovine, human, and rat for subunits b, d, OSCP, and F 6; and between bovine and rat for the DCCD binding proteolipid. Anti-subunit 6 antiserum, before or after immunopurification against the ATP synthase complex, recognized a single polypeptide in the bovine ATP synthase complex and S. cerevisiae mitochondria, but two polypeptides of different M r in bovine SMP, human, and rat mitochondria, and Paracoccus and E. coli membranes.