Redox-dependent accessibility of subunit V of cytochrome oxidase. A novel use of ELISA as a probe of intact membranes.

Abstract

Beef heart cytochrome c oxidase subunit V was isolated by a new one-step procedure. The product was homogeneous to 99% and immunogenically competent. The resulting antibodies inhibited the whole enzyme. A variation of ELISA (enzyme-linked immunosorbent assay) is described which, using a homogeneous antigen, probes the surface of intact membranes to a high degree of specificity. We showed that the subunit is accessible on the surface of inside-out mitochondrial inner membrane particles only when the particles were reduced with ascorbate and N,N,N',N'-tetramethyl-p-phenylene-diamine but is inaccessible on the surface of right-side-out particles regardless of the presence of reductants. These findings have implications with respect to the topography of the enzyme per se and in relation to its neighbors, and with respect to the degree of coupling between electron transport and proton pumping by the enzyme.