Isolation of Precursors of Cytochrome Oxidase from Neurospora crassa: Application of Subunit-Specific Antibodies and Protein A from Staphylococcus aureus

Abstract

A novel immunological procedure has been applied for the isolation of precursors of cytochrome oxidase. It involves antibodies to individual subunits of the oxidase and protein A from Staphylococcus aureus linked to a Sepharose support. Unassembled (free) ‘subunits’ as well as a labile complex containing five polypeptide components of the oxidase were isolated from mitochondrial extracts by this technique. The procedure is superior to the previously used double-immuno-precipitation method, because of its quantitative nature, its sensitivity, rapidity and versatility. Thus, sequential titrations of precursor proteins by addition of various subunit-specific immunoglobulins to an individual extract become feasible. Furthermore, the technique is suitable for the isolation of precursors on a large scale. To avoid contamination of the polypeptide preparations with immunoglobulin, the antibodies were covalently coupled to protein A, which had been previously linked to Sepharose. A radioactive preparation of unassembled subunit 1 of cytochrome oxidase was isolated by such a modified support and its cyanogen-bromide-cleavage products were compared to those of subunit 1 obtained from the assembled enzyme.