Isolation of precursors of cytochrome oxidase from Neurospora crassa: application of subunit-specific antibodies and protein A from Staphylococcus aureus.
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 immuno-globulins 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.
D000818:Animals; D002846:Chromatography, Affinity; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D004792:Enzyme Precursors / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011233:Precipitin Tests; D011817:Rabbits / Q000276:immunology; D013205:Staphylococcal Protein A; D013211:Staphylococcus aureus
D004792:Enzyme Precursors; D013205:Staphylococcal Protein A; D003576:Electron Transport Complex IV