Open AccessIsolation of Precursors of Cytochrome Oxidase from Neurospora crassa : Application of Subunit‐Specific Antibodies and Protein A from Staphylococcus aureus
Author(s) -
WERNER Sigurd,
MACHLEIDT Werner
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12579.x
Subject(s) - neurospora crassa , biochemistry , protein subunit , cytochrome c oxidase , cyanogen bromide , sepharose , oxidase test , cytochrome , biology , staphylococcus aureus , chemistry , microbiology and biotechnology , enzyme , peptide sequence , bacteria , genetics , gene , mutant
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.