Properties and use of an antiserum to cytochrome oxidase from baker's yeast

Abstract

An antiserum against highly purified cytochrome oxidase from the yeast Saccharomyces cerevisiae ( Shakespeare and Mahler (1971) J. Biol. Chem. 246, 7649) has been prepared. Addition of this antiserum to reaction mixtures containing either purified cytochrome oxidase or mitochondrial membranes results in inhibition of the oxidation of reduced cytochrome c competitive to the latter: K m for cytochrome c equals 95 μ m with both preparations; in the presence of 5 μg of antiserum this value is raised to 210 and 250 μ m, with the purified enzyme and the membranes, respectively. The antibody preparation has been labeled with 3H by reductive methylation (condensation with HCHO followed by reduction) and used to study and compare binding to mitochondrial membranes from controls with those from cells in which all cytochrome oxidase synthesis has been stopped by growth in the presence of ethidium bromide. The binding isotherms for the two preparations were quantitatively similar. Thus the two types of membranes appear to contain roughly equivalent amounts of material (s) able to interact with an antibody preparation, itself capable of blocking the cytochrome c binding sites of cytochrome oxidase. The use and limitations of studies with antibodies designed to probe the function and biogenesis of cytochrome oxidase are discussed.