Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.
Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.
D000818:Animals; D000937:Antigen-Antibody Reactions; D001557:Benzenesulfonates / Q000031:analogs & derivatives; D002417:Cattle; D003979:Diazonium Compounds; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000032:analysis*; D005779:Immunodiffusion; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000648:ultrastructure; D010455:Peptides / Q000032:analysis; D013367:Submitochondrial Particles / Q000201:enzymology / Q000648:ultrastructure; D013425:Sulfanilic Acids / Q000031:analogs & derivatives
D001557:Benzenesulfonates; D003979:Diazonium Compounds; D010455:Peptides; D013425:Sulfanilic Acids; D003576:Electron Transport Complex IV; C000469:diazobenzenesulfonic acid