Immunochemical evidence for an inactive form of cytochrome oxidase in mitochondrial membranes of ethanol-fed rats.
Previous studies have established that rats fed ethanol chronically exhibit a 50% decrease in hepatic mitochondrial cytochrome oxidase compared to pair-fed controls, based on both heme aa3 content and specific activity. To determine whether the 'missing' 50% of cytochrome oxidase is present in the membrane but catalytically inactive, or entirely absent, we used immunochemical techniques to determine the content of cytochrome oxidase protein in hepatic submitochondrial particles. Rabbit antiserum against purified rat liver cytochrome oxidase precipitated cytochrome oxidase from detergent-solubilized submitochondrial particles. Immunoinhibition titrations of a fixed amount of anti-oxidase serum with increasing amounts of submitochondrial particle protein showed that similar percentages of added oxidase activity were recovered in supernatants after immunoprecipitation with preparations from both alcoholic and control rats. Similarly, titrations of a fixed amount of submitochondrial particle protein with increasing amounts of antiserum showed comparable decreases in oxidase activity. Equivalent amounts of protein were obtained in immunoprecipitates from both preparations. Immunoprecipitates demonstrated comparable oxidase subunit profiles by electrophoresis, except that one additional band, migrating in the region of oxidase subunit IV, was present in samples from alcoholic rats. The data indicate that cytochrome oxidase immunologic reactivity is quantitatively similar in both types of membranes. The results suggest that the 'missing' cytochrome oxidase is actually present within the membranes of alcoholic animals in an inactive form, apparently devoid of heme aa3.
D000818:Animals; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology*; D007106:Immune Sera; D046911:Macromolecular Substances; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013367:Submitochondrial Particles / Q000201:enzymology
D007106:Immune Sera; D046911:Macromolecular Substances; D000431:Ethanol; D003576:Electron Transport Complex IV