Effects of antibodies to intact cytochrome-c oxidase and its subunit V on the enzymatic activity.
Antibodies have been raised in rabbits against whole beef heart cytochrome-c oxidase and purified subunit V. Antioxidase recognizes nearly all the enzyme subunits but reacts very strongly with subunits II and IV. Antisubunit V is quite specific against subunit V. Inhibition of enzyme activity by antioxidase is typically biphasic in time, indicating populations of both rapidly and slowly reacting molecules. Variation of cytochrome c concentration shows partially competitive kinetics, but the antibody also affects "internal" enzymatic events, including the catalytic turnover induced by N,N,N',N'-tetramethyl-p-phenylenediamine alone and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects can be seen however. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c, but not that with internally trapped cytochrome c. No functionally significant epitopes are detectable on the N side of the membrane in proteoliposomes, although some small effects can be seen with submitochondrial particles. Antisubunit V inhibits the isolated enzyme by at least 60%. The inhibition at high ionic strength induces a biphasic pattern with respect to cytochrome c concentration. Antisubunit V may thus slow the dissociation of cytochrome c from its complex with the enzyme. Antisubunit V has only small effects on the activities of proteoliposomal and submitochondrial particle oxidase in either orientation. On subunit V, some sites, the binding of which can give rise to inhibition, are thus not accessible to antisubunit V when the enzyme is embedded in a functional membrane system.
Nicholls
P
P
Department of Biological Sciences, Brock University, St. Catharines, Ont., Canada.
Cooper
C E
CE
Freedman
J A
JA
Leece
B D
BD
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001667:Binding, Competitive; D002417:Cattle; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000276:immunology*; D009206:Myocardium / Q000201:enzymology; D010101:Oxygen Consumption; D013772:Tetramethylphenylenediamine / Q000494:pharmacology
D000906:Antibodies; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine