title: Antibodies as probes of cytochrome oxidase structure and function.
abstract: Antibodies have been raised in rabbits against whole beef heart cytochrome oxidase and against purified subunit V. Western blot analysis showed that antioxidase was largely composed of anti-II and anti-IV antibodies but some anti-I and antibodies against small subunits were elicited. Similar analysis of anti-V showed it to be relatively specific against subunit V. Three types of anti-V were identified by ELISA with intact enzyme: (i) weak binding and redox independent, (ii) stronger binding, redox-dependent (binding only to reduced or partially reduced enzyme), and membrane-independent, and (iii) moderate-binding, redox-dependent & membrane-dependent. Inhibition of enzyme activity by anti-oxidase was biphasic in time, indicating populations of rapidly- and slowly- reacting molecules. Variation of cytochrome c concentration showed partially competitive kinetics, but the antibody also affected 'internal' enzymatic events including the turnover rate with TMPD and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects could however be seen. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c but not that with internally-trapped cytochrome c. No functionally significant epitopes occur on the N (matrix) side of the membrane. The more strongly binding anti-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. Anti-V may thus slow the dissociation of cytochrome c from its complex with the enzyme. The redox-dependent, membrane-dependent anti-V (reported previously) gave only about 20% inhibition of the enzyme. The effective anti-V antibody had little if any influence on the activity of proteoliposomal oxidase in either orientation. Some sites on subunit V whose binding can give rise to inhibition are not accessible to anti-V when the enzyme is embedded in a functional membrane system.
authors.affiliation: Dept. of Biol. Sciences, Brock Univ., St. Catharines, Ont., Canada.
authors.forename: P
authors.initials: P
authors.lastname: Nicholls
authors.forename: C E
authors.initials: CE
authors.lastname: Cooper
authors.forename: B
authors.initials: B
authors.lastname: Leece
authors.forename: J A
authors.initials: JA
authors.lastname: Freedman
authors.forename: S H
authors.initials: SH
authors.lastname: Chan
mesh_terms: D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D002417:Cattle; D003576:Electron Transport Complex IV / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV