Antibodies as probes of cytochrome oxidase structure and function.
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.
Nicholls
P
P
Dept. of Biol. Sciences, Brock Univ., St. Catharines, Ont., Canada.
Cooper
C E
CE
Leece
B
B
Freedman
J A
JA
Chan
S H
SH
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
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV