Antibodies as probes of cytochrome oxidase structure and function.

P. Nicholls, C. E. Cooper, B. Leece, J. A. Freedman, S. H. Chan

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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.

Original languageEnglish (US)
Pages (from-to)637-651
Number of pages15
JournalProgress in clinical and biological research
StatePublished - 1988

ASJC Scopus subject areas

  • Medicine(all)

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    Nicholls, P., Cooper, C. E., Leece, B., Freedman, J. A., & Chan, S. H. (1988). Antibodies as probes of cytochrome oxidase structure and function. Progress in clinical and biological research, 274, 637-651.