title: Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.
abstract: The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.
authors.affiliation: E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
authors.forename: A B
authors.initials: AB
authors.lastname: Van Kuilenburg
authors.forename: H L
authors.initials: HL
authors.lastname: Dekker
authors.forename: C
authors.initials: C
authors.lastname: Van den Bogert
authors.forename: P
authors.initials: P
authors.lastname: Nieboer
authors.forename: B F
authors.initials: BF
authors.lastname: Van Gelder
authors.forename: A O
authors.initials: AO
authors.lastname: Muijsers
mesh_terms: D000818:Animals; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity
chemical_list: D007106:Immune Sera; D007527:Isoenzymes; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV