Evidence for the presence of di- and triphospho pyridine nucleotide dehydrogenase derivatives as consistent contaminants of purified beef heart cytochrome-c oxidase.
Purified beef heart cytochrome-c oxidase preparations derived by three different laboratories contain NADH-K3 Fe (CN)6, NADH-nitrobluetetrazolium, and NADPH-nitrobluetetrazolium reductases. This is true of preparations exhibiting heme aa3 to protein ratios considered indicative of an excellent purity. An apparent association of cytochrome-c oxidase and one or more of the contaminants persists through immunodiffusion and nondenaturing electrophoresis and, in addition, in one instance copurification of NADH-K3Fe(CN)6 reductase and cytochrome-c oxidase to a constant ratio of specific activities was demonstrated. Cytochrome-c oxidase can be freed of the contaminants by equilibration with an NAD+-affinity matrix. As aconcomitant of equilibration with the matrix, the KM of cytochrome-c oxidase for ferrocytochrome-c is invariably decreased. Rat constants at low ferrocytochrome-c concentrations are consistently enhanced in all oxidase preparations upon equilibration with the NAD+ matrix. However, the effects of such equilibrations on the extrapolated Vmax varies from one preparation to another. Polyacrylamide gel electrophoresis in SDS-urea systems establishes that each of the preparations contains a minimum of three contaminants, each of an apparent formula weight of greater than 40,000 Daltons. NADH-NBT reductase was found to have a formula weight of approximately 46,000 Daltons. Their properties establish that NADH-K3Fe(CN)6 and NADH-NBT reductases are separate proteins; the separate identity of NADPH-NBT reductase has not yet been determined.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004340:Drug Contamination; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D007700:Kinetics; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism
D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV