Evidence for the Presence of Di- and Triphospho Pyridine Nucleotide Dehydrogenase Derivatives As Consistent Contaminants of Purified Beef Heart Cytochrome-c Oxidase

Overview

Journal J Bioenerg Biomembr

Publisher Springer

Specialties Biochemistry
Biology
Endocrinology

Date 1976 Dec 1

PMID 187583

Authors

R PENNIALL

W B Elliott

K J Van Buuren

B F Van Gelder

Affiliations

Soon will be listed here.

Abstract

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.

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