Interaction between cytochrome P-450 (P-450(C21)) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

S. Kominami, H. Hara, Tadashi Ogishima, S. Takemori

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41 Citations (Scopus)

Abstract

The interaction between P-450(C21) and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450(C21) via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450(C21) reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450(C21) and the reductase. The stable complex formation between P-450(C21) and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.

Original languageEnglish
Pages (from-to)2991-2999
Number of pages9
JournalJournal of Biological Chemistry
Volume259
Issue number5
Publication statusPublished - Jan 1 1984
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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