PMID: 42251Jan 1, 1979

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry

Acta biologica et medica Germanica
K P VatsisM J Coon

Abstract

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar...Continue Reading

Related Concepts

NADPH-Ferrihemoprotein Reductase
beta-Naphthoflavone
Cytochrome P450
Benzoflavones
Cytochrome P-450 Oxygenase
Microsomes, Liver
Oxidation
Spectrophotometry, Analyte Not Elsewhere Specified in CPT
Molar Tooth
Spectrophotometry

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