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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Jan;84(1):11–15. doi: 10.1073/pnas.84.1.11

Purification to homogeneity and enzymological characterization of a functional covalent complex composed of cytochromes P-450 isozyme 2 and b5 from rabbit liver.

P P Tamburini, J B Schenkman
PMCID: PMC304131  PMID: 3467342

Abstract

A covalent complex between rabbit hepatic microsomal cytochromes P-450 isozyme 2 (LM2) and b5 was created and purified to greater than 95% homogeneity. The purified complex was largely comprised of the two cytochromes covalently attached at the interface of the functional electron transfer-effector complex as shown by the following evidence. The spin state of the LM2 within the complex was greater than the spin state of free LM2, and the addition of free cytochrome b5 (cyt b5) did not further increase the spin state of the LM2 within the complex. The spectral binding parameters (Kd and delta Amax) for the association of benzphetamine with LM2 in the complex were identical to those observed with free LM2 in the presence of saturating concentrations of free cyt b5 and much different from those observed for LM2 in the absence of cyt b5. Reconstituted monooxygenase activity of the covalent LM2-cyt b5 complex (LM2-cyt b5) in the presence of NADPH-cytochrome P-450 reductase was much higher than the activity of free LM2 and approached the activity of free LM2 in the presence of optimal concentrations of free cyt b5. Furthermore, the Km for the flavoprotein in supporting either free LM2 or LM2-cyt b5-dependent p-nitroanisole demethylation were similar. (iv) Less than 20-25% of the cyt b5 within the complex could be reduced by free NADH-cytochrome b5 reductase (NADH-cyt b5 reductase) albeit at a slow rate. The implications of this data to the current understanding of the mechanism and stoichiometry of protein interactions in the hepatic mixed function oxidase system are discussed.

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Selected References

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