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Molecular Pharmacology, Vol 16, 332-342, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Reconstitution of Cytochrome P-450 and Cytochrome P-450 Reductase into Phosphatidylcholine-Phosphatidylethanolamine Bilayers: Characterization of Structure and Metabolic Activity

B. BÖSTERLING 1, A. STIER 1, A. G. HILDEBRANDT 2, J. H. DAWSON 3, and J. R. TRUDELL 3

1 Max-Planck-Institut fur Biophysikalische Chemie, D-3400 Göttingen, West Germany
2 Institut fur Klinische Pharmakologie der Freien Universitaet, Berlin, Klinikum Steglitz, Hindenburgdamm 30, D-1000 Berlin 45, West Germany
3 Department of Anesthesia, Stanford University, Stanford, California 94305

Cytochrome P-450 LM-2 and NADPH-cytochrome P-450 reductase have been reconstituted into phosphatidylcholine-phosphatidylethanolamine vesicles using a cholate dialysis technique. The structure and composition of the reconstituted system has been characterized by gradient centrifugation, electron microscopy, phosphorous nuclear magnetic resonance, phosphate and protein analysis. The absence of conversion of cytochrome P-450 to cytochrome P-420 during reconstitution has been verified by visible spectroscopy of the reduced CO-bound form. The metabolic activity of the vesicle reconstitution and a nonvesicular preparation has been characterized by NADPH utilization and H2O2 production with and without substrates hexobarbital, cyclohexane, aminopyrine, and benzphetamine as well as by the metabolic production of formaldehyde from aminopyrine and benzphetamine. The reconstitution technique produces vesicles of sufficient diameter and protein content (33% by wt) that each may contain at least two reductase and 10 or more cytochrome P-450 proteins. This vesicle reconstituted system is similar to microsome with regard to bilayer structure, phospholipid composition, surface charge, protein: lipid ratio, cytochrome P-450:NADPH-cytochrome P-450 reductase ratio, and enzymatic coupling. Therefore it may be an excellent system in which to study substrate specificity, electron transport, reductive versus oxidative metabolism, and protein-lipid interactions.

Note:
ACKNOWLEDGMENTS We wish to thank Drs. W. Hubbell, K. Hong, E. Bunnenberg, C. Djerassi, R Sato, T. F. Slater, H. H. Fuldner and S. Finch for many helpful discussions and T. Wronker, U. Wurl, A. Wolff and N. Cline for expert technical assistance.

Submitted on October 17, 1978
Accepted on February 21, 1979




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Drug Metab. Dispos., April 1, 2006; 34(4): 660 - 666.
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