Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes.
Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).
Etter
H U
HU
Institute of Physics, College of Arts and Sciences, University of Tokyo, Japan.
Richter
C
C
Ohta
Y
Y
Winterhalter
K H
KH
Sasabe
H
H
Kawato
S
S
D000818:Animals; D002499:Centrifugation, Density Gradient; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D008968:Molecular Conformation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007074:Immunoglobulin G; D009842:Oligopeptides; D011510:Proteolipids; C020485:proteoliposomes; C071185:N-formylmethionyl-dipropylglycyl-phenylalanine methyl ester; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D004851:Epoxide Hydrolases