Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.
Rotation of cytochrome P-450 was examined in both liver microsomes and reconstituted and phospholipid vesicles. Purified cytochrome P-450 was incorporated into lipid vesicles composed of phosphatidylcholine-phosphatidylethanolamine-Phosphatidylserne. Rotational diffusion was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme. CO complex by a vertically polarized laser flash. No contribution of vesicle tumbling to r(t) was observed over the experimental time range of 0-500 mus for samples in 60% sucrose. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450. In vesicles of a high lipid to protein ratio (=30 by weight), the residual time-independent normalized anisotropy, r (infinity)/r (0), reached a limiting low value, implying that all cytochrome P-450 was rotating. The mean rotational relaxation time, phi 1, was about 95 mus. In contrast, about 35% of cytochrome P-450 was immobilized in vesicles of a low lipid to protein ratio (=1), with phi 1 of about 95 mus for the mobile fraction. The immobile fraction is presumably due to self-aggregation of cytochrome P-450. In rat liver microsomes, 0-50% of cytochrome P-450 was mobile with phi 1 of about 120 mus at 20 degrees C, and the rest was immobile. A significant temperature dependence of r(t) was observed in microsomes. All cytochrome P-450 was immobile below 7 degrees C, and about 50% of the enzyme was mobile at 37 degrees C with phi 1 approximately 60 mus. From the limiting value of r(infinity)/r(0) congruent to 0.12, the tilt angle of the heme plane of cytochrome p-450 from the membrane plane was calculated to be about 40 degrees.
D000818:Animals; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008433:Mathematics; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010714:Phosphatidylethanolamines / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains
D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital