Purification and properties of P-4503b, a constitutive form of cytochrome P-450, from rabbit liver microsomes

Abstract

This laboratory has previously reported the occurrence in rabbit liver microsomes of a non-inducible form of cytochrome P-450, designated P-4503b because of its electrophoretic mobility relative to that of phenobarbital-inducible P-4502 and 5,6-benzoflavone-inducible P-4504. In the present study, P-4503b was purified to electrophoretic homogeneity and a specific content of over 19 nmol per mg of protein by chromatographic procedures carried out in the presence of detergents. The isolated cytochrome has a minimal molecular weight of 52,000 and exhibits absorption maxima at 418, 537, and 571 nm in the oxidized state, 412 and 547 nm in the reduced state, and 451 and 555 nm as the CO complex. In a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine, P-4503b has relatively high activity in the hydroxylation of testosterone in the 6[beta] and 16[alpha] positions as well as significant activity toward a number of other substrates tested. The NADPH oxidase activity of P-4503b is less than half that of P-4502 and 4.