Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver: I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 (2B4) monomers

Abstract

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 oligomers were 14.8 ± 1.7 and 19.2 ± 1.4 nm, respectively. Twentyfour-hour incubation with Emulgen 913 at 4 °C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 oligomers, the particle sizes diminishing to 6.1 ± 1.3 and 5.2 ± 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM 2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM 2 oligomers and the microsomal hemoprotein, cytochrome P450 LM 2 monomers formed complexes with type I and II substrates, but with K d values higher than those of microsomes and cytochrome P450 LM 2 oligomers. Kinetic parameters ( V max and K m ) of H 2O 2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM 2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM 2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.