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Immunization of rats by a homogeneous cytochrome P-450-LM4 from liver microsomes of rabbits pretreated with 3-methylcholanthrene resulted in antibodies to this hemoprotein - anti-P-450-LM4. Using the Ouchterlony method, a reaction of precipitation of anti-P-450-LM4 with cytochrome P-450-LM4 from MeCh- and PB-induced microsomes of rabbit liver has been carried out. The anti-P-450-LM4 at different concentrations does not practically exert any inhibitory action on oxidation of aniline and N,N-dimethylaniline by intact and PB- and MeCh-induced rabbit hepatic microsomes. It is concluded that rabbit liver cytochrome P-450-LM4 does not practically reveal any catalytic activity in ther metabolism of aniline and N,N-dimethylaniline.

Aniline oxidation and oxidative dimethylaniline demethylation with participation of the LM4 form of cytochrome P-450 from rabbit liver microsomes were studied under different conditions: e. g. after incorporation of LM4 form into microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits and after incorporation of this hemoprotein into liposomes in hydroperoxide-dependent reactions. The results obtained suggest that the LM4 form of cytochrome P-450 is catalytically inactive during aniline oxidation and dimethylaniline demethylation in both cases. The absence of catalytic activity of the LM4 form of cytochrome P-450 is due to the structural peculiarities of the active site of this hemoprotein rather than to its environment.

The inhibitory action of divalent copper compounds on hydroperoxide-dependent oxidative demethylation of N,N-demethylaniline involving rabbit liver microsomes and highly purified cytochrome P-450 has been studied. CuCl2 is a non-competitive inhibitor, whereas copper tyrosine and lysine complexes are characterized by a mixed type inhibition. The inhibitory action of copper complexes is based on a decrease of cumene hydroperoxide concentration. The reaction results in formation of RO and RO2 radicals destroying cytochrome P-450 CuCl2 (0,001 M) also destroys cytochrome P-450 in the absence of cumene hydroperoxide; the destruction process is characterized by two phases with different rate constants. The nature of the inhibitory action of CuCl2 on N,N-demethylaniline oxidation by hydroperoxides is discussed.

A systematic study of inhibition by antibodies of dimethylaniline (DMA) and aniline oxidation has been carried out under different conditions: e. g. with participation of intact, phenobarbital- and 3-methylcholantrene-induced microsomes, NADPH and O2; during hydroperoxide-dependent oxidation with three types of microsomes; in a reconstituted system containing cytochrome P-450 LM2, NADPH cytochrome P-450 reductase, NADPH and O2; in systems containing cytochrome P-450 LM2 and cumene hydroperoxide. In all cases the antibodies effectively inhibited oxidation of both substrates. The degree of inhibition increased in the following order: intact less than 3-methylcholantrene- less than phenobarbital-induced microsomes. In the case of hydroperoxide-dependent oxidation of aniline and DMA no complete inhibition was achieved.