Effect Of 3-Methylcholanthrene On Bunitrolol Metabolism - Kinetics And Immunological Studies On 4-Hydroxylation Of Bunitrolol Catalyzed By Two Species Of Cytochromes P450 In Rat Liver Microsomes

Effect of the induction of cytochrome P4501A1 with 3-methylcholanthrene (3-MC) treatment on kinetics of bunitrolol (BTL) 4-hydroxylase activity of rat liver microsomes was investigated, The relationship between the rate and BTL concentration showed monophasic kinetics (K-M = 0.74 +/- 0.13 mu M) when microsomes from untreated rats were used, whereas microsomes from 3-MC-treated rats showed biphasic kinetics (K-M1 = 0.76 +/- 0.13, K-M2 = 646 +/- 16 mu M). Anti-cytochrome P450 (P450) BTL (P4502D subfamily) antiserum inhibited the reaction >90% when low concentrations (similar to 10 mu(M)) of BTL were used in both microsomes. However, at high BTL concentrations (similar to 2,000 mu M), the inhibition was only up to a half of control in microsomes from 3-MC-treated rats, whereas in microsomes from untreated rats, the rates were suppressed >90%. Kinetics observed in microsomes from 3-MC-treated rats changed to nearly monophasic, with a K-M value corresponding to K-M2. Anti-P4501A1 IgG, on the other hand, hardly inhibited the reaction conducted by liver microsomes from 3-MC-treated rats when substrate concentrations were low, whereas at higher concentrations, it inhibited up to 50%, resulting in a monophasic kinetics with a K-M value corresponding to K-M1. These results clearly indicate that the biphasicity of kinetics in BTL metabolism in liver microsomes from 3-MC-treated rats is caused by the involvement of two P450 species: P450 BTL and P4501A1 in the reaction. This is the first direct evidence to the theoretical hypothesis that the biphasic kinetics of the enzyme reaction is caused by the involvement of at least two enzymes with different kinetic parameters.