Use of a monoclonal antibody specific for rabbit microsomal cytochrome P-450 3b to characterize the participation of this cytochrome in the microsomal 6.beta.- and 16.alpha.-hydroxylation of progesterone

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Abstract
A monoclonal antibody was developed that is specific for the 3b electrophoretic class of rabbit liver microsomal cytochrome P-450 as judged by immunoprecipitation and subsequent electrophoretic analysis. The antibody is inhibitory of catalytically distinct, variant forms of P-450 3b prepared from New Zealand White or IIIVO/J rabbits, respectively. Peptide mapping of the immunopurified P-450 3b from NZW and IIIVO/J microsomes indicates that a characteristic difference between the variant forms is exhibited by the antigen. A competitive assay indicates that the binding properties of the antibody do not differ substantially toward the variant forms of P-450 3b. The inhibitory antibody was used to examine the contribution of P-450 3b to the microsomal 16.alpha.- and 6.beta.-hydroxylation of progesterone. The antibody inhibits 40-70% of the 16.alpha.-hydroxylase activity of microsomes from either New Zealand White or IIIVO/J rabbits. It does not inhibit 6.beta.-hydroxylation catalyzed by microsomes prepared from strain IIIVO/J but does inhibit this reaction as catalyzed by microsomes from most New Zealand White rabbits. The antibody also inhibits the increased 16.alpha.-hydroxylase activity of IIIVO/J microsomes observed in the presence of 5.beta.-pregnane-3.beta.,20.alpha.-diol, an allosteric effector of this variant form of P-450 3b. This monoclonal antibody provides a link between the observed properties of the purified, variant forms of P-450 3b and microsomal metabolism. The antibody can be used to phenotype variant forms of P-450 3b in microsomal fractions.

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