REGULATION OF CYP3A9 GENE-EXPRESSION BY ESTROGEN AND CATALYTIC STUDIES USING CYTOCHROME P4503A9 EXPRESSED IN ESCHERICHIA-COLI

Sexual dimorphism in the expression of CYP3A9, a novel form of CYP3A f rom rat brain, is shown for the first time in rat brain as well as in rat liver. CYP3A9 expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males. CYP3A9 gen e expression was inducible by estrogen treatment both in male and in f emale rats. Ovariectomy of adult female rats elicited a drastic reduct ion on the mRNA level of CYP3A9 which could be fully restored by estro gen replacement. These results suggest that estrogen may play an impor tant role in the female-specific expression of the CYP3A9 gene, P450 3 A9 recombinant protein was expressed in Escherichia coli by means of t he pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification, This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P 450 3A9 was purified to electrophoretic homogeneity with a specific co ntent of 10.1 nmol P450/mg protein from solubilized fractions through two chromatographic steps, The purified P450 3A9 protein was active in the metabolism of imipramine, erythromycin, benzphetamine, and ethylm orphine as well as 17 beta-estradiol in a reconstituted system contain ing lipid and rat NADPH-P450 reductase. Of special interest is the fin ding that P450 3A9 can catalyze the formation of desipramine with a tu rnover number of 4.9 nmol/min/nmol P450, suggesting the possible invol vement of this isoform in the metabolism of imipramine in brain, Optim al reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture of L-alpha-dilauroyl phosphatidylcholine, L-alp ha-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH. (C) 1997 Academic Press.