Regulation ofCYP3A9Gene Expression by Estrogen and Catalytic Studies Using Cytochrome P450 3A9 Expressed inEscherichia coli

Abstract

Sexual dimorphism in the expression ofCYP3A9,a novel form ofCYP3Afrom rat brain, is shown for the first time in rat brain as well as in rat liver.CYP3A9expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males.CYP3A9gene expression was inducible by estrogen treatment both in male and in female rats. Ovariectomy of adult female rats elicited a drastic reduction on the mRNA level ofCYP3A9which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of theCYP3A9gene. P450 3A9 recombinant protein was expressed inEscherichia coliby means of the 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 P450 3A9 was purified to electrophoretic homogeneity with a specific content 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 ethylmorphine as well as 17β-estradiol in a reconstituted system containing lipid and rat NADPH-P450 reductase. Of special interest is the finding that P450 3A9 can catalyze the formation of desipramine with a turnover number of 4.9 nmol/min/nmol P450, suggesting the possible involvement of this isoform in the metabolism of imipramine in brain. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture ofl-α-dilauroyl phosphatidylcholine,l-α-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.