Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in Escherichia coli.
Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in Escherichia coli.
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DOI：
10.1006/abbi.1997.0230
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发表时间：
1997-08
影响因子：
3.9
通讯作者：
Huamin Wang;H. Strobel
中科院分区：
文献类型：
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作者：
Huamin Wang;H. Strobel
CYP3A9（一种来自大鼠大脑的新型 CYP3A）表达的性别二态性首次在大鼠大脑和大鼠肝脏中显示。 CYP3A9 在大鼠肝脏中的表达具有雌性特异性，根据其在雌性中的表达量比雄性高 10 倍来判断。雄性和雌性大鼠中雌激素治疗均可诱导 CYP3A9 基因表达。成年雌性大鼠的卵巢切除会导致 CYP3A9 mRNA 水平急剧下降，而这种水平可以通过补充雌激素来完全恢复。这些结果表明雌激素可能在 CYP3A9 基因的女性特异性表达中发挥重要作用。通过pCWOri+表达载体和MALLLAVF氨基末端序列修饰，在大肠杆菌中表达P450 3A9重组蛋白。该构建体具有高水平的表达（130 nmol P450 3A9/升培养物），并且通过两个色谱步骤从溶解的级分中将修饰的 P450 3A9 的重组蛋白纯化至电泳均质，其特定含量为 10.1 nmol P450/mg 蛋白。在含有脂质和大鼠 NADPH-P450 还原酶的重构系统中，纯化的 P450 3A9 蛋白在丙咪嗪、红霉素、苯丙胺和乙基吗啡以及 17β-雌二醇的代谢中具有活性。特别令人感兴趣的是，P450 3A9 可以催化地昔帕明的形成，周转数为 4.9 nmol/min/nmol P450，表明该异构体可能参与脑内丙咪嗪的代谢。 P450 3A9 活性的最佳重构条件需要脂质混合物（L-α-二月桂酰磷脂酰胆碱、L-α-二油酰磷脂酰胆碱和磷脂酰丝氨酸的 1:1:1 混合物）和 GSH。
Sexual dimorphism in the expression of CYP3A9, a novel form of CYP3A from 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 gene 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 of CYP3A9 which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of the CYP3A9 gene. P450 3A9 recombinant protein was expressed in Escherichia coli by 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 17beta-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 of L-alpha-dilauroyl phosphatidylcholine, L-alpha-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.