Waxman D J, Morrissey J J, Leblanc G A
Department of Biological Chemistry, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Mol Pharmacol. 1989 Apr;35(4):519-25.
Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.
垂体决定的激素通过涉及负调控和正调控的过程来调节肝细胞色素P-450的表达。因此，垂体切除术后大鼠肝脏中几种P-450形式的蛋白质水平升高（指定为2a的P-450形式（基因IIIA2）、RLM2（基因IIA2）和PB-4（基因IIB1）），而其他形式的蛋白质水平则受到抑制（例如，P-450 2c（基因IIC11））。在本研究中，发现与这些相同P-450形式相关的微粒体类固醇羟化酶活性在垂体切除术后降低，尽管其中几种的蛋白质水平有所升高。因此，开展了研究以确定微粒体P-450酶比活性降低的生化基础。在使血红素恢复到睾丸P-450的条件下，用促性腺激素对垂体切除的大鼠进行体内治疗，以及用肝匀浆进行的血红素重建实验表明，P-450相关血红素的缺乏不太可能解释所观察到的肝脏P-450酶比活性的降低。然而，对黄素蛋白P-450还原酶的分析显示，从垂体切除的大鼠分离的肝微粒体中，还原酶蛋白及其相关的细胞色素c还原酶活性降低了50%至75%。此外，用外源纯化的P-450还原酶补充分离的肝微粒体，优先刺激了垂体切除大鼠的微粒体类固醇羟化酶活性，使其达到与观察到的P-450蛋白水平变化一致的水平。因此，P-450还原酶的缺乏似乎是垂体切除大鼠中类固醇羟化酶比活性降低的原因，P-450还原酶是许多P-450依赖性羟化反应的限速成分。虽然对垂体切除的大鼠施用生长激素、促肾上腺皮质激素和绒毛膜促性腺激素在恢复肝脏P-450还原酶方面均无效，但用甲状腺素治疗垂体切除的大鼠可使P-450还原酶水平得到实质性恢复。用甲状腺素治疗这些大鼠还将与各个肝脏P-450形式相关的微粒体类固醇羟化酶活性提高到与其各自P-450蛋白水平相当的水平。这些结果表明，肝脏P-450还原酶受到与控制细胞色素P-450表达不同的激素调控，并进一步证明了肝脏类固醇激素代谢内分泌控制的复杂性。
