Naville D, Keeney D S, Jenkin G, Murry B A, Head J R, Mason J I
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, University of Texas, Southwestern Medical Center, Dallas 75235-9051.
Mol Endocrinol. 1991 Aug;5(8):1090-100. doi: 10.1210/mend-5-8-1090.
In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)
在性腺和肾上腺皮质中的类固醇生成途径中，3β-羟基类固醇脱氢酶异构酶（3βHSD）是一种关键酶，它控制着从δ5-3β-羟基类固醇形成δ4-3-酮类固醇。在此，我们使用针对人胎盘3βHSD的抗体和大鼠睾丸3βHSD cDNA探针来研究大鼠肝脏3βHSD mRNA和蛋白质的表达。先前报道大鼠肝脏微粒体3βHSD活性存在显著的性别差异，雄性的活性要高得多。我们已经表明，在雄性大鼠成熟过程中，免疫反应性3βHSD水平呈年龄依赖性增加。免疫反应性蛋白的分子大小与人胎盘和大鼠睾丸3βHSD相似，定位于肝脏的微粒体部分，并集中在中央周围区域。在未成熟（25日龄前）的两性大鼠肝脏或成年雌性肝脏中未检测到免疫反应性蛋白。用大鼠睾丸3βHSD cDNA探针对肝脏和睾丸RNA进行Northern印迹分析显示，成年雄性肝脏中除了主要的2.1千碱基mRNA种类外，还存在一种1.6千碱基的mRNA种类，在未成熟或成年雌性肝脏RNA中均未检测到这两种mRNA。雌性大鼠垂体切除或用睾酮植入物处理可诱导肝脏3βHSD蛋白表达，而对雄性大鼠持续输注生长激素可降低3βHSD蛋白水平。同样，生长激素处理后mRNA种类的水平也降低。使用[3α-3H]脱氢表雄酮作为3βHSD活性的底物，我们测定成年雄性和雌性肝脏中肝脏微粒体NAD(+)-依赖性3βHSD活性的表观Km均为20μM，远大于大鼠睾丸间质细胞瘤3βHSD活性的Km（0.2μM）。肝脏3βHSD活性受到曲洛司坦的抑制，曲洛司坦是一种已证实的性腺和肾上腺3βHSD活性抑制剂。从雄性肝脏cDNA文库中分离出一种大鼠肝脏3βHSD cDNA，它与大鼠卵巢的II型3βHSD形式密切相关，但与III型肝脏3βHSD不同。该cDNA表达后获得的酶具有雄性特异性NAD(+)-依赖性肝脏微粒体3βHSD的特性（即对脱氢表雄酮具有高表观Km），且与高亲和力性腺I型3βHSD的特性不同。（摘要截短至400字）
