Farkash Y, Soreq H, Orly J
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1988 Aug;85(16):5824-8. doi: 10.1073/pnas.85.16.5824.
The enzyme 4-ene-3-ketosteroid-5 alpha-oxidoreductase [5 alpha-reductase; 3-oxo-5 alpha-steroid delta 4-dehydrogenase, 3-oxo-5 alpha-steroid: (acceptor) delta 4-oxidoreductase, EC 1.3.99.5] plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5 alpha-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. We developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of [3H]testosterone revealed the in ovo appearance of active 5 alpha-reductase. Polyadenylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1600- to 2000-base-pair fraction of hepatic poly(A)+ RNA. 5 alpha-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5 alpha-reductase mRNA was found to be a short-lived molecule (t1/2 = 2 hr), whereas its in ovo translatable 5 alpha-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5 alpha-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5 alpha-reductase activities in female rat liver, male rat liver, and prostate homogenates; the ratio of their specific activities was of 2500:630:1, respectively. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5 alpha-reductase expression in rat tissues.
4-烯-3-酮甾体-5α-氧化还原酶[5α-还原酶；3-氧代-5α-甾体δ4-脱氢酶，3-氧代-5α-甾体：（受体）δ4-氧化还原酶，EC 1.3.99.5]在雄激素依赖的靶组织中起关键作用，它催化睾酮转化为具有生物活性的二氢睾酮。由于该酶是一种膜蛋白，在无细胞匀浆中不稳定，因此尚未在分子水平上研究5α-还原酶表达的调控。我们开发了一种灵敏的生物测定法，用于检测注射了大鼠肝脏和前列腺mRNA的非洲爪蟾卵母细胞中表达的酶活性。显微注射后，将完整的卵母细胞在[3H]睾酮存在下孵育，发现卵内出现了活性5α-还原酶。通过蔗糖梯度离心对聚腺苷酸化RNA进行分级分离，结果表明酶活性由肝脏聚（A）+RNA的1600至2000个碱基对的片段编码。当每个卵母细胞注射多达80 ng的RNA时，5α-还原酶mRNA的翻译效率最高。在注射的卵母细胞中，发现5α-还原酶mRNA是一种寿命较短的分子（半衰期=2小时），而其卵内可翻译的5α-还原酶蛋白在超过40小时内表现出稳定的酶活性。此外，在非洲爪蟾卵母细胞中监测到的可翻译的组织特异性5α-还原酶mRNA水平与雌性大鼠肝脏、雄性大鼠肝脏和前列腺匀浆中5α-还原酶活性的变化相关；它们的比活性分别为2500:630:1。总之，这些结果为大鼠组织中5α-还原酶表达的转录调控提供了支持证据。
