Biosynthesis of catalytically active rat testosterone 5 alpha-reductase in microinjected Xenopus oocytes: evidence for tissue-specific differences in translatable mRNA.
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.
Farkash
Y
Y
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Soreq
H
H
Orly
J
J
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D005260:Female; D008099:Liver / Q000378:metabolism; D008297:Male; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism; D009928:Organ Specificity; D011061:Poly A / Q000378:metabolism; D011467:Prostate / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013739:Testosterone / Q000378:metabolism; D014982:Xenopus laevis
D012333:RNA, Messenger; D011061:Poly A; D013739:Testosterone; D012313:RNA; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase