During the past ten years much information has been accumulated concerning the interaction of tritiated estrogens with hormone-dependent tissues. Their striking affinity for estradiol, both in vivol-5 and in vitro,6-9 first suggested that such target tissues as uterus, vagina, and anterior pituitary possess unique components called "estrogen receptors." Strong but reversible association of hormone with receptor, without chemical transformation of the steroid molecule, appears to be a primary step in the uterotrophic process, not affected by such inhibitors of early estrogen response as puromycin or actinomycin D.10 The interaction of estradiol with target tissues involves two distinct phenomenauptake and retention; the latter process, but not the former, becomes saturated in vivo if the hormone administered exceeds the physiological level.1' Centrifugal fractionation experiments'0-'4 demonstrate two sites of estrogen binding in uterine cells. As confirmed by autoradiography,"5 16 most estradiol, both in endometrium and in myometrium, resides in the nuclei, but a certain amount is bound to a macromolecular substance appearing in the supernatant fraction. Toft and Gorski"7 made the important observation that the radioactive estradiol-receptor complex in the supernatant fraction can be characterized by ultracentrifugation in sucrose density gradients where it migrates with a sedimentation coefficient of 9.5S; disruption of the complex by proteases but not by nucleases suggests that the receptor substance is a protein. We have confirmed these findings and have observed further8-20 that a different estradiol-receptor complex, sedimenting at about 5S, can be extracted from uterine nuclei by cold 0.3 M KC1, which solubilizes little uterine DNA.21 Although the 9.5S complex does not appear to be a simple aggregate of the 5S, the two receptor substances show many similar characteristics, discussed in more detail elsewhere.22 An important difference is the ability of the 9.5S complex to form spontaneously on addition of tritiated estradiol to supernatant fraction;9' 20 23 the 5S complex is not produced by adding estradiol to nuclear extract,24 although it can be formed in the whole homogenate. This paper presents evidence that the 9.5S receptor is extranuclear and involved in estrogen uptake, whereas the nuclear 5S complex, probably responsible for hormone retention and growth initiation, is formed from the 9.5S complex by a process which consumes 9.5S receptor and which is retarded at low temperature. Materials and Methods.-These investigations used estradiol-6, 7-H' (spec. act. 57.4 c/mmole), KRH buffer (pH 7.3, Krebs-Ringer-Henseleit-glucose, containing in gm/liter: NaCI, 8.0; KC1, 0.20; Na2HPO4-7H20, 1.73; KH2PO4, 0.20; CaCl2, 0.10; MgCl2, 0.048; and glucose, 1.0), and Tris-EDTA buffer (pH 7.4, 0.01 M 2-amino-2-hydroxymethyl-1,3-propanediol plus 0.0015 M ethylenediaminetetraacetic acid, disodium salt).

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity.

We describe the cloning and sequencing of porcine 17P-estradiol dehydrogenase. The enzyme performs oxidation 360-fold more efficiently than reduction, both measured under optimal conditions. It is localized in specialized vesicles of epithelial cells. The cDNA clones were isolated from a AUNI ZAP XR library of porcine kidney and polymerase-chain-reaction-amplified from templates of uterus epithelium. In both tissues, the same enzyme is coded by a transcript of 2. The open reading frame of 737 amino acids with a predicted molecular mass 79973Da was confirmed by amino acid sequencing of peptides. The 80-kDa translation product is processed to the N-terminal 32-kDa enzyme, part of which is then covalently linked to actin. The estradiol dehydrogenase/actin complex and the 80-kDa translation product comigrate in SDSPAGE.

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Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.