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Characterization of Molecular and Catalytic Properties of Intact and Truncated Human 17ß-Hydroxysteroid Dehydrogenase Type 2 Enzymes: Intracellular Localization of the Wild-Type Enzyme in the Endoplasmic Reticulum¹

Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health (T.J.P., R.M.K., M.H.P., P.V.I., J.P.J.M., R.K.V., P.T.V.) and the Department of Anatomy (J.T.L.), University of Oulu, FIN-90220 Oulu, Finland; the Hauptman-Woodward Medical Research Institute, Inc., and Roswell Park Cancer Institute (D.G.), Buffalo, New York 14203; and the Department of Biosciences, Division of Biochemistry (P.T.V.), FIN-00014 University of Helsinki, Finland

Address all correspondence and requests for reprints to: Prof. Pirkko Vihko, Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: pvihko{at}whoccr.oulu.fi

Human 17ß-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17ß-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5ß-androstan-3,17ß-diol, 5-androstan-3,17ß-diol, and 5-androstan-3ß,17ß-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17ß-hydroxysteroids.

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