J. Steroid Biochem. Molec. Biol. Vol. 46, No. 2, pp. 265-267, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain Pergamon Press Lid SHORT COMMUNICATION CHARACTERIZATION AND SOLUBILIZATION OF TESTOSTERONE 17fl-HYDROXYSTEROID DEHYDROGENASE IN HUMAN HYPERPLASTIC PROSTATE JEAN-Luc CARRE,1 ERIC QUEMENER, l YOLANDEAMET,l BRIGITTESIMON, 1 FRANCOISBERTSOU, PHILIPPE MANGIN, 2 HERV~-HENRI FLOCH ~ a n d JEAN-HERVf/ABALA1N 1. tLaboratoire de Biochimie and 2Service d'Urologie, Facult6 de M6decine, 29200 Brest, France (Received 9 October 1992; accepted 5 April 1993) Summary--17//-Hydroxysteroid dehydrogenase is a membrane-bound enzyme in human prostate. Solubilization of this enzyme can only be obtained in the presence of detergents. The optimal solubilization mixture contained 50 mM Tris-HCl buffer pH 9.0, 20% glycerol, 0.1 M KC1 and 5 mg/ml of the non-ionic detergent N-octyl glucoside. In these conditions, the soluble fraction contained more than 90% of the enzymatic activity. A 2.5-fold increase of specific activity was obtained during solubilization under optimal conditions. INTRODUCTION The main pathway for testosterone metabolism in human prostatic tissue is the formation of 5a-dihydrotestosterone (DHT) [1, 2]. There is extensive experimental evidence to indicate that 5a-DHT acts as an intracelluiar mediator for the different actions testosterone exerts on the prostate [3]. However testosterone can also be converted into less potent or inactive androgens, i.e. 4-ene-androstenedione and 5a- androstanedione [4]. This transformation is a very import- ant step in testosterone action in that it controls the amount of testosterone available in the cells. The enzyme 17#-hy- droxysteroid dehydrogenase (17,O-HSD, E.C.1.1.1.63) catalyzes the interconversion of testosterone and 4-ene-an- drostenedione. Association of 17//-HSD activity with mem- branes in human prostate has been demonstrated [5]. A prerequisite to purification and further characterization is therefore its solubilization from the membranes. The present paper describes the characterization and optimization of conditions for the solubilization of the 17#-HSD from human prostatic tissue. MATERIALS AND METHODS [l'C]Testosterone (52 mCi/mmol) was obtained from New England Corporation (Boston, MA, U.S.A.). The purity of labeled steroids was assessed by TLC on silica gel using benzene-ethanol (9: 1) as a solvent system. Non-radioactive steroids, cofactors and detergents (Triton X-100, Lubrol PX, Na cholate, N-octyl giucoside, Tween 20) were obtained from Sigma (St Louis, MO, U.S.A.). Lubrol WX (Cirrasol AIn Wf was a gift from ICI (Belgium); other chemicals were of analytical grade and bought from Merck (Darmstadt, Germany). All solvents were HPLC grade and were bought from SDS (Solvant Documentation Synthese; Peypin, 13124, France). Hyperplastic prostatic tissue was obtained from men undergoing transurethrai prostatic resection. Ali- *To whom correspondence should be addressed. quots were sent to pathology for histological assessment. Tissues were kept in liquid nitrogen until use. The prostatic tissue was quickly thawed, blotted dry and weighed. All procedures were carried out at 4°C. Slices of the tissue were forced through the 1 mm holes of a disc with an arbor tissue press (Harvard Apparatus, MA, U.S.A.). The minced tissue (0.5 g) was homogenized with a polytron unit (Kinematica, Luzern, Switzerland) in I vol of 50 mM Tris-HCI, pH 9.0, 1 mM EDTA, 5 mM dithiothreitol, 20% glycerol, 100mM KC1 and a detergent. The detergent concentrations used are given in Table 1. 17//-HSD activity was assayed on half the homogenate. The solubilized and membrane bound 17#- HSD activity were separated by immediately centrifuging the homogenate at 105,000g for I h. The supernatants were saved and the pellets were resuspended in 4 ml of homogen- ization buffer containing the same amount of detergent. 17#-HSD activity was assayed on resuspended pellets and on supernatants. Radioactive testosterone (2.4nmol), dissolved in ben- zene-ethanol (9: I), was transferred to an incubation flask and dried under a stream of N2. The basal incubation medium contained the tissue preparation (0.25 g equivalent tissue), substrate steroid, 2 mM NAD, 50 mM Tris-HCl pH 9.0, 1 mM EDTA, 5 mM dithiothreitol, 20% glycerol in a final volume of 5 ml unless otherwise indicated. The enzyme reaction was started by adding the tissue prep- aration. Incubations were carried out at 45°C for 30 rain with constant shaking. The reaction was stopped by ad- dition of 5 ml of cold dichioromethane and the mixture put on ice. The enzyme activity was expressed as pmol of 4-ene-androstenedione formed per 30 rain and per mg pro- tein. All radioactive metabolites were extracted four times with 5 ml of dichioromethane. The extracts were pooled and evaporated under nitrogen. Recovery of steroids was mow itored by counting the radioactivity from an aliquot and was always close to I00%. The organic extracts were redissolved in 70/zl of the following reference steroids: I0/~M testoster- one, 1D#M androstenedione, 10mM DHT, 10mM an- drostanedione in HPLC grade acetonitrile. Radiolabeled 265