3-BETA-HYDROXYSTEROID DEHYDROGENASE-ACTIVITY IN TISSUES OF THE HUMAN FETUS DETERMINED WITH 5-ALPHA-ANDROSTANE-3-BETA, 17-BETA-DIOL AND DEHYDROEPIANDROSTERONE AS SUBSTRATES

3beta-Hydroxysteroid dehydrogenase (3beta-HSD)/DELTA5-->4-isomerase ac tivity in steroidogenic tissues is required for the synthesis of biolo gically active steroids. Previously, by use of dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was establis hed that in addition to steroidogenic tissues 3beta-HSD/DELTA5-->4-iso merase activity also is expressed in extraglandular tissues of the hum an fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3beta-HSD activity. Fo r this purpose, we compared the efficiencies of a 3beta-hydroxy-5-ene steroid (DHEA) and a 3beta-hydroxy-5alpha-reduced steroid (5alpha-andr ostane-3beta,17beta-diol, 5alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K(m)) for 5alpha-A-diol in midtrimes ter placenta, fetal liver, and fetal skin tissues was at least one ord er of magnitude higher than that for DHEA, viz the apparent K(m) of pl acental 3beta-HSD for 5alpha-A-diol was in the range of 18 to 40 mumol /l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme , 17 mumol/l for 5alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human f etal tissues evaluated the maximal velocities obtained with 5alpha-A-d iol as substrate were higher than those obtained with DHEA. A similar finding in regard to K(m)s and rates of product formation was obtained by use of purified placental 3beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5alpha-androstan-17-one (epiandrosterone) as substrates: the K. of 3beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mum ol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein - min and, with epiandrosterone, 127 nmol/mg protein - min. With plac ental homogenate as the source of 3beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [H-3]5alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was o f the mixed type; similar results were obtained with [H-3]DHEA as the substrate at variable concentrations in the presence of a fixed concen tration of 5alpha-A-diol (40 mumol/l). These findings are indicative t hat both steroids bind to a common site on the enzyme, however, the bi nding affinity for these steroids appear to differ markedly as suggest ed by the respective K(m)s. Studies of inactivation of purified placen tal 3beta-HSD/DELTA5-->4-isomerase by an irreversible inhibitor, viz 5 ,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroi dal substrate has a 5alpha-configuration, e.g. epiandrosterone, or a C -5,C-6-double bond, e.g. DHEA or pregnenolone. The lower rates of prod uct formation obtained with placenta and fetal tissues by use of 3beta -hydroxy-5-ene steroids as substrates when compared with those obtaine d with 3beta-hydroxy-5alpha-reduced steroids may be explained by a com bination of factors, including: (i) inhibition of 3beta-HSD activity b y end products of metabolism of 3beta-hydroxy-5-ene steroids, e.g. 4-a ndrostene-3,17-dione formed with DHEA as substrate; (ii) higher bindin g affinity of the enzyme for 3beta-hydroxy-5-ene steroids-and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for th e isomerization step with 5alpha-reduced steroids as substrates, and ( iv) the possible presence in fetal tissues of an enzyme with 3beta-HSD activity only (i.e. no DELTA5-->4-isomerase).