Purification and Properties of Steroid Sulfotransferase in Human Fetal Liver

Abstract

Steroids in fetal circulation are known to exist in a sulfoconjugated form. To investigate the contribution of human fetal liver to the sulfoconjugation of steroids, and the properties of steroid sulfotransferase, an attempt was made to separate the specific sulfotransferases toward estrone (E-ST) and 3β-hydroxysteroid (3β-ST). For the purification procedure, successive chromatography was used:(1) DEAE-Sepharose CL-6B chromatography (2) Chromatofocusing and (3) Adenosine 3', 5'-diphosphateagarose affinity chromatography. Enzymatic activity was obtained with 3H-estrone (E1), 3H-pregnenolone (P) and 3H-dehydroepiandrosterone (DHA) as substrates. Two active fractions of steroid sulfotransferase were collected from the eluate of a CL-6B column. Fractions 88-120 found to be specific for E1, were purified approximately 1, 800 fold by further chromatographic procedures. The affinity column condition used was set at pH 7.2. The apparent Km and Vmax values were 1.66 μM and 35.6 nmol/mg protein/min, respectively. Fractions 130-162 specific for P were further purified approximately 144 fold. The affinity column condition used was set at pH 6.0. The apparent Km and Vmax values were 0.25 μM and 4.36 nmol/mg protein/min, respectively. Both enzymes were found to be a single band and had almost the same molecular weight of approximately 36 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both enzymes equally catalyzed DHA to form DHA-sulfate. In the present study, E-ST and 3β-ST were separated and purified in human fetal liver for the first time, with kinetic analysis for both enzymes.