Reinigung und charakterisierung einer 3′-phosphoadenylylsulfat:steroid-sulfotransferase aus der leber des menschen

Abstract

A 3′-phosphoadenylylsulphate:steroid sulphotransferase is localised in the 150 000 × g supernatant of normal human liver. The enzyme was purified 22-fold by (NH 4) 2SO 4 precipitation, by gel filtration of the precipitate through Sephadex G-200 and by chromatography on DEAE-cellulose. After incubation of the enzyme with dehydroepiandrosterone as substrate, the reaction product isolated was identified as dehydroepiandrosterone sulphate by paper chromatography, various micro-chemical reactions and crystallization with authentic carrier to constant specific activity. The sulphotransferase showed a pH optimum at 7.4 in Tris-HCl buffer. The Michaelis-Menten constants were found to be 6.7·10 −6 M for dehydroepiandrosterone and 4.0·10 −6 M for 3′-phosphoadenylylsulphate. The enzyme lost its activity within 6 days when stored at 4°; however, at −20°, the enzyme was stable for at least 4 months. The steroid sulphotransferase showed a temperature optimum at 50° and an activation energy of 22.2 kcal/mole, within the range of 37–50°. By gel filtration, using Sephadex G-200, the molecular weight of the sulphotransferase was found to be approximately 50 000. The activity of the purified enzyme preparation was not influenced by the addition of cystein or EDTA. In relatively high concentration, SH-blocking agents had an inhibitory effect. The addition of Co 2+, Ni 2+, Ca 2+ and Mn 2+ activated the enzyme, whereas Cu 2+, Zn 2+, Fe 2+ and Hg 2+ inhibited the sulphotransferase. The steroid sulphotransferase from human liver conjugates not only dehydro-epiandrosterone (100%), but also (although to a lower extent) 3α-hydroxy-5β-androstan-17-one (45%), oestriol (31%), oestradiol-17β (15%) and oestrone (8%). Testosterone and cholesterol were not conjugated by the enzyme.