No abstract

Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.