TOPOGRAPHY OF HUMAN PLACENTAL 3-BETA-HYDROXYSTEROID DEHYDROGENASE DELTA(5-4) ISOMERASE IN MICROSOMAL MEMBRANE - A STUDY USING LIMITED PROTEOLYSIS AND IMMUNOBLOTTING

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase Delta(54 ) isomerase (3 beta-HSD) catalyzes the formation of Delta(4)-3-ketoste roids from Delta(5)-3 beta-hydroxysteroids in placental, adrenal, test icular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3 beta-HSD within the human placental m icrosome membranes employing immune-replica analysis in combination wi th surface specific proteolysis. The crucial domains of the enzyme for the dehydrogenase and isomerase reactions are inactivated by proteina se treatments under conditions where latency of hexose-6-phosphate deh ydrogenase was 95%. The data indicate that these crucial domains face the cytosolic side of the endoplasmic reticulum membrane. Incubation o f the intact microsomes with trypsin produces several immune reactive fragments ranging from 29 to 11 kDa in addition to 42 kDa native enzym e, one of them being shielded by the membrane structure and/or by othe r intrinsic and peripheral membrane proteins. Carboxypeptidase Y degra ded the C terminus of the 42 kDa native 3 beta-HSD in intact and deter gent-disrupted microsomes, preserving partially a fragment of 31 kDa. The results from the carboxypeptidase Y digestion indicate that the ca rboxy terminal end of the 3 beta-HSD enzyme is located on the cytoplas mic surface of the endoplasmic reticulum and that only a small fragmen t of approx. II kDa could be removed easily without affecting the enzy me activity. From these data and the predicted hydropathy analysis fro m the literature, we tried to assign a transmembrane arrangement to th e human placental 3 beta-HSD. Our results support a topology model in which practically all the structural 3 beta-HSD enzyme is exposed to t he cytoplasmic side of the membrane with one NH2-terminal-anchoring se gment and all the 3 beta-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.