Topography of human placental 3 B-hydroxysteroid dehydrogenase/A 5-4 isomerase in microsomal membrane. A study using limited proteolysis and immunoblotting

ALVAREZ, C; GENTI-RAIMONDI S; PATRITO, L; FLURY, A

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY

Elsevier

Año: 1994 vol. 1207 p. 102 - 108

0167-4838

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase Δ5-4 isomerase (3β-HSD) catalyzes the formation of Δ4-3-ketosteroids from Δ5-3β-hydroxysteroids in placental, adrenal, testicular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3β-HSD within the human placental microsome membranes employing immune-replica analysis in combination with surface specific proteolysis. The crucial domains of the enzyme for the dehydrogenase and isomerase reactions are inactivated by proteinase treatments under conditions where latency of hexose-6-phosphate dehydrogenase was 95%. The data indicate that these crucial domains face the cytosolic side of the endoplasmic reticulum membrane. Incubation of the intact microsomes with trypsin produces several immune reactive fragments ranging from 29 to 11 kDa in addition to 42 kDa native enzyme, one of them being shielded by the membrane structure and/or by other intrinsic and peripheral membrane proteins. Carboxypeptidase Y degraded the C terminus of the 42 kDa native 3β-HSD in intact and detergent-disrupted microsomes, preserving partially a fragment of 31 kDa. The results from the carboxypeptidase Y digestion indicate that the carboxy terminal end of the 3β-HSD enzyme is located on the cytoplasmic surface of the endoplasmic reticulum and that only a small fragment of approx. 11 kDa could be removed easily without affecting the enzyme activity. From these data and the predicted hydropathy analysis from the literature, we tried to assign a transmembrane arrangement to the human placental 3β-HSD. Our results support a topology model in which practically all the structural 3β-HSD enzyme is exposed to the cytoplasmic side of the membrane with one NH2-terminal-anchoring segment and all the 3β-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.