5-Ene-3β-Hydroxysteroid DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

Abstract

Protein moieties of various molecular sizes and possessing 5-ene-3beta-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118 000, with an isoelectric point between 7-2 and 7-4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17alpha-hydroxy-pregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrated specificity.

Evidence for distinct dehydrogenase and isomerase sites within a single 3.beta.-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

“…The activity of 3/3-hydroxysteroid dehydrogenase (EC 1.1.1.145)/steroid 5-ene-4-ene isomerase (EC 5.3.3.1), hereafter called 3/3-HSD, catalyzes the transformation of 5-ene-3/3-hydroxysteroids to the corresponding 4-ene-3-keto configuration (Samuels et al, 1951) and is therefore an essential step in the biosynthesis of all classes of hormonal steroids, namely, progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. While different proteins responsible for these two reactions have been isolated separately from bacterial sources (Talalay & Wang, 1955), the two activities appear to reside within a single protein in mammalian tissues as observed for the enzyme purified from bovine ovaries (Cheatum & Warren, 1955) as well as from human placenta (Luu-The et al, 1988, 1989Lachance et al, 1990), ovine adrenals (Ford & Engel, 1974), rat adrenals (Ishii-Ohba et al, 1987) andtestes (Ishii-Ohba et al, 1986), and bovine adrenals (Eastman & Neville, 1987;Inano et al, 1990).…”