Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes.
7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.
Department of Surgery I, Miyazaki University School of Medicine 5200 Kihara, Kiyotake Miyazaki, 889-1692, Japan.
Hironori
H
Shiotsuki
Yorio
Y
Maeda
Kazuo
K
Chijiiwa
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits
D006913:Hydroxysteroid Dehydrogenases; C031932:7 beta-hydroxysteroid dehydrogenase