Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2.
Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.
Lu
Ming-Liang
ML
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUQ) and Laval University, Québec, Québec G1V 4G2, Canada.
Huang
Yi-Wei
YW
Lin
Sheng-Xiang
SX
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification*; D004092:20-alpha-Dihydroprogesterone / Q000737:chemistry; D000737:Androstenols; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000737:chemistry; D004960:Estradiol Dehydrogenases; D005990:Glycerol / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013739:Testosterone / Q000737:chemistry; D013997:Time Factors
D000737:Androstenols; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D003902:Detergents; D008081:Liposomes; D011994:Recombinant Proteins; D013196:Dihydrotestosterone; D004092:20-alpha-Dihydroprogesterone; D013739:Testosterone; D006639:Histidine; D004958:Estradiol; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; C523763:HSD17B2 protein, human; D005990:Glycerol; D010100:Oxygen; C005749:thiomesterone