Purification, Reconstitution, and Steady-state Kinetics of the Trans-membrane 17β-Hydroxysteroid Dehydrogenase 2

Abstract

Human membrane 17␤-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17␤-hydroxysteroids into their inactive keto forms in a variety of tissues. 17␤-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-␤-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-␤-D-maltoside-destabilized liposomes prepared from L-␣-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 mol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 mol/ 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␤-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K m of NAD being 110 ؎ 10 M and that of NADP 9600 ؎ 100 M using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20␣-dihydroprogesterone as substrates were also determined.

The Contribution of 17beta-Hydroxysteroid Dehydrogenase Type 1 to the Estradiol-Estrone Ratio in Estrogen-Sensitive Breast Cancer Cells

“…[E2]/[E1] ratio was unchanged by the addition of 0.5 mM NAD to T47D and MCF-7-17β-HSD1 cells (with higher expression of 17β-HSD1 and lower expression of 17β-HSD2), whereas it was decreased by the addition of 0.5 mM NADP. The Kcat/Km value for 17β-HSD2 using NAD is 400 times greater than that using NADP, so NAD is the preferred cofactor for its major role in E2 oxidation [15]. In addition, the specificity of 17β-HSD2 to NAD is around 3 times higher than that of 17β-HSD1 [14]–[16].…”

“…The highly active 17β-HSD1 plays an important role in E2 synthesis using NADPH as cofactor, with a Km value of 0.03±0.01 µM [14]. 17β-HSD2 plays an important role in E1 production using NAD as cofactor, with a Km value of 0.35±0.09 µM [15]. In some estrogen-dependent breast cancer cells, 17β-HSD1 is more abundantly expressed than 17β-HSD2 [16]–[18].…”

Role of aldo–keto reductase family 1 (AKR1) enzymes in human steroid metabolism

“…AKR1C3 catalyzes reduction of estrone ( K d = 0.3 nM for ERα and ERβ) to yield the more potent estrogen 17β-estradiol, ( K d = 0.1 nM for ERα and ERβ) and thus controls activation of ERα and ERβ [37]. The reverse reaction is catalyzed by HSD17B2 also designated as SDR9C2 [49, 50]. Thus these reactions represent molecular switches that can finely tune and control ligand occupancy of the ER.…”

Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

“…However, it proved impossible to extract any VGLUT1 from the insect cell membranes using Cholate or CHAPS (data not shown), and no protein was found bound to the Talon beads using these conditions (data not shown). Previous accounts on Baculovirus-expression of membrane proteins have indicated that Cholate (or CHAPS) extract poorly proteins from insect cell membranes (14). Guided by these observations (14), by substituting Cholate/CHAPS with 1% n-decyl-β-d-maltopyranoside, and using otherwise identical conditions to those used for the PC12 based purification, it was found possible to extract VGLUT1 from the insect cell membranes (Fig.…”