Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.
In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.
Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.
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D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D010053:Ovary / Q000201:enzymology*; D011247:Pregnancy; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D015089:20-Hydroxysteroid Dehydrogenases