Purification and properties of a 3α-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs

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Abstract
An NAD(P)-dependent 3.alpha.-hydroxysteroid dehydrogenase was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI [isoelectric point], MW, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source. Since 3.alpha.-hydroxy steroids are by far the most efficient substrates, the enzyme is more appropriately designated a 3.alpha.-hydroxysteroid dehydrogenase. It also promotes the NAD(P)H-dependent reductions of quinones (e.g., 9,10-phenanthrenequinone, 1,4-benzoquinone), aromatic aldehydes (4-nitrobenzaldehyde) and aromatic ketones (4-nitroacetophenone). The dehydrogenase is not inhibited by dicoumarol, disulfiram, hexobarbital or pyrazole. The mechanism of the powerful inhibition of this enzyme by both non-steroidal and steroidal anti-inflammatory drugs was examined with several substrates. Most non-steroidal anti-inflammatory durgs are competitive inhibitors (e.g., Ki for indomethacin, 0.20 .mu.M for 9,10-phenanthrenequinone reduction at pH 6.0 and 0.835 .mu.M for androsterone oxidation at pH 7.0), except for salicylates, which act non-competitively (e.g., Ki for aspirin, 650 .mu.M for androsterone oxidation). The inhibitory potency of these agents falls sharply as the pH is increased from 6 to 9. Most anti-inflammatory steroids are likewise competitive inhibitors, except for the most potent (betamethasone and dexamethasone), which act non-competitively. The enzyme is inhibited competitively by arachidonic acid and various prostaglandins.