Characterization of NADP-dependent 12 beta-hydroxysteroid dehydrogenase from Clostridium paraputrificum.
Clostridium paraputrificum D 762-06 was found to contain an NADP-dependent 12 beta-hydroxysteroid dehydrogenase, already present in uninduced cells. Its specific activity could, however, be enhanced up to about 3-fold by the inclusion of bile acids with a 12-keto group or a 12 beta-hydroxy group in the growth medium. 3 alpha-Hydroxy-12-keto-5 beta-cholanoic acid was the most effective inducer. A pH optimum of 10.0 and a molecular weight of 126,000 were estimated by molecular sieve chromatography. The enzyme preparation reduced 12-keto groups in conjugated and unconjugated bile acids and oxidized a 12 beta-hydroxy function, but oxidative activity was only about 25% of the reductive one. Disubstituted bile acids showed lower Km values than the corresponding trisubstituted ones, the lowest Km values being those observed for 3,12- and 7,12-5 beta-cholanoic acids. No measurable activity against 12 alpha-hydroxyl groups could be detected. The enzyme was found to be heat-labile (95% inactivation at 50 degrees C for 10 min), but the activity was maintained for about 4 weeks when lyophilized preparations were stored at -20 degrees C. 12 beta-Hydroxysteroid dehydrogenase activity was also demonstrated in the membrane fraction after solubilization with Triton X-100, suggesting that it was originally a membrane-bound enzyme.
Edenharder
R
R
Institute of Hygiene, University of Mainz, (F.R.G.).
Pfützner
A
A
D001647:Bile Acids and Salts / Q000378:metabolism / Q000494:pharmacology; D003013:Clostridium / Q000201:enzymology*; D004355:Drug Stability; D004790:Enzyme Induction / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008095:Lithocholic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001647:Bile Acids and Salts; C031930:12-ketolithocholic acid; D009249:NADP; D008095:Lithocholic Acid; D006913:Hydroxysteroid Dehydrogenases; C057347:12 beta-hydroxysteroid dehydrogenase