Characterization of NADP-dependent 12 Beta-Hydroxysteroid Dehydrogenase From Clostridium Paraputrificum

Biochim Biophys Acta. 1988 Oct 14;962(3):362-70. doi: 10.1016/0005-2760(88)90266-4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bile Acids and Salts / metabolism
  • Bile Acids and Salts / pharmacology
  • Clostridium / enzymology*
  • Drug Stability
  • Enzyme Induction / drug effects
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Hydroxysteroid Dehydrogenases / metabolism*
  • Kinetics
  • Lithocholic Acid / analogs & derivatives
  • Lithocholic Acid / metabolism
  • Lithocholic Acid / pharmacology
  • Molecular Weight
  • NADP / pharmacology*
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Bile Acids and Salts
  • 12-ketolithocholic acid
  • NADP
  • Lithocholic Acid
  • Hydroxysteroid Dehydrogenases
  • 12 beta-hydroxysteroid dehydrogenase