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Journal of Lipid Research, Vol. 20, 234-239, February 1979
Copyright © 1979 by Lipid Research, Inc.

12-Hydroxysteroid dehydrogenase from Clostridium group P strain C48-50 ATCC #29733: partial purification and characterization

Ian A. MacDonald , Joanne F. Jellett , and David E. Mahony

Departments of Medicine and Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada

The growth of Clostridium group P strain C48-50 [an anaerobe that contains 12-hydroxysteroid dehydrogenase (12-HSDH) in the absence of other dehydrogenases active upon bile salts] is greatly enhanced by the addition of 2.0% d-fructose or d-glucose to the growth medium. Other sugars were less effective. The production of NADP-dependent 12-HSDH paralleled the growth of the organism which was optimal at 72 hr. Growth (and enzyme production) were suppressed by the addition of bile salt to the medium; the order of suppression was deoxycholate > chenodeoxycholate >> cholate; 1 mM of either of the dihydroxy-bile salts inhibited 96% of the growth and 100% of the enzyme production. Kinetic studies on cell-free preparations of 12-HSDH revealed a pH optimum of 7.8 with greater linearity of NADP evolution with time occurring only at more alkaline pH values (9-10). Lineweaver-Burke plots revealed Michaelis constant (K_m) values in the range of 3-5 x 10^-4 M for deoxycholate and its glycine and taurine conjugates, while higher values were found for cholate and conjugates (K_m value for taurocholate was 3 x 10^-3 M). Although there was no activity with NAD, 12-HSDH was shown to bind onto both NAD- and NADP-Sepharose columns, with stronger binding on the latter. The enzyme was purified 20-fold by NAD-Sepharose chromatography. The molecular weight was estimated at 100,000 by Sephadex G-200 and a series of molecular weight markers. Substrate specificity studies showed that a variety of bile salts containing 12-OH groups reacted; notably, the 3-sulfates of cholate and deoxycholate were nonsubstrates.—Macdonald, I. A., J. F. Jellett and D. E. Mahony. 12-Hydroxysteroid dehydrogenase from Clostridium Group P strain C48-50 #29733: partial purification and characterization.

Supplementary key words 12-hydroxyl group oxidation • bile salt oxidoreductase

Submitted on March 28, 1978
Accepted on August 1, 1978

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