There is substantial evidence that microbially generated secondary bile acids (deoxychollic and lithocholic acid) are co-carcinogenic and these steroids may facilitate the development, growth and spread of large bowel cancer. Moreover, bile acids have recently been shown to specifically stimulate the formation of fecal mutagens (unsaturated glyceryl ether lipids) by intestinal anaerobic bacteria. The concentration of secondary bile acids in the large bowel appears to be a function of dietary habits and bile acids 7-dehydroxylase activity of the intestinal microflora.
The first aim of this grant proposal is the purification, characterization and regulation of bile acid 7-dehydroxylase from Eubacterium sp. V.P.I. 12708. Our current approach is to purify the bile acid inducible polypeptides (23,500, 27,000 56,000 and 77,000 daltons) using SDS-PAGE as a detection technique. These polypeptides are specifically induced by primary bile acids and co-purify (except 23,500) with 7-dehydroxylase activity during HPLC-gel filtration chromatography. Bile acid inducible polypeptides will be purified by (NH4)2SO4 fractionation, HPLC-DEAE chromatography (2 PH values), HPLC gel filtration and HPLC-reverse phase (C3) chromatography. Purified polypeptides will be used for physical characterization studies.
The second aim of this proposal is to raise antibody against bile acid inducible polypeptides. Specific antibody will be used in immunoprecipitation studies aimed at determining how many polypeptides make up bile acid 7-dehydroxylase and if 7Alpha, 7Beta and Delta6-reductase activities all reside in the same enzyme complex.
The third aim will be the purification and characterization of bile acid binding proteins and to determine their role in bile acid 7-dehydroxylation.
The fourth aim of this proposal will be to clone bile acid 7-dehydroxylase from Eubacterium sp. V.P.I. 12708 into E. coli using the expression vector PKG 1800.
The final aim will be to determine the mechanism of conversion of 3Alpha-sulfolithocholic to Delta3-cholenoic acid and 5Beta-cholanoic acid by Clostridum sordellii and to develop a """"""""cell-free"""""""" system that will catalyze this bile acid biotransformation(s) so that the enzymology and mechanism of the reaction can be studied in detail.
