Subcellular distribution of cholic acid:coenzyme A ligase and deoxycholic acid:coenzyme A ligase activities in rat liver

Abstract

Cholic acid:CoA ligase and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent Vmax for the 2 substrates are similar, the Km for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Km for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.