Abstract
An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid N-acyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions. Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. Choloyl-CoA synthetase is associated with the microsomal membranes and bile acid-CoA:amino acid N-acyltransferase activity with the postmicrosomal supernatant. No significant amino acid N-acyltransferase activity is present in the lysosome fraction. These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids.
Highlights
An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid Nacyltransferase activity (E.C. 2.3.1) are described
Several authors have published evidence indicating that the intrahepatic synthesis of the amino acid conjugates of bile acids is the result of two independent enzymic reactions with a bile acid coenzyme A thioester intermediate [1,2,3]
Bremer and Gloor [6] noted in rats that, while both the microsome fraction and postmicrosome supernatant were required for conjugation of bile acids, boiled supernatant could substitute for native supernatant
Summary
An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid Nacyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. N o significant amino acid N-acyltransferase activity is present in the lysosome fraction These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids. Bremer and Gloor [6] noted in rats that, while both the microsome fraction and postmicrosome supernatant were required for conjugation of bile acids, boiled supernatant could substitute for native supernatant They concluded that the enzymic proteins responsible for both steps were present in microsomes. Schersten, et al [7] found that conjugation of bile
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