Characterization of microsomal choloyl-coenzyme A synthetase

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Abstract
Choloyl-CoA synthetase (EC 6.2.1.7) [guinea pig liver] was characterized for the 1st time under appropriate assay conditions. The pH optimum for the reaction is pH 7.2-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The .**GRAPHIC**. (apparent Km) for CoA, extrapolated from kinetic data, is 50 .mu.M, but in fact the rate of reaction is increased little by concentrations of CoA above 25 .mu.M. The .**GRAPHIC**. for ATP is 600 .mu.M. High concentrations of ATP appear to cause substrate inhibition. The .**GRAPHIC**. for cholate was 6 .mu.M. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.