Kimura M, Okuno E, Inada J, Ohyama H, Kido R
Hoppe Seylers Z Physiol Chem. 1983 Jun;364(6):637-45. doi: 10.1515/bchm2.1983.364.1.637.
Amino-acid N-choloyltransferase was purified from human liver. The procedure resulted in about 100-fold enriched activity and glycine- and taurine-dependent activities co-purified and did not separate to any extent in any of the steps. The final enzyme preparation had an apparent molecular mass of 100 kDa by gel filtration, 118 kDa by sucrose density gradient centrifugation and was composed of two identical subunits which had a molecular mass of 52 kDa, as judged by dodecyl sulfate polyacrylamide disc gel electrophoresis. The glycine- and taurine-dependent activities showed optima at pH 7.2 and pH 6.5, respectively. Apparent Km values of purified enzyme were 3.2 and 0.6mM for glycine and taurine, respectively. The Km value for choloyl-CoA was 50 microM for the glycine-dependent activity and 87 microM for the taurine-dependent activity. Bile acid derivatives and cholesterol had an inhibitory effect on both glycine- and taurine-dependent activity in vitro; on the other hand, the reaction was stimulated by the addition of glutathione, EDTA and L-cysteine. Amino acid substrate specificity was restricted to glycine, taurine, beta-alanine and D-alpha-alanine. As well as choloyl-CoA, its deoxy derivatives were also good substrates for the enzyme.
从人肝脏中纯化出氨基酸N - 胆酰基转移酶。该纯化过程使酶活性提高了约100倍，且依赖甘氨酸和牛磺酸的活性共同纯化，在任何步骤中都没有明显分离。通过凝胶过滤法测得最终酶制剂的表观分子量为100 kDa，通过蔗糖密度梯度离心法测得为118 kDa，经十二烷基硫酸钠聚丙烯酰胺圆盘凝胶电泳判断，其由两个分子量为52 kDa的相同亚基组成。依赖甘氨酸和牛磺酸的活性分别在pH 7.2和pH 6.5时表现出最佳活性。纯化酶对甘氨酸和牛磺酸的表观Km值分别为3.2 mM和0.6 mM。对于依赖甘氨酸的活性，胆酰辅酶A的Km值为50 μM，对于依赖牛磺酸的活性为87 μM。胆汁酸衍生物和胆固醇在体外对依赖甘氨酸和牛磺酸的活性均有抑制作用；另一方面，添加谷胱甘肽、EDTA和L - 半胱氨酸可刺激该反应。氨基酸底物特异性仅限于甘氨酸、牛磺酸、β - 丙氨酸和D - α - 丙氨酸。除胆酰辅酶A外，其脱氧衍生物也是该酶的良好底物。
