Yoshida K, Arai K, Kobayashi N, Saitoh H
Department of Urology, Saitama Medical Center, Medical School, Japan.
Andrologia. 1990 May-Jun;22(3):239-46. doi: 10.1111/j.1439-0272.1990.tb01973.x.
Gamma-glutamyl transpeptidase was purified to apparent homogeneity from human testis by DEAE cellulose, acetone, precipitation, fractionation by ammonium sulphate, Sephacryl S-200 chromatography, Q-Sepharose chromatography and S-Sepharose chromatography following solubilization of the enzyme by Triton X-100. The purified enzyme had an apparent molecular weight of 54 KDa by Sephacryl S-200 gel filtration. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, two submits of molecular weight 38 KDa and 14 KDa were obtained. The purified enzyme showed a single band with pI 6.0. The Km value and the optimal pH of the enzyme for L-gamma-glutamyl-3-carboxy-4-nitroanilide were found 1.09 mmol/l and 8.2-8.5, respectively. Serial lectin binding study with various lectin columns showed that the majority of the asparagine-linked oligosaccharides of the enzyme was complex-types. However, complex-types with bisecting N-acetylglucosamine residue were not recognized.
通过用Triton X-100溶解酶后，经DEAE纤维素、丙酮沉淀、硫酸铵分级分离、Sephacryl S-200层析、Q-Sepharose层析和S-Sepharose层析从人睾丸中纯化γ-谷氨酰转肽酶至表观均一。通过Sephacryl S-200凝胶过滤，纯化后的酶表观分子量为54 kDa。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，得到了分子量为38 kDa和14 kDa的两个亚基。纯化后的酶在等电聚焦上呈现单一的pI为6.0的条带。发现该酶对L-γ-谷氨酰-3-羧基-4-硝基苯胺的Km值和最适pH分别为1.09 mmol/l和8.2 - 8.5。用各种凝集素柱进行的系列凝集素结合研究表明，该酶的大多数天冬酰胺连接的寡糖为复合型。然而，带有平分型N-乙酰葡糖胺残基的复合型未被识别。
