利用免疫亲和层析法对大鼠肝脏微粒体和溶酶体β-葡萄糖醛酸酶进行纯化及特性分析。

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

作者信息

Himeno Mnishimura Y, Tsuji H, Kato K

出版信息

Eur J Biochem. 1976 Nov 15;70(2):349-59. doi: 10.1111/j.1432-1033.1976.tb11024.x.

DOI:10.1111/j.1432-1033.1976.tb11024.x

Abstract

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.

摘要

利用将纯化的抗大鼠包皮腺β-葡萄糖醛酸酶特异性免疫球蛋白G偶联到琼脂糖2B上制备的亲和层析和等电聚焦技术，分别从微粒体和溶酶体组分中纯化大鼠肝脏β-葡萄糖醛酸酶（EC 3.2.1.31），其相对于匀浆的纯化倍数约为9500倍，且产率很高。纯化后的酶在聚丙烯酰胺凝胶电泳中呈现均一性，分子量约为310000。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳中，微粒体β-葡萄糖醛酸酶呈现一条对应分子量为79000的条带，而溶酶体β-葡萄糖醛酸酶有三条不同的条带，分别由一条主要条带和两条次要条带组成，分子量分别为79000、74000和70000。微粒体和溶酶体β-葡萄糖醛酸酶均观察到一条宽泛的pH活性曲线，在pH 4.4处有一个单一的最佳值。用兔抗大鼠肝脏溶酶体β-葡萄糖醛酸酶抗血清进行免疫凝胶扩散技术检测，结果显示两种酶具有相同或非常相似的抗原决定簇。