用木瓜蛋白酶或 Triton X-100 增溶后从大鼠肾脏中纯化的 γ-谷氨酰转肽酶的大小和物理性质比较。

Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

作者信息

Hughey R P, Curthoys N

出版信息

J Biol Chem. 1976 Dec 25;251(24):8763-70.

Abstract

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.

摘要

γ-谷氨酰转肽酶与肾近端直小管细胞的刷状缘膜相关。用木瓜蛋白酶或 Triton X-100 处理可使其定性溶解。这两种方法均不影响其催化活性，但酶的两种产物形式在物理性质上有很大差异。木瓜蛋白酶溶解的转肽酶可溶于水性缓冲液，纯化了 430 倍。其 s20,w 为 4.9 S，斯托克斯半径为 36 Å，计算分子量为 69,000。通过沉降平衡离心法（Mr = 66,700）显示其为均一的。相比之下，Triton 溶解的转肽酶仅在有去污剂存在时可溶，纯化了 300 倍。这种酶形式的斯托克斯半径为 70 Å，但 s20,w 仅为 4.15 S。在 Triton X-100 的临界胶束浓度以下酶的聚集以及其结合 1.16 mg Triton X-100 - 蛋白质复合物的能力经计算为 169,000，但复合物的糖蛋白部分占总质量的 52%（87,000）。Triton X-100 的质量（82,000）与其报道的胶束分子量一致。用木瓜蛋白酶或菠萝蛋白酶处理 Triton 纯化的转肽酶会产生一种在各方面都与木瓜蛋白酶纯化的酶相同的酶形式。Triton 和木瓜蛋白酶纯化的转肽酶在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上均显示两条蛋白带。两种形式的较小亚基看起来相同（Mr = 27,000），而 Triton 和木瓜蛋白酶纯化的酶的较大亚基的表观分子量分别为 54,000 和 51,000。这些数据表明，γ-谷氨酰转肽酶较大亚基中的一个肽段（3,000 至 19,000）负责其与 Triton 胶束的结合，可能还负责将酶固定在刷状缘膜中。