Evidence that the hydrophobic domain of rat renal gamma-glutamyltransferase spans the brush border membrane.
Evidence that the hydrophobic domain of rat renal gamma-glutamyltransferase spans the brush border membrane.
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大鼠肾γ-谷氨酰转移酶的疏水结构域跨越刷状缘膜的证据。
DOI：
10.1016/0005-2736(82)90323-6
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Curthoys,NP
中科院分区：
文献类型：
--
作者：
Tsao,B;Curthoys,NP
用乳过氧化物酶和葡萄糖氧化酶催化的~(125)I-碘标记分离的大鼠肾刷状缘膜两侧的囊泡。总膜蛋白的放射自显影表明实现了不对称标记。从用Triton X-100或木瓜酶增溶的囊泡中分离到已建立的跨膜蛋白氨基肽酶M和γ-谷氨酰基转移酶的特异性免疫沉淀物。在电泳和放射自显影之后，来自外部标记囊泡的每种酶的两种溶解形式的免疫共沉淀物显示出相同的标记强度。在这些实验中，γ-谷氨酰转移酶的小亚基被优先标记，这表明与大亚基相比，它更暴露在膜的外表面。对于来自内部标记的囊泡的样品，每个酶的Triton溶解形式被强烈标记，而木瓜酶溶解形式包含少量的放射性。因此，违禁膜标记的程度是最小的。在这些实验中，γ-谷氨酰转移酶的大亚基被优先标记。氨基肽酶M和γ-谷氨酰基转移酶标记模式的相似性表明，这两种两亲性酶的疏水结构域是从内表面选择性标记的，γ-谷氨酰基转移酶也可能是一种跨膜蛋白。
Lactoperoxidase and glucose oxidase catalyzed125I-iodination was used to specifically label isolated rat renal brush border membrane vesicles from either side of the membrane. Autoradiography of total membrane proteins demonstrated that asymmetric labeling was achieved. Specific immunoprecipitates of aminopeptidase M, an established transmembrane protein, and of γ-glutamyltransferase were isolated from vesicles solubilized with Triton X-100 or with papain. Following electrophoresis and autoradiography, the immunoprecipitates of the two solubilized forms of each enzyme derived from externally labeled vesicles exhibited the same intensity of labeling. In these experiments, the small subunit of the γ-glutamyltransferase was preferentially labeled suggesting that, compared to the large subunit, it is more exposed on the external surface of the membrane. With the samples derived from internally labeled vesicles, the Triton-solubilized form of each enzyme was intensely labeled, whereas the papain-solubilized forms contained insignificant amounts of radioactivity. Thus, the extent of contramembrane labeling was minimal. In these experiments, the large subunit of the γ-glutamyltransferase was preferentially labeled. The similarity of the labeling patterns obtained for aminopeptidase M and γ-glutamyltransferase suggests that the hydrophobic domain of the two amphipathic enzymes are selectively labeled from the internal surface and that the γ-glutamyltransferase may also be a transmembrane protein.