TOPOGRAPHY AND MICROFILAMENT CORE ASSOCIATION OF A CELL-SURFACE GLYCOPROTEIN OF ASCITES TUMOR-CELL MICROVILLI
TOPOGRAPHY AND MICROFILAMENT CORE ASSOCIATION OF A CELL-SURFACE GLYCOPROTEIN OF ASCITES TUMOR-CELL MICROVILLI
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DOI：
10.1002/jcb.240400406
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发表时间：
1989-08-01
影响因子：
4
通讯作者：
CARRAWAY, KL
中科院分区：
文献类型：
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作者：
SHENG, Z;VANDERPUYE, OA;CARRAWAY, KL
正在从 13762 只大鼠乳腺腹水肿瘤细胞中分离出的微绒毛中研究膜-微丝相互作用。这些微绒毛被唾液粘蛋白复合物覆盖，该复合物由唾液粘蛋白腹水唾液酸糖蛋白-1 (ASGP-1) 和相关的伴刀豆球蛋白 A (Con A) 结合糖蛋白 ASGP-2 组成。微绒毛的有限蛋白水解从微绒毛释放大的、高度糖基化的 ASGP-1 片段，并增加 ASGP-2 与 Triton 不溶性微绒毛微丝核心的结合 (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988)。为了分析膜中 ASGP-2 的形貌及其与微丝核心的关联，用蛋白酶 K 处理微绒毛一段时间并离心。用 Triton X-100 提取沉淀的微绒毛，以制备微丝核心和 Triton 可溶性蛋白质，或用 0.1 M 碳酸盐（pH 11）提取，以制备去除外周膜蛋白的微绒毛膜。通过十二烷基硫酸盐凝胶电泳、Con A 和 L-植物血凝素凝集素印迹以及抗 ASGP-2 免疫印迹分析这些微绒毛部分。该过程中最早的主要蛋白水解产物是 70 kDa 的膜结合片段。在较长时间下，观察到 60 kDa 释放片段、30-40 kDa Triton 可溶片段以及 25-30 kDa 膜和微丝相关片段。微丝相关蛋白在速度沉降梯度上的鬼笔环肽位移分析表明，25-30 kDa 片段与微丝核心密切相关。从这些研究中，我们提出 ASGP-2 在 15-20 kDa 片段上有一个与膜附近的微丝核心间接关联的位点。
Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanvalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the mirofilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.