Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.
Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.
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艰难梭菌毒素 A 的兔回肠受体的表征。受体偶联 G 蛋白的证据。
DOI：
10.1172/jci115267
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Dickey,BF
中科院分区：
文献类型：
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作者：
Pothoulakis,C;LaMont,JT;Eglow,R;Gao,N;Rubins,JB;Theoharides,TC;Dickey,BF
本研究的目的是表征表面受体的毒素A，肠毒素艰难梭菌，兔肠刷状缘（BB）和大鼠嗜碱性白血病（RBL）细胞。使用改良的Bolton-Hunter方法对纯化的毒素A进行放射性标记，以分离2 microCi/微克，保留全部生物活性。~ 3 H-毒素A与兔BB和RBL细胞上的一类受体特异性结合，解离常数分别为5.4 × 10 ~（-8）M和3.5 × 10 ~（-8）M。RBL细胞对毒素A高度敏感（细胞变圆），每个细胞有180，000个特异性结合位点，而IMR-90成纤维细胞对毒素A的敏感性低得多，缺乏可检测的特异性结合位点。暴露BB胰蛋白酶或糜蛋白酶显着降低3 H-毒素A的特异性结合。BB与Bandeirea simplicifolia（BS-1）凝集素的预孵育也降低了特异性结合，CHAPS增溶的受体可以用WGA-琼脂糖固定。加入100 nM毒素A加速了35 S-GTP γ S与兔回肠BB的结合，BB与GTP类似物GTP γ S或Gpp（NH）p的预孵育显著降低了3 H-毒素A的特异性结合。我们的数据表明，毒素A的膜受体是一种含有半乳糖和N-乙酰葡糖胺的糖蛋白，它似乎与G蛋白偶联。图片
The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.Images