Isolation and localization of H, K-ATPase in colonic epithelial cell.

结肠上皮细胞中 H,K-ATP 酶的分离和定位。

基本信息

批准号：
02454114
负责人：
SUZUKI Yuichi
金额：
$ 3.97万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

1)Solubilization of H,K-ATPase: Approximately 30% of the ouabain-sensitive H,K-ATPase activity in crude membrane fraction of epithelial cells from guinea pig distal colon has been solubilized with 0.05% n-Dodecyl-b-D-maltoside. 2)Histochemistry: Cerium-based cytochemical method failed to reveal the localization of the ouabainsensitive H,K-ATPase because the reaction product of ATPase activity was not blocked by ouabain. 3)cDNA cloning: cDNAs encoding the colonic H,K-ATPase were isolated and its nucleotide sequence was determined. 155 b Hpall fragment of the rat Na,K-ATPase a1 cDNA was used as a probe to screen a cDNA library constructed in l_<zap>ll from poly(A)+ RNA isolated from the mucosa of the guinea pig distal colon. The cDNA is 4242 nucleotides in length and contains 144 bp of 5'-untranslated region, 3102 bp of an open reading frame and 996 bp of 3'-untranslated region. The deduced amino acid sequence predicts a protein of 1033 residues which belongs to the P-type family of membrane-transporting ATPases and exhibits 67% amino acid identity to the rat Na,K-ATPase a1, and 64% identity to the rat gastric H,K-ATPase a subunit. In immunoblotting, a polyclonal antibody raised against a synthetic peptide (residues 10-24) identified a membrane protein of 94 kd in the distal, but not in the proximal colon. These findings suggest that the cloned cDNA represents the putative H,K-ATPase in the distal colon.

1) H,K-ATP酶的溶解：豚鼠远端结肠上皮细胞粗膜部分中哇巴因敏感的H,K-ATP酶活性约30%已被0.05%正十二烷基-b-D-麦芽糖苷溶解。 2)组织化学：基于铈的细胞化学方法未能揭示哇巴因敏感的H,K-ATP酶的定位，因为ATP酶活性的反应产物不被哇巴因阻断。 3)cDNA克隆：分离编码结肠H,K-ATP酶的cDNA并测定其核苷酸序列。使用大鼠Na，K-ATP酶a1 cDNA的155b Hpall片段作为探针来筛选在1_11中由分离自豚鼠远端结肠粘膜的poly(A)+RNA构建的cDNA文库。该cDNA长度为4242个核苷酸，包含144bp的5'-非翻译区、3102bp的开放阅读框和996bp的3'-非翻译区。推导的氨基酸序列预测蛋白质有 1033 个残基，属于 P 型膜转运 ATP 酶家族，与大鼠 Na,K-ATPase a1 具有 67% 的氨基酸同一性，与大鼠胃 H 具有 64% 的氨基酸同一性,K-ATP酶的一个亚基。在免疫印迹中，针对合成肽（残基 10-24）产生的多克隆抗体在远端结肠中鉴定出 94 kd 的膜蛋白，但在近端结肠中未检测到。这些发现表明克隆的 cDNA 代表远端结肠中假定的 H,K-ATP 酶。