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

1) H， k - atp酶的增溶：在豚鼠结肠远端上皮细胞的粗膜组分中，约30%的瓦苦因敏感H， k - atp酶活性已被0.05%的n-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瓦巴因敏感的H， k - atp酶的定位，因为atp酶活性的反应产物没有被瓦巴因阻断。3)cDNA克隆：分离编码结肠H、k - atp酶的cDNA，确定其核苷酸序列。以大鼠Na，K-ATPase a1 cDNA的155b Hpall片段为探针，从豚鼠远端结肠粘膜分离的poly(a)+ RNA中筛选l_<zap>ll构建的cDNA文库。cDNA全长4242个核苷酸，包含144 bp的5'-未翻译区，3102 bp的开放阅读框和996 bp的3'-未翻译区。推导出的氨基酸序列预测了一个1033个残基的蛋白质，属于p型膜转运atp酶家族，其氨基酸与大鼠Na， k - atp酶a1的同源性为67%，与大鼠胃H， k - atp酶a的同源性为64%。在免疫印迹中，针对合成肽（残基10-24）的多克隆抗体在远端结肠中鉴定出94kd的膜蛋白，而在近端结肠中没有。这些发现表明克隆的cDNA代表了远端结肠中假定的H， k - atp酶。

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.