Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic.
Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic.
复制标题
胃 H,K-ATP 酶拓扑：氨基酸 888-907 位于细胞质。
DOI：
10.1016/s0006-291x(05)81345-2
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发表时间：
1991
影响因子：
3.1
通讯作者：
Swiger,R
中科院分区：
文献类型：
--
作者：
Smolka,A;Alverson,L;Fritz,R;Swiger,K;Swiger,R
胃酸化是由胃壁细胞顶端膜的一种组成蛋白H，K-ATP酶介导的。H，K-ATP酶α亚基一级结构的亲水性分析预测了8个跨膜（TM）结构域，而奥美拉唑结合数据根据10个TM结构域进行解释（Mercieret al.（1991）FASEB J. 5，A749）。本研究用胰蛋白酶水解胃粘膜微粒体，得到一组与H，K-ATP酶的高度特异性探针单克隆抗体HK 12.18结合的肽。抗H，K-ATP酶α亚基C端的抗血清结合相同的肽和一个较小的肽。结合数据表明HK 12.18的推定表位，并合成了包含该位点的20-mer肽。该肽直接与HK 12.18结合，将HK 12.18从微粒体H，K-ATP酶中置换，并阻断HK 12.18对胃壁细胞的免疫染色。此外，完整的胃微粒体竞争性抑制HK 12.18与肽-BSA缀合物的结合。总之，这些数据将HK 12.18表位置于氨基酸888-907之间，并将该结构域鉴定为胞质。这一结果明确排除了H，K-ATP酶的第六和第七个TM α螺旋之间的一对TM结构域，并支持具有8个TM结构域的二级结构模型。
Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase α subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercieret al.(1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the C-terminus of H,K-ATPase α subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitope for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888–907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM α helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.