Biochemical Studies of Structure, Function and Regulation of Gastric (H^+ + K^+)-ATPase

克隆了人胃(H++K+)-ATPase基因(15kb)，并测定了其核苷酸序列。该基因有22个外显子，编码1,035个残基的蛋白质，包括启动子蛋氨酸(Mr=114,047)。在大鼠和猪酶中发现的磷酸化位点和5‘-磷酸吡哆醛和异硫氰酸荧光素结合残基在人类酶中也是保守的。人(H^++K^+)-ATPase基因内含子的位置与人(Na++K^+)-ATPaseα和αIII亚基的位置基本相同。这两个ATPase基因在组织结构上的相似性以及它们的蛋白质一级结构的同源性(约60%)表明这两个基因来自一个共同的祖先基因。然而，(H^++K^+)-ATPase基因的5‘侧翼区和(Na++K^+)-ATPaseα(+)亚基的5’-侧翼区没有明显的序列同源性，表明它们的转录调控是不同的。快速抽动肌浆网Ca^&lt；2+&Gt；-ATPase基因的控制区与(H^++K+)-ATPase基因的序列也没有同源性。(H^++K^+)-ATPase基因的5‘侧翼区含有RNA聚合酶II和多种转录调控因子的潜在结合部位，以及几个可能对该基因在胃壁细胞中的特异和调控表达起重要作用的正向和反向重复序列。我们还克隆了大鼠胃(H^++K^+)-ATPase基因。ITS 5‘-上游区与人类基因相应部分的比较表明，存在高度保守的序列。来自胃粘膜的核蛋白而不是来自肝脏的核蛋白识别这些序列。由于Northern印迹杂交显示(H++K+)-ATPase基因仅在胃粘膜中转录，因此这些蛋白必须正向调节胃特异性转录。

The human gastric (H^+ + K^+)-ATPase gene (15 kilobases) was cloned, and its nucleotide sequence was determined. The gene has 22 exons and codes a protein of 1,035 residues including the initiator methionine (Mr=114,047). The phosphorylation site and pyridoxal 5'-phosphate- and fluorescein isothiocyanate-binding residues found in the rat and pig enzymes are also conserved in the human enzymes. The positions of introns in the human (H^+ + K^+)-ATPase gene are essentially the same as those in the human (Na^+ + K^+)-ATPase alpha and alpha III subunits. The similarity in organization of these two ATPase genes and the homology in the primary structures of their proteins (about 60%) suggest that these two genes were derived from a common ancestral gene. However, the 5'-flanking regions of the genes for (H^+ + K^+)-ATPase and the (Na^+ + K^+)-ATPase alpha (+) subunit show no apparent sequence homology, indicating that their transcriptions are requlated differently. The control region of the fast-twitch sarcoplasmic reticulum Ca^<2+>-ATPase gene also showed no sequence homology to that of (H^+ + K^+)-ATPase. The 5'-flanking region of the (H^+ + K^+)-ATPase gene contains potential binding sites for RNA polymerase II and various transcriptional regulation factors and several direct and inverted repeat sequences which may be important for specific and controlled expression of the genesin gastric parietal cells. We also cloned the rat gastric (H^+ + K^+)-ATPase gene. Comparison of its 5'-upstream region with the corresponding part of the human gene indicated the presence of highly conserved sequences. Nuclear proteins from gastric mucosa but not from liver recognizes these sequences. Such proteins must positively regulate gastric specific transcription, since Northern blot hybridization indicated that the (H^+ + K^+)-ATPase gene was transcribed only in gastric mucosa.