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

克隆了人胃（H^+ + K^+）-ATP酶基因（15个酶），并测定了其核苷酸序列。该基因有22个外显子，编码包括起始甲硫氨酸在内的1，035个残基的蛋白质（Mr= 114，047）。在大鼠和猪酶中发现的磷酸化位点和吡哆醛5 '-磷酸盐-和异硫氰酸荧光素-结合残基在人酶中也是保守的。人类（H^+ + K^+）-ATP酶基因中内含子的位置与人类（Na^+ + K^+）-ATP酶α和α III亚基中的位置基本相同。这两个ATP酶基因在组织结构上的相似性和它们蛋白质一级结构上的同源性（约60%）表明这两个基因来源于一个共同的祖先基因。然而，（H^+ + K^+）-ATP酶和（Na^+ + K^+）-ATP酶α（+）亚基基因的5 '侧翼区没有显示出明显的序列同源性，表明它们的转录调节方式不同。快缩肌浆网Ca^2+-ATPase基因的控制区也与（H^+ + K^+）-ATPase基因的控制区没有序列同源性。（H^+ + K^+）-ATP酶基因的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位点，以及几个可能对该基因在胃壁细胞中特异性和受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^+）-ATP酶基因。将其5 '上游区与人基因的相应部分进行比较，表明存在高度保守的序列。来自胃粘膜的核蛋白识别这些序列，而来自肝脏的核蛋白不识别这些序列。由于北方印迹杂交表明（H^+ + K^+）-ATP酶基因仅在胃粘膜中转录，因此这些蛋白质肯定正调控胃特异性转录。

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.