Mechanism of Ion Transport by H^+-ATPase and H^+/K^+-ATPase

H^+ -ATP酶通过ATP水解使H^+偶联易位。我们研究了三种类型的ATP酶：大肠杆菌质膜上的H^+ -ATP合成酶（ATP合成酶）（类似于线粒体酶）；突触囊泡或神经分泌胃壁细胞的H^+ - atp酶，负责向胃中分泌酸。所得主要结果总结如下。1. 大肠杆菌H^+ -ATP酶的甘氨酸富序列（Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr，残基149 ~ 156）位于ATP的γ磷酸段附近，对催化作用具有重要意义。尤其是Thr-156对催化作用至关重要。2. 大肠杆菌H^+ -ATP酶γ亚基的羧基末端对ATP水解和H^+ -易位之间的偶联至关重要。3. 我们发现突触囊泡中约20%的膜蛋白是H^+ - atp酶，这种atp酶形成了神经递质运输所必需的H^+的电化学梯度。4. 我们获得了大鼠和人胃H^+/K^+ - atp酶的cDNA和基因，发现该基因仅在胃壁细胞中转录。此外，我们还发现了两个胃特异性核苷酸序列基序和特异性结合基序的蛋白质。化学修饰研究也发现了H^+/K^+ - atp酶催化位点的重要残基。对H^+易位atp酶的比较研究有助于理解复杂的载体酶。

The H^+ -ATPase translocates H^+ coupling with hydrolysis of ATP. We studied three type ATPases : H^+ -ATPase (ATP synthase) of Escherichia coli plasma membrane (similar to mitochondrial enzyme) ; H^+ -ATPase of synaptic vesicles or neurosecretory gastric parietal cells responsible for acid secretion into stomach. Major results obtained are summarized as follows. 1. Glycine rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149 - 156) of E. Coli H^+ -ATPase was found to be near the gamma phosphate moiety of ATP, and important for catalysis. Especially Thr-156 is essential for catalysis. 2. The carboxyl terminal moiety of the gamma subunit of E. Coli H^+ -ATPase was found to be essential for coupling between ATP hydrolysis and H^+ -translocation. 3. We found that about 20 % of the membrane protein in synaptic vesicles was H^+ -ATPase, and this ATPase forms electrochemical gradient of H^+ essential for transport of neurotransmitters. 4. We obtained the cDNA and gene for gastric H^+/K^+ -ATPase from rat and human, and found this gene was transcribed only in gastric parietal cells. Furthermore we found two gastric specific nucleotide sequence motifs and proteins specifically bound to the motifs. Chemical modification studies also showed important residues in the catalytic site of H^+/K^+ -ATPase. Comparative studies on H^+ translocating ATPases have been useful for understanding complicated vectorial enzymes.