[胃质子泵的结构-功能研究]

Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Japan.

Yakugaku Zasshi. 1997 Jul;117(7):379-93. doi: 10.1248/yakushi1947.117.7_379.

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.

H⁺，K⁺ - ATP酶是一种负责胃酸分泌的质子泵。它通过ATP水解，主动转运质子和钾离子，导致壁细胞膜两侧形成10⁶倍的质子梯度。该泵属于P型ATP酶家族，其中包括钠泵（Na⁺，K⁺ - ATP酶）和钙泵（Ca²⁺ - ATP酶）。本综述通过使用功能性抗体、特异性抑制剂、荧光试剂和定点突变体，重点研究该质子泵的结构 - 功能关系。首先，我们制备了改变H⁺，K⁺ - ATP酶功能的单克隆抗体。其中一种抗体HK2032抑制了H⁺，K⁺ - ATP酶活性以及通过S - S交联打开的胃小泡中的氯电导，这表明氯通道存在于H⁺，K⁺ - ATP酶分子中，并且H⁺，K⁺ - ATP酶是一种多功能分子。另一种抗体HK4001通过抑制其磷酸化步骤来抑制H⁺，K⁺ - ATP酶活性。利用这种抗体，我们在兔远端结肠中发现了一种H⁺，K⁺ - ATP酶同工型。其次，我们发现巴拉圭传统草药的主要成分scopadulcic acid B是一种对H⁺，K⁺ - ATP酶具有特异性的抑制剂。该化合物通过稳定酶的钾离子形式来抑制H⁺，K⁺ - ATP酶活性。第三，我们通过观察FITC标记酶的荧光来研究H⁺，K⁺ - ATP酶的构象变化。H⁺，K⁺ - ATP酶不利用乙酰磷酸代替ATP作为主动转运的能量来源，这表明能量转导系统在P型ATP酶中并不常见。最后，我们在人肾细胞中构建了H⁺，K⁺ - ATP酶的功能性表达系统。通过将该功能性表达系统与定点诱变相结合，我们研究了催化中心（一个磷酸化位点和一个ATP结合位点）和假定的阳离子结合位点中氨基酸残基的重要性。我们新发现了决定对阳离子亲和力的位点。