Structural aspects of the gastric H,K-ATPase.
Structural aspects of the gastric H,K-ATPase.
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胃 H,K-ATP 酶的结构。
DOI：
10.1007/bf00768850
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发表时间：
1992
影响因子：
3
通讯作者：
Hersey,S
中科院分区：
文献类型：
--
作者：
Sachs,G;Besancon,M;Shin,JM;Mercier,F;Munson,K;Hersey,S
胃H， k - atp酶是α， β异二聚体。在猪酶的情况下，大催化亚基由1033个氨基酸组成，而β亚基由大约291个氨基酸组成，并且严重糖基化。α亚基的膜拓扑结构很难用亲水性分析来预测。用荧光素-5-马来酰亚胺对完整的囊泡进行胰蛋白酶水解，然后用半胱氨酸进行标记，为α亚基在残基104和162 （M1/M2）、291和358 （M3/M4）、776和835 （M5/M6）以及853和946 （M7/M8）之间的8膜跨越模型提供了实验证据。没有证据表明在分子的C端有一对片段（M9/M10），这与Na，K和ca atp酶的预测相反。对完整的囊泡进行碘化处理，然后羧基肽酶Y对C端酪氨酸进行切割，表明α亚基的C端是细胞质的。抗体146的表位位于胞浆外，位于M7/M8之间的残基871 ~ 874之间。K竞争性咪唑吡啶的结合位点SCH28080位于M1和M2之间的胞质外环，而奥美拉唑在酸转运囊泡中酸活化产生的共价SH试剂的结合位点分别位于连接M5/M6和M7/M8的胞质外环813（或822）和892的2个半胱氨酸上。β亚基仅在破裂的囊泡中水解。在这些条件下，只有在还原剂存在的情况下，从236位开始的片段才被释放出来，这表明半胱氨酸210和263是二硫连接的。这个亚基似乎只有一个跨膜段，正如疏水性所预测的那样。SCH28080或奥美拉唑与酶的胞质外表面结合都会影响细胞质构象的变化，表明蛋白质形状的变化存在跨膜传递。
The gastric H,K-ATPase is an alpha,beta heterodimer. The large catalytic subunit is composed, in the case of the hog enzyme, of 1033 amino acids, whereas the beta subunit is composed of about 291 amino acids and is heavily glycosylated. The membrane topology of the alpha subunit is difficult to predict using hydropathy analysis. Tryptic hydrolysis of intact, inside out vesicles followed by cysteine labelling with fluorescein-5-maleimide provided experimental evidence for an 8 membrane spanning model for the alpha subunit, between residues 104 and 162 (M1/M2), 291 and 358 (M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). No evidence was found for a pair of segments (M9/M10) towards the C terminal end of the molecule, contrary to predictions for the Na,K- and Ca-ATPases. Iodination of intact vesicles followed by carboxypeptidase Y cleavage of the C terminal tyrosines showed that the C terminal end of the alpha subunit was cytoplasmic. The epitope for antibody 146 was extracytoplasmic and located between residues 871 to 874 between M7/M8. The binding site of the K competitive imidazo-pyridine, SCH28080, was to the extracytoplasmic loop between M1 and M2, whereas the binding of the covalent SH reagent generated from acid activation of omeprazole in acid transporting vesicles was to 2 cysteines at positions 813 (or 822) and 892 predicted to be in the extracytoplasmic loops connecting M5/M6 and M7/M8, respectively. The beta subunit was only hydrolysed in broken vesicles. A fragment beginning at position 236 was liberated under these conditions only in the presence of reducing agents, showing that cysteine 210 and 263 were disulfide linked. It seems that this subunit has only a single membrane spanning segment as predicted by hydrophobicity. Binding of either SCH28080 or omeprazole to the extracytoplasmic face of the enzyme affected cytoplasmic conformational changes, showing that there was transmembranal transmission of changes of shape of the protein.