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首页> 外文期刊>Biochemistry >Correlation between the Activities and the Oligomeric Forms of Pig Gastric H/K-ATPase.
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Correlation between the Activities and the Oligomeric Forms of Pig Gastric H/K-ATPase.

机译：猪胃H / K-ATPase活性与寡聚形式之间的相关性。

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摘要

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.
机译：膜结合的H / K-ATPase被八甘醇十二烷基醚（C（12）E（8））或正辛基葡萄糖苷（nOG）溶解。 H / K-ATPase活性以及原聚和寡聚组分的分布通过高效凝胶色谱（HPGC）和使用全内反射荧光显微镜（TIRFM）的单分子检测进行评估。由C（12）E（8）增溶酶的HPGC证明，尽管C（12）E（8）增溶作用降低，但低聚物的分布是高12％的低聚物，44％的二原型和44％的原型。 H / K-ATPase活性达到膜结合酶的1.8％。 C（12）E（8）溶解的酶的电子显微镜图像显示存在protomer以及两个和多个protomer的组合。尽管nOG增溶的H / K-ATPase保持了与膜结合酶相同的周转率和71％的比活性，但检测到的低聚体含量提高了56％，二聚体含量提高了34％，原聚体含量提高了10％。溶解的荧光素5'-异硫氰酸酯（FITC）修饰的H / K-ATPase在α链的Lys-518处的TIRFM分析显示FITC荧光强度的定量光漂白。对于C（12）E（8）溶解的FITC酶，每个初始相对荧光强度单位分别为4、2和1的分数分别为5％，44％和51％。在nOG增溶的FITC酶的情况下，4个单元和2个单元的每个部分分别为54％和46％，没有可检测的1个单元部分。这代表了水溶液中H / K-ATPase的首次直接观察。 HPGC的H / K-ATPase的低聚形式的酶活性与分布之间的相关性以及对H / K-ATPase的单分子和其他分子的观察表明，H / K-ATPase分子的四原形式代表功能物种在膜上。

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