猪胃黏膜（H⁺ + K⁺）-ATP酶的纯化与特性分析

Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.

作者信息

Hongo T, Nojima S, Setaka M

机构信息

Department of Membrane Biology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.

出版信息

Jpn J Pharmacol. 1990 Feb;52(2):295-305. doi: 10.1254/jjp.52.295.

DOI:10.1254/jjp.52.295

PMID:2156097

Abstract

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

摘要

通过差速离心和重复的7% Ficoll梯度离心相结合的方法，从猪胃黏膜的胃底部获得了富含（H⁺ + K⁺）-ATP酶的膜组分。通过重复的7% Ficoll梯度离心分离得到的微粒体膜组分不含哇巴因敏感的（Na⁺ + K⁺）-ATP酶、5'-核苷酸酶和琥珀酸脱氢酶；并且它高度富含（H⁺ + K⁺）-ATP酶和K⁺刺激的对硝基苯磷酸酶（p-NPPase）。（H⁺ + K⁺）-ATP酶的最适pH为7.4，在此pH下，分别受到Tl⁺、K⁺、Rb⁺和NH₄⁺的刺激，其Ka值分别为0.0667、0.526、0.667和3.03 mM。另一方面，单价阳离子如Na⁺、Li⁺和（CH₃）₄N⁺以及二价阳离子如Cu²⁺、Ca²⁺、Ba²⁺、Sr²⁺和Cd²⁺浓度依赖性地抑制该酶活性。哇巴因和寡霉素没有作用，而特异性的（H⁺ + K⁺）-ATP酶抑制剂奥美拉唑以pH依赖性方式抑制该酶活性。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳显示在97,400道尔顿处有一条主要条带（占蛋白质的90%以上），在Mg²⁺和[γ-³²P]-ATP存在下被磷酸化，在K⁺存在下被去磷酸化。本方法非常简单，通过该方法获得的微粒体组分的（H⁺ + K⁺）-ATP酶活性与通过其他方法如自由流动电泳获得的相比要高得多。