Takaya J, Omori K, Taketani S, Kobayashi Y, Tashiro Y
Department of Pediatrics, Kansai Medical University, Osaka.
J Biochem. 1987 Oct;102(4):903-11. doi: 10.1093/oxfordjournals.jbchem.a122131.
The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.
通过蔗糖密度梯度离心从猪胃黏膜制备的富含（H⁺，K⁺）ATP酶的微粒体部分，用乳化剂有效增溶，酶活性明显保留，然后通过聚乙二醇分级分离、蓝色琼脂糖CL - 6B和氨基己基琼脂糖层析对ATP酶进行高度纯化。纯化后的酶在SDS - PAGE上呈现单一条带，表观分子量约为94 kDa，并表现出K⁺ - ATP酶和K⁺刺激的对硝基苯磷酸酶（pNPPase）活性。ATP酶活性的最适pH为7.0。对纯化酶的氨基酸分析表明，它含有大量疏水氨基酸（42%）以及少量氨基葡萄糖和半乳糖胺。在双向免疫扩散和蛋白质免疫印迹试验中，对ATP酶具有单特异性的兔抗体显著抑制了K⁺ - ATP酶和K⁺ - pNPPase活性。
