Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes.
Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes.
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猪胃微粒体 (H, K)ATP 酶的溶解、纯化和表征。
DOI：
10.1093/oxfordjournals.jbchem.a122131
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发表时间：
1987
影响因子：
2.7
通讯作者：
Y. Tashiro
中科院分区：
文献类型：
--
作者：
J. Takaya;K. Omori;S. Taketani;Y. Kobayashi;Y. Tashiro
猪胃粘膜微粒体经蔗糖密度梯度离心后，经Emulgen有效增溶（H ~+，K ~+）ATP酶，保留酶活性，再经聚乙二醇分级分离、Blue Sepharose CL-6 B和氨基己基Sepharose柱层析，得到高纯度的ATP酶。纯化的酶在SDS-PAGE上显示单一条带，表观分子量约为94 kDa，并且具有K+-ATP酶和K+-刺激的-p-硝基苯磷酸酶（pNPPase）活性。ATP酶活性的最适pH为7.0。氨基酸分析表明，该酶含有大量的疏水性氨基酸（42%）和少量的氨基葡萄糖和氨基半乳糖。在Ouchterlony免疫双扩散和Western印迹试验中，ATP酶的兔单特异性抗体显著抑制K+-ATP酶和K+-pNPPase活性。
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.