Sun Y, Ball W J
Department of Pharmacology, College of Medicine, University of Cincinnati, Ohio 45267-0575.
Am J Physiol. 1992 Jun;262(6 Pt 1):C1491-9. doi: 10.1152/ajpcell.1992.262.6.C1491.
While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.
虽然蛋白质印迹分析清楚地显示了钠钾ATP酶的α和β亚基在多种大鼠组织中的存在，但在肝脏中却不易检测到β亚基。这一观察结果与之前的一份报告一致，该报告表明从大鼠肝脏免疫沉淀的钠钾ATP酶没有明确证据显示存在β亚基（休伯特等人，《生物化学》25: 4156 - 4163，1986年）。然而，对密度梯度纯化的羔羊和大鼠肝脏微粒体进行蛋白质印迹分析显示，存在一种分子量约为42 kDa的蛋白质，它与β特异性多克隆抗体以及β定向单克隆抗体发生免疫反应。用N - 糖苷酶F对该蛋白质进行去糖基化处理后产生了一种核心蛋白（βc，分子量约为32,000），其电泳迁移率与纯化的肾脏酶的βc蛋白相同。使用亚型特异性单克隆抗体和合成肽定向多克隆抗体来证明肝脏中仅存在α1和β1蛋白，以及大鼠脑中存在β2蛋白。功能研究随后表明，尽管大鼠和羔羊肝脏的酶对强心苷抑制的敏感性与其相应的肾脏酶相似，但羔羊肝脏的酶对Na +、K +和ATP的亲和力高于肾脏酶。
