Yamashita T, Kawakita M
Zoological Institute, Faculty of Science, University of Tokyo.
J Biochem. 1993 Feb;113(2):136-40. doi: 10.1093/oxfordjournals.jbchem.a124016.
In order to analyze the localization and pharmacological characteristics of the Na+/H(+)-antiporter of bovine kidney, renal cortex membranes were fractionated by 35-48% continuous sucrose density gradient centrifugation. Both the Na+/H(+)-antiporting activity and the 110-kDa peptide cross-reactive with a polyclonal antibody against a peptide representing the C-terminal 22 amino acid residues of human Na+/H(+)-antiporter (NHE1) were found in the same fractions as alkaline phosphatase, a marker enzyme of the brush-border membrane. Most of the Na+/H(+)-antiporter distributed among the fractions was found to be of the low amiloride-sensitive type. Brush-border membranes were solubilized with sodium cholate and the solubilized proteins were applied to an immunoaffinity matrix (Protein A Sepharose CL-4B coupled with the above antibody). The 110 kDa protein was adsorbed to the affinity matrix and then recovered in the fraction eluted from the column at pH 11.5. After reconstitution into proteoliposome, the fraction containing the 110 kDa protein was highly active in Na+/H(+)-antiport. A 125-fold increase in the specific activity of the Na+/H+ antiport reaction was attained with the present immunoaffinity purification procedure. This represents the highest degree of purification of the native Na+/H(+)-antiporter so far attained.
为了分析牛肾Na⁺/H⁺逆向转运体的定位和药理学特性，采用35%-48%连续蔗糖密度梯度离心法对肾皮质膜进行分级分离。Na⁺/H⁺逆向转运活性以及与针对代表人Na⁺/H⁺逆向转运体（NHE1）C末端22个氨基酸残基的肽段的多克隆抗体发生交叉反应的110 kDa肽段，均与刷状缘膜的标记酶碱性磷酸酶在相同的级分中被发现。发现大部分分布在这些级分中的Na⁺/H⁺逆向转运体属于低阿米洛利敏感性类型。用胆酸钠溶解刷状缘膜，将溶解的蛋白质应用于免疫亲和基质（与上述抗体偶联的蛋白A琼脂糖CL-4B）。110 kDa蛋白质吸附到亲和基质上，然后在pH 11.5从柱上洗脱的级分中回收。重新组装到蛋白脂质体中后，含有110 kDa蛋白质的级分在Na⁺/H⁺逆向转运中具有高活性。通过目前的免疫亲和纯化程序，Na⁺/H⁺逆向转运反应的比活性提高了125倍。这代表了迄今为止对天然Na⁺/H⁺逆向转运体所达到的最高纯化程度。
