Yamashita T, Kawakita M
Zoological Institute, Faculty of Sciences, University of Tokyo.
J Biochem. 1992 Feb;111(2):162-7. doi: 10.1093/oxfordjournals.jbchem.a123731.
Bovine renal brush-border membranes were solubilized by 1.6% sodium cholate. Na+/H(+)-antiporter was recovered in the supernatant after centrifugation at 160,000 x g for 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H(+)-antiporter showed a pH-gradient dependent and amiloride-sensitive 22Na+ uptake very similar to that of brush-border membrane vesicles. Factors affecting the efficiency of reconstitution as well as the stability of the solubilized antiporter at various temperatures were studied. Sodium cholate-solubilized brush-border membrane proteins were fractionated by Sephacryl S-400 and DEAE-Toyopearl chromatography, and fractions containing reconstitutively active Na+/H(+)-antiporter were identified. A 110 kDa peptide cross-reactive with a polyclonal antibody against a C-terminal peptide (22-amino acid residues) of human Na+/H(+)-antiporter was consistently found on the immunoblot of the active fractions. A closely similar peptide was also detected in human placental membranes by this antibody. These results strongly suggest that the 110 kDa protein is responsible for Na+/H(+)-antiporter activity.
用1.6%的胆酸钠溶解牛肾刷状缘膜。在160,000×g离心1小时后，Na⁺/H⁺逆向转运体在上清液中被回收，并通过胆酸盐透析法成功地重组成蛋白脂质体。重组的Na⁺/H⁺逆向转运体表现出与刷状缘膜囊泡非常相似的pH梯度依赖性和氨氯地平敏感性²²Na⁺摄取。研究了影响重组效率的因素以及溶解的逆向转运体在不同温度下的稳定性。用Sephacryl S - 400和DEAE - Toyopearl色谱法对胆酸钠溶解的刷状缘膜蛋白进行分级分离，并鉴定出含有具有重组活性的Na⁺/H⁺逆向转运体的级分。在活性级分的免疫印迹上始终发现一种110 kDa的肽，它与针对人Na⁺/H⁺逆向转运体C末端肽（22个氨基酸残基）的多克隆抗体发生交叉反应。用该抗体在人胎盘膜中也检测到一种非常相似的肽。这些结果有力地表明，110 kDa的蛋白质负责Na⁺/H⁺逆向转运体的活性。
