Identification of the human NHE-1 form of Na(+)-H+ exchanger in rabbit renal brush border membranes.
Identification of the human NHE-1 form of Na(+)-H+ exchanger in rabbit renal brush border membranes.
复制标题
兔肾刷状缘膜中人 NHE-1 形式 Na()-H 交换剂的鉴定。
DOI：
10.1172/jci116433
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Shenolikar,S
中科院分区：
文献类型：
--
作者：
Weinman,EJ;Steplock,D;Corry,D;Shenolikar,S
为了研究人Na（+）-H+交换器（NHE-1）与肾刷状缘膜（BBM）Na（+）-H+交换器之间的关系，制备了抗人NHE-1胞浆外结构域（Ab-E）和胞浆内结构域（Ab-I）的多克隆抗体。Western免疫印迹分析表明，这两种抗体识别97 kD的蛋白在兔肾BBM，但不基底外侧膜（BLM）。辛基葡糖苷提取的兔肾BBM蛋白也含有97 kD多肽，从阴离子交换柱用0.2 M NaCl洗脱的级分（级分A）也含有97 kD多肽。在0.2和0.4 M NaCl之间洗脱的级分（级分B）不含该蛋白。先前的重构研究表明，组分B中的Na（+）-H+交换活性显著高于组分A。NH_4Cl对家兔给药3 - 7天，这种刺激可增加肾BBM Na（+）-H~+交换活性，但未导致肾BBM或BLM中97-kD蛋白表达的变化。结果表明，亲和纯化的多克隆抗体的两个独立的域的人Na（+）-H+交换识别97 kD的蛋白质在兔肾BBM，但不BLM。使用抗体识别97-kD蛋白质与溶解的BBM蛋白质的色谱分离后和施用NH4Cl后天然BBM中的大多数功能性Na（+）-H+交换活性之间的解离表明兔肾BBM含有不止一种形式的Na（+）-H+交换剂。
To study the relation between the human Na(+)-H+ exchanger (NHE-1) and the renal brush border membrane (BBM) Na(+)-H+ exchanger, polyclonal antibodies to synthetic peptides representing a putative external (Ab-E) and an internal cytosolic domain (Ab-I) of human NHE-1 were generated in rabbits. Western immunoblot analyses indicated that both antibodies recognized a 97-kD protein in rabbit renal BBM but not basolateral membranes (BLM). Octyl glucoside-extracted rabbit renal BBM proteins also contained the 97-kD polypeptide as did a fraction eluted from an anion-exchange column with 0.2 M NaCl (fraction A). A fraction eluting between 0.2 and 0.4 M NaCl (fraction B) did not contain this protein. Prior reconstitution studies have indicated that Na(+)-H+ exchange activity is higher significantly in fraction B than fraction A. Administration of NH4Cl for 3-7 d to rabbits, a stimulus known to increase renal BBM Na(+)-H+ exchange activity, did not result in a change in expression of the 97-kD protein in either renal BBM or BLM. The results indicate that affinity-purified polyclonal antibodies to two separate domains of the human Na(+)-H+ exchanger recognize a 97-kD protein in rabbit renal BBM but not BLM. The dissociation between recognition of the 97-kD protein using antibodies and the majority of functional Na(+)-H+ exchange activity after chromatographic fractionation of solubilized BBM proteins and in native BBM after administration of NH4Cl suggest that rabbit renal BBM contains more than one form of Na(+)-H+ exchanger.Images