Silva N L, Haworth R S, Singh D, Fliegel L
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1995 Aug 22;34(33):10412-20. doi: 10.1021/bi00033a013.
We expressed the carboxyl-terminal 178 amino acids of the rabbit cardiac Na+/H+ exchanger as a fusion protein with glutathione-S-transferase. The fusion protein (PCR178) was found in the supernatant of extracts of E. coli and was purified using Glutathione-Sepharose affinity chromatography. Affinity-purified antibodies raised against the carboxyl-terminal region of the Na+/H+ exchanger identified the resultant protein. PCR178 copurified with a 70 kDa protein. Amino-terminal sequencing of the 70 kDa protein identified it as dnaK, the bacterial equivalent of the mammalian 70 kDa heat shock protein (hsp70). DnaK was dissociated from the Na+/H+ exchanger fusion protein by the addition of MgATP. When purified PCR178 was coupled to a cyanogen bromide-activated Sepharose column, bovine hsp70 bound to the column and was eluted with MgATP. Nondenaturing polyacrylamide gel electrophoresis showed that, in the absence of MgATP, hsp70 formed a complex with PCR178. The complex was dissociated by the addition of MgATP. GST alone did not form a complex with hsp70. Immunoprecipitation of the Na+/H+ exchanger with antiexchanger antibodies resulted in coprecipitation of hsp70 protein from antiporter containing cells. Cells that overexpress the Na+/H+ exchanger had increased amounts of hsp70 which coprecipitated with antiexchanger antibody. The results show that heat shock protein complexes with the mammalian Na+/H+ exchanger.
我们将兔心脏钠氢交换体的羧基末端178个氨基酸表达为与谷胱甘肽-S-转移酶的融合蛋白。融合蛋白（PCR178）存在于大肠杆菌提取物的上清液中，并通过谷胱甘肽-琼脂糖亲和层析进行纯化。针对钠氢交换体羧基末端区域产生的亲和纯化抗体可识别所得蛋白。PCR178与一种70 kDa的蛋白共纯化。对该70 kDa蛋白进行氨基末端测序，确定其为dnaK，即哺乳动物70 kDa热休克蛋白（hsp70）的细菌对应物。通过添加MgATP，dnaK从钠氢交换体融合蛋白上解离。当将纯化的PCR178偶联到溴化氰活化的琼脂糖柱上时，牛hsp70会结合到柱上并通过MgATP洗脱。非变性聚丙烯酰胺凝胶电泳显示，在没有MgATP的情况下，hsp70与PCR178形成复合物。添加MgATP后，该复合物解离。单独的谷胱甘肽-S-转移酶不与hsp70形成复合物。用抗交换体抗体对钠氢交换体进行免疫沉淀，导致从含有反向转运体的细胞中共沉淀出hsp70蛋白。过表达钠氢交换体的细胞中，与抗交换体抗体共沉淀的hsp70量增加。结果表明，热休克蛋白与哺乳动物钠氢交换体形成复合物。
