Margulis B A, Welsh M
Department of Medical Cell Biology, Uppsala University, Sweden.
Biochem Biophys Res Commun. 1991 Jul 15;178(1):1-7. doi: 10.1016/0006-291x(91)91771-4.
It has been proposed that the members of the hsp70 major heat shock protein family in non-stressed cells participate in the intracellular transport of newly synthesized proteins and in maintaining such proteins in an unfolded state. Specific binding of other cellular proteins to hsp70 may play a role in hsp70 function. A simple method for isolation of hsp70 binding proteins using hsp70-sepharose column chromatography is described. The column binds hsp70 antibodies and thermodenatured a2-interferon in an ATP-dependent manner. Low-ionic strength extracts and actomyosin preparations from bovine muscle were passed through the column and eluates in ATP and 2M NaCl were analyzed by electrophoresis. Two proteins that eluted with ATP were identified by immunoblotting as hsp70 and actin. The polypeptides that eluted with 2 M NaCl, among which 51 and 58 kDa proteins were prominent, are suggested to bind tightly by hydrophobic interaction to hsp70. An antiserum raised against total hsp70-binding protein recognized a polypeptide with a mass of 40 kDa as determined both by immunoblotting and immunoprecipitation. Hsp70 binding proteins may thus participate in the function of hsp70 with regards to hsp70s role in protein targeting, unfolding and transport.
有人提出，非应激细胞中hsp70主要热休克蛋白家族的成员参与新合成蛋白质的细胞内运输，并使这些蛋白质保持未折叠状态。其他细胞蛋白与hsp70的特异性结合可能在hsp70的功能中起作用。本文描述了一种使用hsp70-琼脂糖柱色谱法分离hsp70结合蛋白的简单方法。该柱以ATP依赖的方式结合hsp70抗体和热变性的α2-干扰素。将来自牛肌肉的低离子强度提取物和肌动球蛋白制剂通过该柱，并对ATP和2M NaCl中的洗脱物进行电泳分析。通过免疫印迹鉴定出两种与ATP一起洗脱的蛋白质为hsp70和肌动蛋白。用2M NaCl洗脱的多肽（其中51kDa和58kDa的蛋白质最为突出）被认为通过疏水相互作用与hsp70紧密结合。通过免疫印迹和免疫沉淀测定，针对总hsp70结合蛋白产生的抗血清识别出一种质量为40kDa的多肽。因此，就hsp70在蛋白质靶向、展开和运输中的作用而言，hsp70结合蛋白可能参与hsp70的功能。
