Isolation of hsp70-binding proteins from bovine muscle.

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.