Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.
Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis; D006360:Heat-Shock Proteins / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins