Analysis of protein binding to heat shock protein 70 in pancreatic islet cells exposed to elevated temperatures or interleukin 1 beta.
To elucidate a role for heat shock proteins in islet function, isolated pancreatic islets were labeled with [35S]methionine after control, heat shock, or interleukin 1 beta (IL-1 beta) treatment, extracted in the presence of detergent, and then passed over affinity columns with antibodies against heat shock protein 70 (hsp 70), hsp 70 itself, or ATP conjugated to the columns. In control or IL-1 beta-treated islets, the antibody column efficiently absorbed hsp 70 together with two other proteins of molecular masses 46 and 53 kDa. In extracts from heat-shocked cells, the binding of cellularly synthesized hsp 70 to the antibody column was inefficient but improved by the addition of unlabeled partially purified hsp 70 to the extracts. When assessing the binding of proteins in the extracts to the hsp 70 column, hsp 70 and the 46- and 53-kDa proteins among others all bound to the column. No differences in the patterns of binding to the hsp 70 column between extracts from the different islet exposures were noticed. The 46-kDa protein was identified as actin by immunoblot analysis. ATP-agarose column chromatography revealed a pattern of binding similar to that of the hsp 70 column. It is concluded that hsp 70 contains at least two functional domains, one adjacent to the epitope recognized by the antibody and active in restoring cellular function after heat shock, whereas the other has the ability to bind the 46- and 53-kDa and possibly other proteins. Furthermore, the stress induced by heat shock differs significantly from that after IL-1 beta treatment with respect to the functional behavior of hsp 70.
Margulis
B A
BA
Department of Medical Cell Biology, Uppsala University, Sweden.
Welsh
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000276:immunology / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D006360:Heat-Shock Proteins; D007375:Interleukin-1; D011506:Proteins; D000255:Adenosine Triphosphate