Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.
A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002466:Cell Nucleolus / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells / Q000032:analysis; D006360:Heat-Shock Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008845:Microinjections; D051381:Rats
D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate