Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

Abstract

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 microin'ected cells maintained at 37C 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.An apparent defense mechanism which cells utilize when confronted with adverse changes in their local environment has been termed the heat-shock or stress response. The response is characterized by the rapid, preferential synthesis and accumulation of the so-called heat-shock or stress proteins. This changeover in the pattern of protein synthesis is accompanied by a sharp curtailment of transcription and translation of genes which were active before the environmental insult. The exposure of cells to a number of different and seemingly unrelated agents, including heat-shock treatment, amino acid analogs, and heavy metals to name just a few, results in the synthesis and accumulation of the stress proteins. Although the function of the various stress proteins is still not clear, their accumulation in the cell collectively appears to afford the cell protection, especially upon subsequent stress situations (for general reviews, see references 1, 16, and 20).Considerable work from many laboratories has focused on the identification, characterization, and localization of the stress proteins, with the ultimate aim being to dissect the function of these proteins. In our laboratory, the proteins synthesized at elevated levels after physiological stress of mammalian cells are referred to, according to their apparent size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as the 28-, 72-, 73-, 80-, 90-, 100-, and 110-kilodalton (kDa) proteins (20). All of these proteins, with the exception of the 72-kDa species, are present in appreciable levels in normal, unstressed 37°C cells (25).Hence, it has been suggested by us and othe...

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

“…Attempts to isolate Hsc66 using ATP-affinity chromatography were not successful. In contrast to DnaK (Zylicz et al, 1987) and bovine hsc7O (Welch & Feramisco, 1985) Hsc66 does not bind to agarose columns linked to C-8 of ATP via a 9-carbon spacer; we have not investigated this further, and the structural basis for the failure to bind to the immobilized ATP is not known. Cells overproducing Hsc2O were lysed by French press, and the protein was purified by anion-exchange chromatography followed by size exclusion chromatography.…”

“…ATP-affinity chromatography of Hsc66 and DnaK was carried out as described by others (Welch & Feramisco, 1985;Zylicz et al, 1987) using ATP coupled at C-8 to agarose through a 9-atom spacer (Sigma Chem. Co., A2767).…”

Programmed cell death: Dying cells synthesize a co‐ordinated, unique set of proteins in two different episodes of cell death

“…The striking similarity of the patterns at pupal ecdysis, when the anteriormost and posteriormost muscles are destroyed, and at adult ecdysis, when the abdominal muscles are destroyed, suggests that the co-ordinate synthesis of these proteins is a major factor in involution. Since the cells are under stress at this time and it is known that several factors, including both hypoxia and inhibition of oxidative phosphorylation, and even increases in calcium levels, can induce the synthesis of stress proteins [18][19][20], this possibility is currently being explored. The proteins appear to be too alkaline to be heat-shock proteins.…”

Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism

“…Antibodies against the major ORPs are being prepared to localise these proteins. One possibility is localisation of some ORPs on the nucleolus to protect RNA production similar to heat shock proteins 70 and 110 (Welch & Feramisco, 1985;Subjeck et al, 1983).…”