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Effects of Heat Shock Proteins (M_r 70,000) on Protein and DNA Synthesis at Elevated Temperatures in Vitro¹

Department of Radiation Research, City of Hope National Medical Center, Duarte, California 91010

The protective effect of M_r 70,000 heat shock protein (HSP-70) during thermotolerance has been previously observed. However, it is not known what cellular processes or components may be protected by this protein during the tolerance state. In the studies reported here, the protective effects of purified HSP-70, the nonspecific heat-stable proteins fetuin and trypsin inhibitor (ovomucoid), and other proteins and agents such as bovine serum albumin, D₂O, or glycerol on protein and DNA synthesis during heating were investigated in vitro. In vitro protein synthesis at 30, 40, and 42°C was measured by globin mRNA translation. Protein synthesis was inhibited 40 to 70% when incubated for 60 min at 40 and 42°C. However, protein synthesis was protected when either fetuin or ovomucoid was present during protein synthesis at elevated temperatures. The protection was concentration dependent. The HSP-70 purified from Chinese hamster (HA-1) cells was also able to confer protection to the translation system, but at much lower concentrations than either fetuin or ovomucoid. Other proteins, such as bovine serum albumin, or other agents, such as D₂O or glycerol which are known protectors of cellular survival during heating, did not protect the translation system.

Similar experiments were performed with DNA synthesis in vitro. Purified DNA polymerase was added to the activated calf thymus DNA in an in vitro replication system. A temperature of 46°C for 60 min inhibited replication by 40%. Addition of heat-stable proteins, purified HSP-70, bovine serum albumin, D₂O, or glycerol did not confer protection to the replication system.

These studies provide new evidence that HSP-70 may confer protection to a component of the protein synthesis machinery during thermotolerance.

¹ This work was supported by Bio-Medical Research Support Grants, NIH 2S07-RR05471-25 and NIH CA 33572.

² To whom requests for reprints should be addressed, at City of Hope National Medical Center, Dept. of Radiation Research, 1500 E. Duarte Road, Duarte, CA 91010.

Received 7/19/88. Accepted 12/ 9/88.