CALF THYMUS HSC70 PROTEIN PROTECTS AND REACTIVATES PROKARYOTIC AND EUKARYOTIC ENZYMES

The heat-shock 70 protein (Hsp 70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form on e of the cellular systems for the prevention and restoration of heat-i nduced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymu s to apparent homogeneity, This protein was classified as an eukaryoti c Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 reco gnized it, (ii) its amino terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase acti vity that was stimulated by various peptide substrates, We show that t his calf thymus Hsc70 protein protected calf thymus DNA polymerases al pha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-ina ctivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation, In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E. coli but not from eukaryotic cells, Fi nally, the addition of calf thymus DnaJ cochaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.