Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.
The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated 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. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.
Ziemienowicz
A
A
Department of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Zylicz
M
M
Floth
C
C
Hübscher
U
U
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D006358:Hot Temperature; D013950:Thymus Gland / Q000737:chemistry*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli