Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1±0.1 µM and 1.4±0.7 mM respectively at 25 °C. This corresponds to ΔG0´ = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, ΔH0´ was calculated to -36±2 kJ/mol. This gives ΔS0´ = 6.7 J/mol per K. Adenosine 5´-[γ-thio]triphosphate, ADP, adenosine 5´-[β,γ-imino]triphosphate and adenosine 5´-[β,γ-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 µM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
Research Article| September 15 1996
Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein)
Engelbert BUXBAUM *
1School of Biological Science, Division of Biochemistry, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
*To whom correspondence should be sent, at present address: Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 318, Leicester LE1 9HN, U.K.
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Engelbert BUXBAUM, Philip G WOODMAN; Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein). Biochem J 15 September 1996; 318 (3): 923–929. doi: https://doi.org/10.1042/bj3180923
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