In previous work we found that bovine brain hsp70 has a single binding site for nucleotide, and that, with ATP at this site, the rates of association and dissociation of clathrin from hsp70 are fast, whereas with ADP at this site, these rates are unmeasurably slow. In the present study we show, first, that peptide C, cytochrome c peptide, and RNase S peptide bind competitively with clathrin, suggesting that they bind to the same site on hsp70, although RNase S peptide binds an order of magnitude more weakly than peptide C and cytochrome c peptide. Second, we show that, with ADP bound to hsp70, as occurs with clathrin, the rate constant for dissociation of peptide markedly decreases compared to the rate constant observed in ATP. In contrast, ADP only slightly decreases the rate of association of peptide. Based on these data we propose a model in which substrates of hsp70 bind to and dissociate from the ATP form of the enzyme, while, following ATP hydrolysis, they are locked onto the ADP form of the enzyme, unable to dissociate until ADP is released and ATP rebinds.

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