CHARACTERIZATION OF NUCLEOTIDE-FREE UNCOATING ATPASE AND ITS BINDING TO ATP, ADP, AND ATP ANALOGS

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previo us work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystall ographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be r emoved by dialysis or treatment with ethylenediaminetetraacetic acid ( EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this stu dy, the strongly bound ADP was removed by first replacing it with 5'-a denylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-c oated vesicles, even after 10 days of storage at 4 degrees C. Therefor e, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotid e-free uncoating ATPase, we were able to accurately measure the dissoc iation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnit ude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.