Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues.
The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed 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 study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl 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-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation 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 magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.
Gao
B
B
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Greene
L
L
Eisenberg
E
E
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism*
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate