CHARACTERIZATION OF THE NUCLEOTIDE-BINDING PROPERTIES AND ATPASE ACTIVITY OF RECOMBINANT HAMSTER BIP PURIFIED FROM BACTERIA

HSP70 family proteins bind ATP and hydrolyze it, but the precise role of these activities in their in vivo chaperoning function has not been determined. In this report, we characterized wild-type hamster BiP is olated hom bacteria in terms of its ATP binding and ATPase activities. Recombinant BiP behaved essentially the same as endogenous BiP in ter ms of oligomeric status, protease digestion patterns, and ATPase prope rties. By engineering a Factor Xa cleavable site following the His tag which was used for affinity purification, we demonstrated that the si x histidines had no effect on either the structural or ATPase properti es of recombinant BiP. We also found that bacteria-synthesized BiP had a tightly bound ADP that was resistant to dialysis. Removal of the bo und nucleotide allowed us to directly measure the binding affinity of ATP and ADP to BiP (K-d of 0.2 mu M for ATP and 0.29 mu M for ADP) by equilibrium dialysis. Careful characterization of wild-type BiP will a llow us to use this system to characterize BiP ATP binding site mutant s that can be used to probe the role of ATP binding and ATPase activit y in BiP functions.