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 isolated from bacteria in terms of its ATP binding and ATPase activities. Recombinant BiP behaved essentially the same as endogenous BiP in terms of oligomeric status, protease digestion patterns, and ATPase properties. By engineering a Factor Xa cleavable site following the His tag which was used for affinity purification, we demonstrated that the six histidines had no effect on either the structural or ATPase properties of recombinant BiP. We also found that bacteria-synthesized BiP had a tightly bound ADP that was resistant to dialysis. Removal of the bound nucleotide allowed us to directly measure the binding affinity of ATP and ADP to BiP (Kd of 0.2 microM for ATP and 0.29 microM for ADP) by equilibrium dialysis. Careful characterization of wild-type BiP will allow us to use this system to characterize BiP ATP binding site mutants that can be used to probe the role of ATP binding and ATPase activity in BiP functions.
Wei
J
J
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Hendershot
L M
LM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D004926:Escherichia coli; D015951:Factor Xa / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006639:Histidine; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D015951:Factor Xa; D000251:Adenosine Triphosphatases