Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp 70.
We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70.ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KDof hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.
Gross
M
M
Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA.
Hessefort
S
S
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D001798:Blood Proteins; D018840:HSP70 Heat-Shock Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases; D000251:Adenosine Triphosphatases