Abstract
Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.
Highlights
From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutesof Health, Bethesda, Maryland 20892 and the §Department of Physiology, Medical Collegeof Oita, Oita, 879-56, Japan
The equilibrium dialysis experiments shown in the firstwo ase-We investigated the number of nucleotide binding lines of Table I support the view that ADP is bound very sites presenton the uncoating ATPaseby equilibrium dialysis
Purified uncoating ATPase with 0.85 bound ADPper enzyme molecule was precipitated with ammonium sulfate and resuspended in buffer A at protein concentration of about 10 p~ and dialyzed in the same buffer for 20 h at 4 "C
Summary
From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutesof Health, Bethesda, Maryland 20892 and the §Department of Physiology, Medical Collegeof Oita, Oita, 879-56, Japan. In this study we examined the nucleotide bindinginprlyospo-somes (Terlecky et al, 1992) Sincemany of these erties of purified bovine brain uncoating ATPase, onfeunctions of the hsp7Os have been found tobe affected by the of the constitutively expressed members of the hsp7O binding of ATP, it is important togain an understanding of family. Perhaps the most thoroughly studied constitutivehsp is ADP molecule bound per enzyme molecule which could the bovine brain uncoating ATPase which is able to uncoat not be removedby extensivedialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociationbut rather to strong bindionfgthe nucleotide to the uncoating ATPase.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
