Gao B, Emoto Y, Greene L, Eisenberg E
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Apr 25;268(12):8507-13.
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.
70 kDa热休克蛋白（hsp70s）的许多功能似乎都受结合核苷酸的调控。在本研究中，我们检测了纯化的牛脑脱衣ATP酶的核苷酸结合特性，该酶是hsp70家族组成型表达成员之一。我们发现，通过ATP-琼脂糖柱层析纯化的脱衣ATP酶每个酶分子保留一个结合的ADP分子，通过广泛透析无法去除该分子。由于这种结合的ADP能与游离的ADP或ATP快速交换，无法去除结合核苷酸并非由于解离缓慢，而是由于核苷酸与脱衣ATP酶的强结合。为证实这一观点，平衡透析实验表明ADP和ATP的解离常数均小于0.1 μM。施密德等人（施密德，S. L.，布雷尔，W. A.，和罗斯曼，J. E.（1985年）《生物化学杂志》260，10057 - 10062）提出脱衣ATP酶有两个核苷酸结合位点，一个对ATP具有特异性，另一个既能结合ATP和ATP类似物，又不能结合ADP。相比之下，我们发现结合了ADP的酶不会进一步结合腺苷5'-（β，γ-亚氨基）三磷酸或dATP，即使在200 μM游离ATP存在的情况下，每个酶分子也不会结合超过一个ATP分子。这些结果有力地表明该酶只有一个核苷酸结合位点。在稳态ATP水解过程中，该位点结合的核苷酸中85%被确定为ATP，15%为ADP；这与上述交换实验中测定的ADP释放速率一致，在该实验中发现ADP释放速率比ATP水解的总体速率快六倍。
