Chang X B, Hou Y X, Riordan J R
S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, USA.
J Biol Chem. 1998 Sep 11;273(37):23844-8. doi: 10.1074/jbc.273.37.23844.
Membrane vesicles prepared from cells expressing the multidrug resistance-associated protein (MRP) transport glutathione S-conjugates of hydrophobic substrates in an ATP dependent manner. Purified MRP possesses ATPase activity which can be further stimulated by anticancer drugs or leukotriene C4. However, the detailed relationship between ATP hydrolysis and drug transport has not been established. How the ATPase activity of MRP is regulated in the cell is also not known. In this report, we have examined the effects of different nucleotides on the ATPase activity of purified MRP. We have found that pyrimidine nucleoside triphosphates have little effect on enzymatic activity. In contrast, purine nucleotides dATP, dGTP, and adenosine 5'-(beta,gamma-imido)triphosphate function as competitive inhibitors. Somewhat unexpectedly, low concentrations of all the nucleoside diphosphates (NDPs) tested, except UDP, stimulate the ATPase activity severalfold. ADP or GDP at higher concentrations was inhibitory, reflecting NDP binding to the substrate site. On the other hand, the enhancement of hydrolysis at low NDP concentrations must reflect interactions with a separate site. Therefore, we postulate the presence of at least two types of nucleotide binding sites on the MRP, a catalytic site(s) to which ATP preferentially binds and is hydrolyzed and a regulatory site to which NDPs preferentially bind and stimulate hydrolysis. Interestingly, the stimulatory effects of drugs transported by MRP and NDPs are not additive, i.e. drugs are not able to further stimulate the NDP-activated enzyme. Hence, the two activation pathways intersect at some point. Since both nucleotide binding domains of MRP are likely to be required for drug stimulation of ATPase activity, the two sites that we postulate may also involve both domains.
从表达多药耐药相关蛋白（MRP）的细胞制备的膜泡以ATP依赖的方式转运疏水性底物的谷胱甘肽S-共轭物。纯化的MRP具有ATP酶活性，抗癌药物或白三烯C4可进一步刺激该活性。然而，ATP水解与药物转运之间的详细关系尚未确立。MRP的ATP酶活性在细胞中是如何调节的也不清楚。在本报告中，我们研究了不同核苷酸对纯化的MRP的ATP酶活性的影响。我们发现嘧啶核苷三磷酸对酶活性影响很小。相反，嘌呤核苷酸dATP、dGTP和腺苷5'-（β，γ-亚氨基）三磷酸作为竞争性抑制剂起作用。有点出乎意料的是，除UDP外，所测试的所有核苷二磷酸（NDP）的低浓度均能将ATP酶活性刺激数倍。较高浓度的ADP或GDP具有抑制作用，这反映了NDP与底物位点的结合。另一方面，低NDP浓度下水解的增强必定反映了与另一个位点的相互作用。因此，我们推测MRP上至少存在两种类型的核苷酸结合位点，一个是催化位点，ATP优先与之结合并水解，另一个是调节位点，NDP优先与之结合并刺激水解。有趣的是，由MRP转运的药物和NDP的刺激作用不是相加的，即药物不能进一步刺激NDP激活的酶。因此，这两条激活途径在某一点相交。由于MRP的两个核苷酸结合结构域可能都是药物刺激ATP酶活性所必需的，我们推测的这两个位点可能也涉及这两个结构域。
