Leier I, Jedlitschky G, Buchholz U, Cole S P, Deeley R G, Keppler D
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
J Biol Chem. 1994 Nov 11;269(45):27807-10.
The multidrug resistance-associated protein (MRP) is the product of an ATP-binding cassette transporter gene overexpressed in some tumor cells resistant to antineoplastic agents. We studied the transport function of MRP in membrane vesicles prepared from HeLa cells transfected with an MRP expression vector and overexpressing this 190-kDa membrane glycoprotein. ATP-dependent primary-active transport into the vesicles was demonstrated for leukotriene C4 (LTC4), LTD4, LTE4, and S-(2,4-dinitrophenyl)glutathione with relative rates, at a substrate concentration of 50 nM, of 1.0, 0.27, 0.14, and 0.16, respectively. The endogenous glutathione conjugate LTC4 had the highest affinity for this transporter with a Km of 97 nM. The Km for ATP was 19 microM. Direct photoaffinity labeling with [3H]LTC4 labeled a 190-kDa membrane protein predominantly in the MRP-transfected HeLa cells. ATP-dependent LTC4 transport was effectively inhibited by the LTD4 receptor antagonist MK 571, whereas cyclosporin A and, particularly, its analog PSC 833 were much less potent. The respective Ki values were 0.6, 5, and 27 microM, respectively. In addition, MK 571 preferentially inhibited photoaffinity labeling of the 190-kDa protein in the MRP transfectants. Our results provide direct evidence that the MRP gene encodes a primary-active ATP-dependent export pump for conjugates of lipophilic compounds with glutathione and several other anionic residues. We conclude that the biosynthetic release of LTC4 from cells is mediated by the 190-kDa product of the MRP gene.
多药耐药相关蛋白（MRP）是一种ATP结合盒转运蛋白基因的产物，在一些对抗肿瘤药物耐药的肿瘤细胞中过度表达。我们研究了从转染MRP表达载体并过度表达这种190 kDa膜糖蛋白的HeLa细胞制备的膜囊泡中MRP的转运功能。在底物浓度为50 nM时，白三烯C4（LTC4）、LTD4、LTE4和S -（2,4 -二硝基苯基）谷胱甘肽的ATP依赖性原发性主动转运进入囊泡得到证实，相对速率分别为1.0、0.27、0.14和0.16。内源性谷胱甘肽结合物LTC4对该转运蛋白具有最高亲和力，Km为97 nM。ATP的Km为19 μM。用[3H]LTC4进行直接光亲和标记主要标记了MRP转染的HeLa细胞中的一种190 kDa膜蛋白。LTD4受体拮抗剂MK 571有效抑制了ATP依赖性LTC4转运，而环孢素A，尤其是其类似物PSC 833的效力则低得多。各自的Ki值分别为0.6、5和27 μM。此外，MK 571优先抑制MRP转染细胞中190 kDa蛋白的光亲和标记。我们的结果提供了直接证据，表明MRP基因编码一种原发性主动ATP依赖性输出泵，用于亲脂性化合物与谷胱甘肽及其他几种阴离子残基的结合物。我们得出结论，细胞中LTC4的生物合成释放是由MRP基因的190 kDa产物介导的。
