Rebbeor J F, Connolly G C, Henson J H, Boyer J L, Ballatori N
Department of Environmental Medicine, University of Rochester School of Medicine, NY 14642, USA.
Am J Physiol Gastrointest Liver Physiol. 2000 Aug;279(2):G417-25. doi: 10.1152/ajpgi.2000.279.2.G417.
Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.
多药耐药相关蛋白1和2（Mrp1和Mrp2）被认为介导还原型谷胱甘肽（GSH）的低亲和力ATP依赖性转运，但该假说尚无直接证据。本研究检测了小斑鳐（Raja erinacea）肝脏是否表达Mrp2同源物，以及斑鳐肝细胞膜囊泡是否具有ATP依赖性GSH转运活性。针对哺乳动物Mrp2特异性表位的抗体在斑鳐肝细胞膜上标记出一条180 kDa的蛋白带，并通过免疫荧光对胆小管进行染色，表明斑鳐肝脏表达一种同源蛋白。使用（3）H标记的S - 二硝基苯基谷胱甘肽（DNP - SG）进行Mrp转运活性的功能测定。DNP - SG通过ATP依赖性和ATP非依赖性机制在斑鳐肝细胞膜囊泡中积累。ATP依赖性DNP - SG摄取具有相对较高的亲和力[米氏常数（K（m））= 32±9μM]，并被Mrp2的已知底物和GSH顺式抑制。有趣的是，在囊泡中还检测到了（3）H标记的S - 乙基谷胱甘肽和（3）H标记的GSH的ATP依赖性转运。ATP依赖性GSH转运由低亲和力途径介导（K（m）= 12±2 mM），该途径被Mrp2转运体的底物顺式抑制，但不受膜电位或pH梯度解偶联剂的影响。这些结果为肝细胞膜囊泡中GSH的ATP依赖性转运提供了首个直接证据，并支持了哺乳动物细胞中GSH外排由Mrp蛋白家族成员介导的假说。
