Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, Sugiyama Y
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
J Biol Chem. 1998 Jan 16;273(3):1684-8. doi: 10.1074/jbc.273.3.1684.
Transport of many organic anions across the bile canalicular membrane is mediated by the canalicular multispecific organic anion transporter (cMOAT). Previously, we cloned cDNA that may encode cMOAT from Sprague-Dawley rat liver (Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1997) Am. J. Physiol. 272, G16-G22). In the present study, the function of this cloned cDNA was investigated by examining the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) into membrane vesicles isolated from an NIH/3T3 cell line transfected with an expression vector containing the cloned cDNA. Although the membrane vesicles from the control NIH/3T3 cells exhibited endogenous activity in transporting DNP-SG and leukotriene C4 in an ATP-dependent manner, the transfection of cMOAT cDNA resulted in a significant increase in the transport activity for these ligands. The uptake of DNP-SG into membrane vesicles was osmotically sensitive and was stimulated to some extent by other nucleotide triphosphates (GTP, UTP, and CTP) but not by AMP or ADP. The K(m) and Vmax values for the uptake of DNP-SG by the membrane vesicles were 0.175 +/- 0.031 microM and 11.0 +/- 0.73 pmol/min/mg protein, respectively, for the transfected rat cMOAT and 0.141 +/- 0.036 microM and 3.51 +/- 0.39 pmol/min/mg protein, respectively, for the endogenous transporter expressed on control NIH/3T3 cells. These results suggest that the product of the previously cloned cDNA has cMOAT activity being able to transport organic anions in an ATP-dependent manner. Alternatively, it is possible that the cDNA product encodes an activator of endogenous transporter since the K(m) value for DNP-SG was comparable between the vector- and cMOAT-transfected cells. The transport activity found in the control NIH/3T3 cells may be ascribed to mouse cMOAT since Northern blot analysis indicated the presence of a transcript that hybridyzed to the carboxyl-terminal ATP-binding cassette sequence of the murine protein.
许多有机阴离子通过胆小管膜的转运是由胆小管多特异性有机阴离子转运体（cMOAT）介导的。此前，我们从Sprague-Dawley大鼠肝脏中克隆了可能编码cMOAT的cDNA（伊藤，K.，铃木，H.，广桥，T.，久米，K.，清水，T.，以及杉山，Y.（1997年）《美国生理学杂志》272卷，G16 - G22页）。在本研究中，通过检测S -（2,4 -二硝基苯基）-谷胱甘肽（DNP - SG）以ATP依赖方式进入从转染了含有该克隆cDNA的表达载体的NIH/3T3细胞系中分离出的膜囊泡的摄取情况，来研究该克隆cDNA的功能。尽管来自对照NIH/3T3细胞的膜囊泡在以ATP依赖方式转运DNP - SG和白三烯C4方面表现出内源性活性，但cMOAT cDNA的转染导致这些配体的转运活性显著增加。DNP - SG进入膜囊泡的摄取对渗透压敏感，并且在一定程度上受到其他三磷酸核苷酸（GTP、UTP和CTP）的刺激，但不受AMP或ADP的刺激。对于转染的大鼠cMOAT，膜囊泡摄取DNP - SG的K(m)值和Vmax值分别为0.175±0.031微摩尔和11.0±0.73皮摩尔/分钟/毫克蛋白，而对于对照NIH/3T3细胞上表达的内源性转运体，K(m)值和Vmax值分别为0.141±0.036微摩尔和3.51±0.39皮摩尔/分钟/毫克蛋白。这些结果表明，先前克隆的cDNA的产物具有cMOAT活性，能够以ATP依赖方式转运有机阴离子。或者，也有可能该cDNA产物编码一种内源性转运体的激活剂，因为在载体转染细胞和cMOAT转染细胞之间，DNP - SG的K(m)值相当。在对照NIH/3T3细胞中发现的转运活性可能归因于小鼠cMOAT，因为Northern印迹分析表明存在与鼠蛋白的羧基末端ATP结合盒序列杂交的转录本。
