Akerboom T P, Narayanaswami V, Kunst M, Sies H
Institut für Physiologische Chemie I, Universität Düsseldorf, Federal Republic of Germany.
J Biol Chem. 1991 Jul 15;266(20):13147-52.
Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.
在ATP存在的情况下，大鼠肝脏胆小管质膜囊泡对硫醚S-(2,4-二硝基苯基)谷胱甘肽(DNPSG)的摄取增强，并且DNPSG会出现过冲现象，短暂积累5.5倍。基于指示染料氧杂萘邻酮V的反应，ATP的刺激不是由膜电位的产生引起的。ATP依赖性摄取对DNPSG的表观Km为71 microM，Vmax为0.34 nmol·min-1·mg囊泡蛋白-1。如DNPSG转运对巯基试剂的敏感性所示，蛋白质巯基基团对转运活性至关重要。它与其他硫醚存在竞争性抑制，如S-己基谷胱甘肽(Ki = 66 microM)、光亲和标记物S-(4-叠氮苯甲酰基)谷胱甘肽(Ki = 56 microM)，以及与谷胱甘肽二硫化物(Ki = 0.44 mM)和胆汁酸牛磺胆酸盐(Ki = 0.61 mM)。2 mM的谷胱甘肽(GSH)或0.4 mM的胆酸盐不产生抑制作用。谷胱甘肽二硫化物和牛磺胆酸盐在胆小管膜囊泡中均显示出ATP依赖性转运，且被DNPSG抑制。未发现GSH有ATP依赖性转运。DNPSG的转运也受到α-萘基-β-D-葡萄糖醛酸苷(Ki = 0.42 mM)的竞争性抑制，但不受2 mM的α-萘基硫酸盐的抑制，并且依布硒仑和对硝基苯酚的葡萄糖醛酸苷有显著抑制作用。结果表明，DNPSG的胆小管转运系统由ATP直接驱动，其他类化合物的胆汁转运也可能通过该系统进行。
