Kast C, Stieger B, Winterhalter K H, Meier P J
Department of Medicine, University Hospital, Zurich, Switzerland.
J Biol Chem. 1994 Feb 18;269(7):5179-86.
To investigate whether electrogenic and ATP-dependent taurocholate transport activities are both mediated by the same bile acid-transporting polypeptide in rat liver, we further purified isolated canalicular membrane vesicles by free flow electrophoresis. Removal of most of the contaminating endoplasmic reticulum resulted in a complete loss of electrogenic taurocholate transport from an ecto-ATPase-enriched canalicular membrane subfraction. In contrast, ATP-dependent taurocholate transport remained associated with both an ecto-ATPase-enriched and an ecto-ATPase-free canalicular membrane subfraction. Microsomes containing 64% of total endoplasmic reticulum exhibited saturable electrogenic (Km approximately 270 microM), but no ATP-dependent taurocholate uptake. Golgi membrane vesicles were devoid of any taurocholate transport activity. These results indicate that electrogenic taurocholate transport resides entirely in the endoplasmic reticulum, whereas ATP-dependent bile acid transport is an intrinsic function of the canalicular membrane as well as of a so far unidentified intracellular membrane bound compartment. Hence, the two transport activities are most probably mediated by two different bile acid transporting polypeptides. Furthermore, the finding of ATP-dependent taurocholate transport in virtually ecto-ATPase-free vesicles argues against the concept of primary active bile acid transport being exclusively mediated by the canalicular ecto-ATPase.
为了研究大鼠肝脏中电生性和ATP依赖性牛磺胆酸盐转运活性是否均由同一种胆汁酸转运多肽介导，我们通过自由流动电泳进一步纯化了分离的胆小管膜囊泡。去除大部分污染的内质网后，富含外向ATP酶的胆小管膜亚组分中电生性牛磺胆酸盐转运完全丧失。相比之下，ATP依赖性牛磺胆酸盐转运仍与富含外向ATP酶和不含外向ATP酶的胆小管膜亚组分相关。含有64%总内质网的微粒体表现出可饱和的电生性（Km约为270 microM），但没有ATP依赖性牛磺胆酸盐摄取。高尔基体膜囊泡没有任何牛磺胆酸盐转运活性。这些结果表明，电生性牛磺胆酸盐转运完全存在于内质网中，而ATP依赖性胆汁酸转运是胆小管膜以及迄今未鉴定的细胞内膜结合区室的固有功能。因此，这两种转运活性很可能由两种不同的胆汁酸转运多肽介导。此外，在几乎不含外向ATP酶的囊泡中发现ATP依赖性牛磺胆酸盐转运，这与原发性主动胆汁酸转运仅由胆小管外向ATP酶介导的概念相悖。
