Berr F, Meier P J, Stieger B
Department of Medicine II, Hospital Grosshadern, University of Munich, Federal Republic of Germany.
J Biol Chem. 1993 Feb 25;268(6):3976-9.
In the present study we used the water-soluble short chain phosphatidylcholine analogue L-alpha-dibutyryl-glycero-3-phosphatidylcholine (diC4PC) to investigate the mechanism involved in the canalicular secretion of phospholipids in rat liver. Uptake of 14C-labeled di-C4PC was studied in isolated microsomes as well as in basolateral (sinusoidal) and canalicular plasma membrane vesicles. Saturable uptake of diC4PC into an osmotically active space was observed in microsomes and canalicular membrane vesicles. In contrast, diC4PC uptake into basolateral membrane vesicles could be accounted for by cross-contamination with endoplasmic reticulum and canalicular membrane vesicles. Whereas the Km values for diC4PC uptake (37 degrees C) were similar in microsomes (7.4 +/- 2.6 mM) and canalicular membrane vesicles (8.2 +/- 2.0 mM), the Vmax values were approximately 2-fold higher in canalicular membrane vesicles (29.6 +/- 2.7 nmol/mg of protein x min) than in microsomes (16.7 +/- 2.1 nmol/mg of protein x min). Furthermore, Pronase treatment of the membrane vesicles reduced diC4PC uptake by 34-54% in both subfractions, whereas the D-[14C]glucose-accessible water space was only reduced by approximately 20%. These data provide direct evidence for the presence of a protein-mediated phosphatidylcholine translocating activity in the canalicular membrane of rat hepatocytes. This canalicular "flippase" has kinetic properties similar to those described previously in microsomes and provides a potential pathway for the translocation of bile salt dissolvable biliary phospholipids to the exoplasmic leaflet of the canalicular membrane.
在本研究中，我们使用水溶性短链磷脂酰胆碱类似物L-α-二丁酰甘油-3-磷脂酰胆碱（二C4PC）来研究大鼠肝脏胆小管磷脂分泌所涉及的机制。在分离的微粒体以及基底外侧（窦状）和胆小管质膜囊泡中研究了14C标记的二C4PC的摄取。在微粒体和胆小管膜囊泡中观察到二C4PC可饱和摄取到渗透活性空间中。相比之下，二C4PC摄取到基底外侧膜囊泡中可能是由于内质网和胆小管膜囊泡的交叉污染所致。虽然微粒体（7.4±2.6 mM）和胆小管膜囊泡（8.2±2.0 mM）中二C4PC摄取的Km值相似，但胆小管膜囊泡（29.6±2.7 nmol/mg蛋白质×分钟）中的Vmax值比微粒体（16.7±2.1 nmol/mg蛋白质×分钟）高约2倍。此外，用链霉蛋白酶处理膜囊泡可使两个亚组分中的二C4PC摄取减少34 - 54%，而D-[14C]葡萄糖可进入的水空间仅减少约20%。这些数据为大鼠肝细胞胆小管膜中存在蛋白质介导的磷脂酰胆碱转运活性提供了直接证据。这种胆小管“翻转酶”具有与先前在微粒体中描述的类似的动力学特性，并为胆汁盐可溶解的胆汁磷脂转运到胆小管膜的外质小叶提供了一条潜在途径。
