von Dippe P, Levy D
J Biol Chem. 1983 Jul 25;258(14):8896-901.
The taurocholate transport system in normal and transformed hepatocytes has been characterized using transport kinetics and photoaffinity labeling procedures. A photoreactive diazirine derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino [ 1,2-3H ]ethanesulfonic acid (7-ADTC), which has been shown to be a substrate for the bile acid carrier system, was photolyzed in the presence of intact hepatocytes, hepatoma tissue culture (HTC) cells, and plasma membranes derived from the hepatocyte sinusoidal surface. Irradiation of membranes in the presence of 7-ADTC resulted in the incorporation of the photoprobe into two proteins with Mr = 68,000 and 54,000. The specificity of labeling was confirmed by the significant inhibition of labeling observed when photolysis was carried out in the presence of taurocholate. The 68,000-Da protein was easily extracted with water and was shown to exhibit electrophoretic properties identical with rat serum albumin. The 54,000-Da protein required Triton X-100 for solubilization, indicating a strong association with the plasma membrane. Labeling of intact hepatocytes also resulted in specific labeling of the 54,000-Da protein. In contrast to hepatocytes, HTC cells derived from Morris hepatoma 7288C as well as H4-II-E cells derived from Reuber hepatoma H-35 exhibited a total loss of mediated bile acid uptake. Photolysis of 7-ADTC in the presence of HTC cells did not result in the labeling of any proteins, a result consistent with the loss of transport activity, and further supporting the specificity of the labeling reaction. The anion transport inhibitor N-(4-azido-2-nitrophenyl)-2-aminoethyl-[ 35S ]sulfonate, which has been shown to be a substrate for the bile acid carrier system also labeled the 54,000-Da plasma membrane protein when photolyzed in the presence of intact hepatocytes. These results suggest that the 54,000-Da protein is a component of the hepatocyte bile acid transport system and that the activity of this system is greatly reduced in several hepatoma cell lines.
已利用转运动力学和光亲和标记程序对正常和转化肝细胞中的牛磺胆酸盐转运系统进行了表征。牛磺胆酸盐的一种光反应性重氮衍生物，即(7,7 - 偶氮 - 3α,12α - 二羟基 - 5β - 胆烷 - 24 - 酰基)-2 - 氨基[1,2 - ³H]乙烷磺酸盐(7 - ADTC)，已被证明是胆汁酸载体系统的一种底物，在完整肝细胞、肝癌组织培养(HTC)细胞以及源自肝细胞膜窦状表面的质膜存在的情况下进行光解。在7 - ADTC存在的情况下对膜进行照射导致光探针掺入两种分子量分别为68,000和54,000的蛋白质中。当在牛磺胆酸盐存在的情况下进行光解时观察到标记显著受到抑制，从而证实了标记的特异性。68,000道尔顿的蛋白质很容易用水提取，并显示出与大鼠血清白蛋白相同的电泳特性。54,000道尔顿的蛋白质需要用Triton X - 100溶解，这表明它与质膜有很强的结合。对完整肝细胞进行标记也导致对54,000道尔顿蛋白质的特异性标记。与肝细胞不同，源自莫里斯肝癌7288C的HTC细胞以及源自鲁伯肝癌H - 35的H4 - II - E细胞表现出介导的胆汁酸摄取完全丧失。在HTC细胞存在的情况下对7 - ADTC进行光解不会导致任何蛋白质的标记，这一结果与转运活性的丧失一致，并进一步支持了标记反应的特异性。阴离子转运抑制剂N - (4 - 叠氮基 - 2 - 硝基苯基)-2 - 氨基乙基 - [³⁵S]磺酸盐，已被证明是胆汁酸载体系统的一种底物，当在完整肝细胞存在的情况下进行光解时也标记了54,000道尔顿的质膜蛋白。这些结果表明，54,000道尔顿的蛋白质是肝细胞胆汁酸转运系统的一个组成部分，并且该系统的活性在几种肝癌细胞系中大大降低。
