Simon F R, Sutherland J, Sutherland E
Hepatobiliary Research Center, University of Colorado School of Medicine, Denver.
Am J Physiol. 1990 Sep;259(3 Pt 1):G394-401. doi: 10.1152/ajpgi.1990.259.3.G394.
Intestinal absorption of bile salts occurs by passive processes throughout the length of the small intestine, whereas active carrier-mediated uptake is localized to the ileum. Although previous studies have extensively characterized brush-border transport of bile acids, their extrusion across the basolateral membrane is less well understood. Because previous reports had failed to show specific bile acid binding sites except with the use of photolabeled bile salt derivatives, we sought to identify and characterize the binding parameters of the physiological bile salt taurocholate in ileal and jejunal plasma membrane subfractions. Brush-border membrane (BBM) and basolateral membrane (BLM) fractions were rapidly and simultaneously isolated from the small intestinal mucosa. BBM fractions were isolated with enrichments of 50- to 54-fold for leucine aminopeptidase, whereas the basolateral membrane enrichment of Na(+)-K(+)-ATPase, its specific marker enzyme, was 22- to 25-fold. Contamination from intracellular organelles was minimal. Binding of [14C]taurocholate was demonstrated in both jejunal as well as ileal plasma membrane fractions. However, only ileal binding demonstrated saturation, reversibility, and susceptibility to proteolytic enzymes. [14C]taurocholate binding to BBM fractions also showed competition with bile acids but was not altered by pH or alkylating agents. In contrast, binding of taurocholate to the basolateral membrane showed optimal pH between 6.5 and 7.5 and was inhibited by thiol and alkylating agents. Kinetic analysis of specific ileal BBM and BLM binding showed the parameters for BBM as 288 +/- 70 microM and 2.4 +/- 0.6 nmol/mg protein and for BLM as 6.6 +/- 0.7 microM and 0.56 +/- 0.01 nmol/mg protein for dissociation constant and maximum binding capacity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
胆汁盐在小肠全长通过被动过程进行肠吸收，而由载体介导的主动摄取则局限于回肠。尽管先前的研究已广泛描述了胆汁酸在刷状缘的转运，但它们跨基底外侧膜的外排情况却了解较少。由于先前的报告除了使用光标记胆汁盐衍生物外未能显示特定的胆汁酸结合位点，我们试图鉴定并表征生理胆汁盐牛磺胆酸盐在回肠和空肠质膜亚组分中的结合参数。从小肠黏膜快速同时分离出刷状缘膜（BBM）和基底外侧膜（BLM）组分。分离得到的BBM组分中亮氨酸氨肽酶富集了50至54倍，而其特定标记酶钠钾ATP酶在基底外侧膜的富集倍数为22至25倍。细胞内细胞器的污染极少。在空肠和回肠质膜组分中均证实了[14C]牛磺胆酸盐的结合。然而，只有回肠结合表现出饱和性、可逆性以及对蛋白水解酶的敏感性。[14C]牛磺胆酸盐与BBM组分的结合也显示出与胆汁酸的竞争性，但不受pH或烷基化剂的影响。相比之下，牛磺胆酸盐与基底外侧膜的结合在pH 6.5至7.5之间显示出最佳状态，并受到硫醇和烷基化剂的抑制。对回肠BBM和BLM特异性结合的动力学分析表明，BBM的解离常数和最大结合容量参数分别为288±70微摩尔和2.4±0.6纳摩尔/毫克蛋白，BLM的分别为6.6±0.7微摩尔和0.56±0.01纳摩尔/毫克蛋白。（摘要截断于250字）
