Kramer W, Girbig F, Gutjahr U, Kowalewski S, Jouvenal K, Müller G, Tripier D, Wess G
Hoechst Aktiengesellschaft, Frankfurt am Main, Germany.
J Biol Chem. 1993 Aug 25;268(24):18035-46.
The anatomical localization of the Na+/bile acid cotransport system from rabbit small intestine was determined using brush border membrane vesicles prepared from eight different segments of the small intestine. Na(+)-dependent transport activity for bile acids, both for [3H]taurocholate and [3H]cholate, was found in the distal segment 8 only representing the terminal 12% of the small intestine. In contrast, the Na(+)-dependent D-glucose transporter and the H(+)-dependent oligopeptide transporter were found over the whole length of rabbit small intestine in all segments. Photoaffinity labeling with 7,7-azo- and 3,3-azo-derivatives of taurocholate with subsequent fluorographic detection of labeled polypeptides after one- and two-dimensional gel electrophoresis showed that an integral membrane polypeptide of M(r) 87,000 is present in the entire small intestine, whereas an integral membrane protein of M(r) 93,000 together with a peripheral membrane protein of M(r) 14,000 are exclusively expressed in the distal small intestine correlating with Na(+)-dependent bile acid transport activity. Photoaffinity labeling with the cationic bile acid derivative 1-(7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta[3 beta-3H]cholan-24-oyl)-1,2- diaminoethane hydrochloride and 7,7-azo-3 alpha,12 beta-dihydroxy-5 beta[12 alpha-3H]cholan-24-oic acid did not result in a specific labeling of the above mentioned proteins, demonstrating their specificity for physiological bile acids. Photoaffinity labeling of the 93- and 14-kDa bile acid-binding proteins was strongly Na(+)-dependent. Significant labeling of the 93- and 14-kDa proteins occurred only in the presence of Na+ ions with maximal labeling above 100 mM [Na+] showing a parallel [Na+] dependence to transport activity. Inactivation of Na(+)-dependent [3H]taurocholate uptake by treatment of ileal brush border membrane vesicles with 4-nitrobenzo-2-oxa-1,3-diazol chloride led to a parallel decrease in the extent of photoaffinity labeling of both the 93- and 14-kDa protein. Sequence analysis of the membrane-bound 14-kDa bile acid-binding protein surprisingly revealed its identity with gastrotropin, a hydrophobic ligand-binding protein exclusively found in the cytosol from ileocytes and thought to be involved in the intracellular transport of bile acids from the brush border membrane to the basolateral pole of the ileocyte. In conclusion, the present studies suggest that both an integral 93- and a peripheral 14-kDa membrane protein, identified as gastrotropin, and both exclusively expressed in the terminal ileum, are essential components of the Na+/bile acid cotransport system in rabbit terminal ileum.
利用从兔小肠八个不同节段制备的刷状缘膜囊泡，确定了兔小肠中Na⁺/胆汁酸共转运系统的解剖定位。仅在代表小肠末端12%的远端节段8中发现了对[³H]牛磺胆酸盐和[³H]胆酸盐的Na⁺依赖性胆汁酸转运活性。相比之下，在兔小肠的所有节段的全长中都发现了Na⁺依赖性D - 葡萄糖转运体和H⁺依赖性寡肽转运体。用牛磺胆酸盐的7,7 - 偶氮和3,3 - 偶氮衍生物进行光亲和标记，随后在一维和二维凝胶电泳后对标记多肽进行荧光显影检测，结果表明，在整个小肠中存在一种相对分子质量为87,000的整合膜多肽，而相对分子质量为93,000的整合膜蛋白以及相对分子质量为14,000的外周膜蛋白仅在远端小肠中表达，这与Na⁺依赖性胆汁酸转运活性相关。用阳离子胆汁酸衍生物1 - (7,7 - 偶氮 - 3α,12α - 二羟基 - 5β[3β - ³H]胆烷 - 24 - 酰基) - 1,2 - 二氨基乙烷盐酸盐和7,7 - 偶氮 - 3α,12β - 二羟基 - 5β[12α - ³H]胆烷 - 24 - 酸进行光亲和标记，并未导致上述蛋白质的特异性标记，证明了它们对生理性胆汁酸的特异性。93 kDa和14 kDa胆汁酸结合蛋白的光亲和标记强烈依赖于Na⁺。仅在存在Na⁺离子的情况下，93 kDa和14 kDa蛋白才会有显著标记，在[Na⁺]高于100 mM时标记达到最大值，显示出与转运活性平行的[Na⁺]依赖性。用4 - 硝基苯 - 2 - 恶唑 - 1,3 - 二氮杂茂氯化物处理回肠刷状缘膜囊泡，使Na⁺依赖性[³H]牛磺胆酸盐摄取失活，导致93 kDa和14 kDa蛋白的光亲和标记程度平行降低。对膜结合的14 kDa胆汁酸结合蛋白的序列分析令人惊讶地发现，它与促胃泌素相同，促胃泌素是一种仅在回肠细胞胞质溶胶中发现的疏水性配体结合蛋白，被认为参与胆汁酸从刷状缘膜向回肠细胞基底外侧极的细胞内转运。总之，本研究表明，一种被鉴定为促胃泌素的相对分子质量为93 kDa的整合膜蛋白和一种相对分子质量为14 kDa的外周膜蛋白，两者都仅在回肠末端表达，是兔回肠末端Na⁺/胆汁酸共转运系统的重要组成部分。
