Wenzel U, Jouvenal K, Tripier D, Ziegler K
Institute of Pharmacology and Toxicology, Justus-Liebig-University, Frankfurt a.M., Germany.
Biochem Pharmacol. 1995 Feb 14;49(4):479-87. doi: 10.1016/0006-2952(94)00451-q.
The hydrophobic cyclohexapeptide cyclo(Phe-Thr-Lys-Trp-Phe-DPro) (008), an analog of somatostatin with retro sequence, was previously shown to competitively inhibit the uptake of cholate and taurocholate into isolated rat liver cells. Conversely, the competitive uptake inhibition of 008 into isolated rat hepatocytes by bile acids confirmed the observation of common binding and transport sites by bile acids and cyclosomatostatin. Furthermore the transport characteristics of 008 uptake revealed a significant and rapid binding to cell membranes. In this context it was of special interest to investigate the specificity of the binding component since specific binding of the substrate to membrane proteins could be responsible for the low Km of 008-transport. Therefore, the cyclohexapeptide 008 could be used as the ligand in affinity chromatography in order to isolate such binding proteins. The gel matrix used did not interact non-specifically with octylglucoside-solubilized proteins from isolated rat liver plasma membranes. In affinity chromatography of octylglucoside-solubilized plasma membranes, two dominant proteins with apparent molecular masses of 60 and 58 kDa bound specifically to the 008 ligand. When used as ligands in affinity chromatography, these membrane-associated 60 and 58 kDa proteins bound exclusively to aromatic cyclopeptides, e.g. cyclosomatostatin 008, but not to linear peptides or taurocholate derivatives. The amino acid sequences of tryptic digests of the 008-affinity-purified 58 kDa protein were identical to the sequence of a microsomal pI6.1 carboxylesterase. Immunofluorescence of intact hepatocytes showed that this xenobiotic metabolizing enzyme is also located in sinusoidal rat liver plasma membranes and could therefore account for the extensive and specific binding of the cyclosomatostatin to sinusoidal plasma membranes of rat liver.
疏水性环六肽环（苯丙氨酸 - 苏氨酸 - 赖氨酸 - 色氨酸 - 苯丙氨酸 - D - 脯氨酸）（008）是一种具有反向序列的生长抑素类似物，先前已证明它能竞争性抑制胆酸盐和牛磺胆酸盐进入分离的大鼠肝细胞。相反，胆汁酸对008进入分离的大鼠肝细胞的竞争性摄取抑制作用证实了胆汁酸和环生长抑素存在共同的结合和转运位点。此外，008摄取的转运特性显示其与细胞膜有显著且快速的结合。在这种情况下，研究结合成分的特异性特别有趣，因为底物与膜蛋白的特异性结合可能是008转运低Km值的原因。因此，环六肽008可用作亲和色谱中的配体，以分离此类结合蛋白。所用的凝胶基质与从分离的大鼠肝细胞膜中用辛基葡糖苷增溶的蛋白质无非特异性相互作用。在对辛基葡糖苷增溶的质膜进行亲和色谱时，两种表观分子量分别为60 kDa和58 kDa的主要蛋白质特异性结合到008配体上。当用作亲和色谱中的配体时，这些与膜相关的60 kDa和58 kDa蛋白质仅与芳香环肽结合，例如环生长抑素008，但不与线性肽或牛磺胆酸盐衍生物结合。经008亲和纯化的58 kDa蛋白质的胰蛋白酶消化产物的氨基酸序列与微粒体pI6.1羧酸酯酶的序列相同。完整肝细胞的免疫荧光显示，这种外源性代谢酶也位于大鼠肝血窦质膜中，因此可以解释环生长抑素与大鼠肝血窦质膜的广泛且特异性结合。
