ANALYSIS OF NEW PHYSIOLOGICAL FUNCTIONS OF BILE ACIDS BY HIGHLY ACCURATE MOLECULAR RECOGNITION SYSTEM

最近，我们在大鼠脑的细胞质部分发现了三种胆汁酸，它们与一些蛋白质结合。此外，我们还证明，在高危人群中可能作为结肠癌促进剂的脱氧胆酸盐，通过酰基腺苷不可逆转地优先与组蛋白H3上Lys4的e-氨基结合。我们试图通过高选择性的分子识别系统来阐明胆汁酸的新的生理功能。首先，我们开发了一种新的特异性提取方法，用于从生物体液中提取小分子结合蛋白。我们利用可切割的亲和凝胶通过二硫键固定鹅去氧胆酸，提取了大鼠脑组织、大脑、中脑、小脑、脑干、海马体和脑下垂体中的鹅去氧胆酸结合蛋白。捕获的蛋白质经SDS-PAGE分离，凝胶内胰酶消化后用MALDI-TOF MS进行分析。从所有组织中提取的组分包括α-，…更多的β-微管蛋白、β-肌动蛋白和14-3-3蛋白作为鹅去氧胆酸结合蛋白。此外，在脑垂体提取物中还观察到两条丰富的蛋白质条带，分别鉴定为血清白蛋白和生长激素。鹅去氧胆酸与生长激素的亲和标记表明，Lys55、Lysl92、Lysl96和Lys205可被鹅去氧胆酸酰基腺苷特异性标记。我们在人黑质中发现了一个新的胆汁酸转运蛋白。此外，我们还利用鹅去氧胆酸盐的NBD衍生物荧光探针，建立了一种新的转运活性分析方法。在细胞内，胆汁酸酰基腺苷与多种蛋白质反应生成不可逆的蛋白质加合物，并通过MALDI-TOF MS和NanoLC/ESI-MS/MS鉴定所有用抗鹅去氧胆酸盐抗体检测到的蛋白质加合物。较少

Recently, we found three bile acids, which were bound with some proteins, in the cytoplasmic fraction of the rat brain. Also, we demonstrated that deoxycholate, which may act as colon tumor promoters in high-risk populations, irreversibly and preferentially bound to e-amino group of Lys4 on histone H3 via acyl adenylate. We tried to clarify new physiological functions of bile acids by the highly selective molecular recognition system.First, we developed a new specific extraction method for capturing small molecule-binding proteins from biological fluids by the cleavable affinity gel. We extracted the chenodeoxycholate-binding proteins in the rat brain tissue, cerebrum, midbrain, cerebellum, brainstem, hippocampus, and pituitarium, by using the cleavable affinity gel immobilized chenodeoxycholate via the disulfide linker. The captured proteins were separated by SDS-PAGE, and analyzed by MALDI-TOF MS following in-gel tryptic digestion. The extracted fraction from all tissues included α-, … More β-tubulin, β-actin, and 14-3-3 protein as chenodeoxycholate-binding proteins. Moreover, two abundant protein bands were observed in the extraction fraction from pituitarium, and identified as serum albumin and growth hormone. The affinity labeling of chenodeoxycholate onto growth hormone demonstrated that Lys55, Lysl92, Lysl96, and Lys205 were specifically labeled by chenodeoxycholate acyl adenylate.We found a new bile acid-transporting protein in human substantia nigra. In addition, we developed a new analytical method of transporting activity by using the fluorescent probe, a NBD derivative of chenodeoxycholate.In cells, bile acid acyl adenylate reacted with many proteins to produce irreversible protein adducts, and we identified all protein adducts detected by western blot analysis using anti-chenodeoxycholate antibody by MALDI-TOF MS and nanoLC/ESI-MS/MS following in-gel digestion.We developed a new specific analytical method for detection of phosphorylated sites of proteins by using derivatization reagent containing Br. Less