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

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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