IIDA Takashi NIHON UNIVERSITY, COLLEGE OF HUMANITIES & SCIENCES, PROFESSOR, 文理学部, 教授 (60060125)
MANO Nriyasu TOHOKU UNIVERSITY, GRADUATE SCHOOL OF PHARMACEUTICAL SCIENCES, ASSOCIATE PREOFESSOR, 大学院・薬学研究科, 助教授 (50323035)
KOBAYASHI Norihiro KOBE PHARMACEUTICAL UNIVERSITY, FACULTY OF PHARMACEUTICAL SCIENCES, PROFESSER, 薬学部, 教授 (90205477)
|Budget Amount *help
¥15,200,000 (Direct Cost : ¥15,200,000)
Fiscal Year 2003 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 2002 : ¥12,400,000 (Direct Cost : ¥12,400,000)
Bile acids are synthesized in the liver from cholesterol. In the intestinal lumen, they assist lipolysis and the absorption of fats, and then return to the liver upon absorption in the ileum and proximal colon. Because of their efficient hepatic uptake, bile acids have low concentrations in the peripheral blood. Using LC/ESI-MS, we have recently found three unconjugated bile acids in the rat brain cytoplasmic fraction. Chenodeoxycholic acid (CDCA) was detected only upon extraction with high concentrations of guanidine hydrochloride, indicating that it is bound non-covalently to protein. In the present study, we demonstrated the enzyme activity from 3β-hydroxy-5-cholenoic acid to CDCA in rat brain tissue, and we identified the candidates of CDCA-binding proteins.
To distinguish from endogenous contaminants, ^<18>O-hydroxy-5-cholenoic acid, 3β,7α-dihydroxy-5-cholenoic acid, and 7α-hydroxy-3-oxo-4-cholenoic acid were prepared and used as substrates for the in vitro incubation. The results
suggested that 3β-hydroxy-5-cholenoic acid was NADPH-dependently converted to 3β,7α-dihydroxy-5-cholenoic acid in microsomal preparation. And 7α-hydroxy-3-oxo-4-cholenoic acid was NAD^+-dependently produced from 3β,7α-dihydroxy-5-cholenoic acid in microsomal preparation, and finally, Δ4-3-keto form was NADPH-dependently converted to CDCA by rat brain cytosolic enzyme. These findings indicated the presence of the enzyme activity from 3β-hydroxy-5-cholenoic acid to CDCA in rat brain tissue.
Affinity gel was prepared by immobilization of CDCA. The rat brain cytoplasmic fraction was applied to the gel, and proteins were extracted by using high concentrations of guanidine hydrochloride. The protein mixture was separated by two-dimensional gel electrophoresis, and the protein map was compared with the control protein map. Several proteins were specifically enriched by the affinity extraction. The peptide fragment mixtures obtained from in-gel tryptic digestion was analyzed by MALDI-TOFMS, and the protein identification was performed by peptide mass fingerprinting method., Nine proteins were identified, and they have included tubulin α and β, 14-3-3 protein ζ, which may relate to Alzheimer's disease. Less