STUDIES ON EXPROITATION OF UNKNOWN ORPHAN PROTEIN AND THEIR STRUCTURE CHARACTERIZATION

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The aim of this study was to develop the principles for the exploitation of the unknown orphan proteins and their structural determination. To exploit the unknown orphan proteins, characteristic protein concerning the formation of bile acid acyl-adenylates was examined in rat liver subcellular fraction. There was an enrichment of the functional protein with high affinity for lithocholic acid (LCA) under the optimum pH 7 and the preferential requirement of Mg^<2+> as a bivalent cation in the microsomal fraction. Although many questions around the structure and function of this protein remain to be solved, it seems that the produced acyl-adenylates reacts with protein. Therefore, analysis of the protein adducts formed from lysozyme by in vitro incubation of lithocholyl-adenylate was done by MALDI- TOFMS and western blots using custom-made monoclonal antibody which recognizes LCA residues anchored on lysozyme. The monoclonal antibody will be useful for monitoring the formation and localiz … More ation of protein-bound LCA, which may be concern with the activity of LCA as a colon cancer promoter. For the analysis of the interaction between orphan proteins and their ligands, an affinity labeling method combined with MS has been carried out by the development of two novel labeling reagents, acyl-adenylate and 2-deoxyglucuronide of deoxycholic acid (DCA), and the usefulness of the analytical method was confirmed by employing monoclonal antibody having high affinity to DCA. To determine the post-translationally modified proteins, a method to isolate the modified peptides had to be developed. Therefore, we have been developed an immunoaffinity extraction with a highly specific and selective immobilized antibody for the group separation of peptides by using substance P bound (R)- and (5)-ibuprofen as model. The established enrichment procedure was very useful for separation of modified peptides from complex peptide fragment mixtures obtained by protein digestion. The antibody generated in this study was also useful for immunobloting to identify glutathione-S-transferase modified with (S)-ibuprofen in complex protein mixtures. Less