STUDIES ON EXPROITATION OF UNKNOWN ORPHAN PROTEIN AND THEIR STRUCTURE CHARACTERIZATION

本研究的目的是为开发未知孤儿蛋白及其结构测定提供理论依据。为了开发未知的孤儿蛋白，在大鼠肝亚细胞组分中检测了与胆汁酸酰基腺苷酸形成有关的特征蛋白。在最适pH为7的条件下，微粒体组分中富集了对石胆酸（LCA）具有高亲和力的功能蛋白，并且优先需要Mg^<2+>作为二价阳离子。虽然围绕该蛋白的结构和功能的许多问题仍有待解决，但似乎产生的酰基腺苷酸与蛋白质反应。因此，通过体外孵育石胆酰-腺苷酸由溶菌酶形成的蛋白质加合物的分析通过MALDI-TOFMS和蛋白质印迹使用定制的单克隆抗体进行，所述单克隆抗体识别锚定在溶菌酶上的LCA残基。该单克隆抗体可用于监测肿瘤的形成和定位， ...更多信息 蛋白结合的LCA的活化，这可能与LCA作为结肠癌促进剂的活性有关。为了分析孤儿蛋白与配体之间的相互作用，通过开发脱氧胆酸（DCA）的酰基腺苷酸和2-脱氧葡萄糖醛酸苷两种新的标记试剂，建立了一种结合质谱的亲和标记方法，并通过使用与DCA具有高亲和力的单克隆抗体证实了该分析方法的有效性。为了确定后修饰的蛋白质，必须开发分离修饰的肽的方法。因此，我们已经开发了一种具有高度特异性和选择性的固定化抗体的免疫亲和萃取，用于以P物质结合的（R）-和（5）-布洛芬为模型的肽的基团分离。建立的富集程序是非常有用的分离修饰的肽从复杂的肽片段的混合物获得的蛋白质消化。本研究中产生的抗体也可用于免疫印迹，以鉴定复杂蛋白质混合物中用（S）-布洛芬修饰的谷胱甘肽-S-转移酶。少

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