STUDIES ON EXPROITATION OF UNKNOWN ORPHAN PROTEIN AND THEIR STRUCTURE CHARACTERIZATION

未知孤儿蛋白的利用及其结构表征研究

• 批准号: 13672266
• 负责人: IKEGAWA Shigeo
• 金额: $ 2.24万
• 依托单位: KINKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672266/
• 关键词: Orphan protein; Acyl-adenylates; Protein adduct; Monoclonal antibdy; Lysozyme; Immunobloting; Affinity extraction; TOF; MS; ELISA; リトコール酸; 抗体

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

本研究的目的是制定未知孤儿蛋白的开发原理及其结构测定。为了开发未知的孤儿蛋白，在大鼠肝亚细胞部分中检查了与胆汁酸酰基腺苷酸形成有关的特征蛋白。在最适pH 7和微粒体级分中优先需要Mg 2+ 作为二价阳离子的情况下，对石胆酸(LCA)具有高亲和力的功能蛋白得到富集。尽管有关该蛋白质的结构和功能的许多问题仍有待解决，但所产生的酰基腺苷酸似乎与蛋白质发生反应。因此，通过 MALDI-TOFMS 和蛋白质印迹法，使用识别锚定在溶菌酶上的 LCA 残基的定制单克隆抗体，对通过体外培养石胆酰腺苷酸而由溶菌酶形成的蛋白质加合物进行分析。该单克隆抗体将可用于监测蛋白质结合 LCA 的形成和定位，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其配体之间的相互作用，通过开发两种新型标记试剂，酰基腺苷酸和脱氧胆酸(DCA)的2-脱氧葡萄糖醛酸苷，进行了结合MS的亲和标记方法，并通过使用对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。为了确定翻译后修饰的蛋白质，必须开发一种分离修饰肽的方法。因此，我们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提取方法，以 P 物质结合的 (R)- 和 (5)- 布洛芬为模型，用于肽的基团分离。所建立的富集程序对于从蛋白质消化获得的复杂肽片段混合物中分离修饰肽非常有用。本研究中生成的抗体还可用于免疫印迹，以识别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。较少的

项目成果

池川 繁男: "生物薬科学実験講座 3巻 脂質"廣川書店. 382 (2002)

池川茂夫：《生物制药科学实验教程第3卷脂质》广川书店382（2002）。

Nariyasu Mano, Masashi Uchida, Hiroko Okuyama, Bcuko Sasaki, Shigeo Ikegawa and Junichi Goto: "Simultaneous detection of cholyl adenylate and chloyl coenyme A thioester utilizing liquid chromatography/ electrospray ionization mass spectrometry."Anal Sci..

Nariyasu Mano、Masashi Uchida、Hiroko Okuyama、Bcuko Sasaki、Shigeo Ikekawa 和 Junichi Goto：“利用液相色谱/电喷雾电离质谱法同时检测胆酰腺苷酸和氯酰辅酶 A 硫酯。”《Anal Sci..》

Takashi Iida, Yuka Kasuga, Masayuki Arakawa, Kumiko Mushiake, Shigeo Ikegawa, Junichi Goto and Toshio Nambara: "Complete ^1H and ^<13>C resonance assignments of glycosidic conjugates of nonamidated and amidated bile acids."Magn. Reson. Chem.. 39. 749-756

Takashi Iida、Yuka Kasuga、Masayuki Arakawa、Kumiko Mushiake、Shigeo Ikekawa、Junichi Goto 和 Toshio Nambara：“非酰胺化和酰胺化胆汁酸的糖苷缀合物的完整 ^1H 和 ^13C 共振分配。”Magn。

Shigeo Ikegawa: "Bile acids, Experimental Manuals for Bio-Pharmaceutical Sciences, Vol.3., Lipids[III], Keizo Inoue and Yasuhito Nakagawa (eds.)"Hirokawa Publishing Co.. 73-84 (2002)

池川茂夫：“胆汁酸，生物制药科学实验手册，第 3 卷，脂质[III]，井上敬三和中川康仁（编辑）”广川出版公司.. 73-84 (2002)

Nariyasu Mano: "Simultaneous detection of cholyl adenylate and coenzyme A thioester Utilizing liquid chromatography/electrospray ionization mass"Anal.Sci.. 17. 1037-1042 (2001)

Nariyasu Mano：“利用液相色谱/电喷雾电离质量同时检测胆酰腺苷酸和辅酶 A 硫酯”Anal.Sci. 17. 1037-1042 (2001)