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研究生(外文):Hsin-Hui Wu
論文名稱(外文):Spectroscopic and Biochemical Characterization of Human Membrane-associated Progesterone Receptor Component 1 (PGRMC1_ Human)
指導教授(外文):Kelvin Huang-Chou Chen
外文關鍵詞:progesteroneprogesterone membrane-associated component 1anti-apopticmetabolized steroid5-aminolevulinic acid
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 人體孕酮受體膜蛋白(PGRMC1_Human)與Dap1p、inner zone antigen (IZA)及小鼠孕酮受體膜蛋白質(mPGRMCp)皆與假定的孕酮受體膜蛋白(MAPR)有高度的保留性。最近的研究指出此類蛋白質會與血紅素(Heme)結合。由胺基酸序列分析顯露出此家族蛋白有類似cytochrome b5 motif的序列,但卻沒有以兩個組胺酸(histidines)來與血紅素結合。儘管其被命名為孕酮受體膜蛋白(PGRMC1),但其與cytochrome b5的同源性相對高於固醇類受體蛋白,且唯一可確認的生化活性是與血紅素結合。對於孕酮受體膜蛋白(PGRMC1),目前所知的生化特性就是會與血紅素結合,誘發抗細胞凋零(anti-apoptic)訊號或代謝固醇(metabolized steroid)訊息。因此,人體孕酮受體膜蛋白中的血紅素是否能夠完全負載是非常重要的。人體孕酮受體膜蛋白在大腸桿菌(E. coli)系統上成功的被轉殖及大量表現。在大腸桿菌培養液中加入血紅素的前驅物5-氨基乙醯丙酸鹽可以提升人體孕酮受體膜蛋白中血紅素的含量,從微量提高至87%。本論文藉由原二色光譜(CD)、原子吸收光譜儀(AA) 、紫外光-可見光光譜(UV-Vis)、小角度散射(SAXS)及一般的生化方法去了解人體孕酮受體蛋膜白與血紅素結合的特性及其基質結合區的化學環境。原二色光譜指出人體孕酮受體膜蛋白有α-螺旋及β-摺板,所以不是因為不正確的蛋白質摺疊造成人體孕酮受體膜蛋白中不同程度血紅素的含量。由紫外光-可見光吸收光譜證實人體膜關聯孕酮受體蛋白是一種具有高自旋氧化態鐵離子(Ⅲ)鍵結的紫質鍵結蛋白,與cytochrome b5的低自旋氧化態鐵離子鍵結的紫質鍵結蛋白不同。
Human progesterone membrane-associated component 1 (PGRMC1_Human) protein belongs to a highly conserved class of putative membrane-associated progesterone binding proteins (MAPR), which Dap1p and inner zone antigen (IZA), the rat homologue of mouse progesterone receptor membrane-associated component 1 protein (mPGRMC1p), recently being reported to bind heme. While primary structure analysis reveals similarities to the cytochrome b5 motif, neither of the two axial histidines responsible for ligation to the heme is present in any of the MAPR proteins. In spite of its name, PGRMC1_Human shares homology with cytochrome b5-related proteins rather than hormone receptors, and heme binding is the sole biochemical activity of PGRMC1_Human. The only known biochemical function of PGRMC1_Human is binding to heme and inducing anti-apoptic signaling or metabolized steroid. So Heme of PGRMC1_Human can be fully loaded is important. The PGRMC1_Human gene was cloned, and the gene product was over-expressed in Escherichia coli. An addition of the heme precursor 5-aminolevulinic acid (ALA) to the medium increased heme content of PGRMC1_Human. The heme loading percentage for PGRMC1_Human can be increased to 87% form trace. In the thesis, CD, UV-Vis, small angle X-ray scattering (SAXS) and general biochemical methods have been used to characterize the nature of heme binding in PGRMC1_Human. CD indicate a well –ordered structure, suggesting the different level of heme loading is probably not due to improperly folded protein. UV-Vis spectrum confirmed a high-spin Fe (Ⅲ) for PGRMC1_Human, indicating one axial amino acid ligand, in contrast to the low-spin Fe (Ⅱ) of cytochrome b5.
研究背景.......................... 3
類固醇作用機制...................... 3
膜孕酮受體........................ 5
圓二色光譜 ........................ 9
實驗............................ 14
藥品試劑......................... 15
實驗方法 ........................16
結果與討論......................... 24
結論............................ 35
參考文獻.......................... 37
附錄............................ 42
著作............................ 43
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