亞砷酸鈉與Galectin-1蛋白交互作用之研究; The study of interaction of arsenite and galectin-1

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題名:	亞砷酸鈉與Galectin-1蛋白交互作用之研究;The study of interaction of arsenite and galectin-1
作者:	黃琦斐;Chi-Fei Huang
貢獻者:	生命科學研究所
關鍵詞:	亞砷酸鈉;固相結合試驗;solid phase binding assay;ITC;arsenite;galectin-1;CD
日期:	2002-07-08
上傳時間:	2009-09-22 10:16:47 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	Galectin-1蛋白是一種b-半乳糖苷結合蛋白，其參與細胞貼附、分化及生長調控。Gal-1具有6個還原態的硫氫基，此官能基和蛋白質之lectin活性有直接的關係，但是Gal-1在形成二聚體的過程，並無硫氫基的參與。由近年的研究指出，氧化態Gal-1喪失其lectin活性，但在低濃度下，可促使神經細胞再生及使細胞轉化。由實驗室初步結果顯示，As3+可和Gal-1上的硫氫基結合，並且造成Gal-1的氧化，而此種氧化態Gal-1，運用其生物性功能來影響CHOA細胞；而轉殖Gal-1基因的哺乳類動物細胞，會累積且吸收較多的砷化物。我們利用大腸桿菌系統純化Gal-1和Gal-As3+；並以CD觀察蛋白質，發現gal-As3+在200~205 nm之間有一負值訊號，而gal-1則在205~210nm之間有一負值訊號。由CD值位移的結果間接顯示，亞砷酸鈉 (As3+)和gal-1結合，導致蛋白質二級結構的改變。而由恆溫滴定微卡計的試驗結果得知，亞砷酸鈉滴入gal-1蛋白溶液中的吸附焓介於-55~-69 (kJ/mole As3+)之間。顯示亞砷酸鈉和gal-1之間的作用能量已超過配位鍵的範圍 (~21 kJ/mole)。由初步的結果得知，gal-As3+失去部分lectin活性，且影響HFW細胞的存活率。 Galectin-1 (gal-1), a b-galactoside binding protein, functions in cell adhesion, development, and growth regulation. Gal-1 has six free sulfhydryl groups, which are essential for their lectin property and do not involve in the formation of dimeric gal-1. Recent studies suggest that oxidized gal-1 loses the lectin activity, however, promotes axonal regeneration and cell transformation. We propose that As+3 binds gal-1 through sulfhydryl groups and results in oxidation of gal-1; thereafter, this oxidized gal-1 exerts its biological effect in CHOA cells. Our current results also suggest that gal-1-transfected mammalian cells uptake and accumulate more As3+. The gal-1 and gal-As3+ complexes were prepared from the cell extracts of gal-1-transfected E. coli. The circular dichroism (CD) spectrum of gal-As3+ exhibited a negative signal around 200-205 nm, whereas that of the gal-1 showed a negative signal around 205-210 nm. This shift in the CD spectrum is indicative of a substantial change in secondary structure resulted from the binding of As3+ to gal-1 proteins. Furthermore, the Isothermal titration microcalometric (ITC)studies showed that the absorption enthalpy (△Habs) of As3+ titration into gal-1 protein solution is around –55~-69 kJ/mole As3+. These adsorption enthalpy is high enough for As3+ and gal-1 to form a coordination compounds (~21 kJ/mole). The current results also showed that the gal-As3+ complexes lose partially the lectin activity and effect the cell survival of HFW cells.
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