Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20894
標題: 魚腥藻CH1穀胺醯胺合成酵素純化研究
Purification and Properties of Glutamine Syntetase from Cyanobacterium
作者: 高嘉霠
Kao, Jia-Yin
關鍵字: Anabaena
魚腥藻
glutamine synthetase
cyanobacterium
穀胺醯胺合成酵素
藍藻
出版社: 植物學系
摘要: 穀胺醯胺合成酵素(Glutamine synthetase,GS)對絲狀固氮型藍藻魚腥藻 CH1的氮代謝是不可或缺的重要酵素。本實驗以四個純化步驟可將GS部份 純化,依序為硫酸銨沈澱、diethylaminoethyl(DEAE)-Sepharose CL-6B 陰離子交換色層分析法、Sephacryl S-200 分子篩色層分析法,和Matrex Gel Red A 親和性色層分析法。經由此純化步驟所純化之GS較未純化之GS 比活性增加35倍,而回收率為19%。經由一系列的電泳分析與Ferguson plots的計算,估計魚腥藻CH1的GS的分子量約為619 kDa。其由十二個相 同的次單元所組成,每個次單元的分子量經電泳分析的結果約為53.4 kDa 。GS的活性可用三種方法測定:g-glutamyltransfer、g- glutamylsynthetic和biosynthetic assays。以此三種方法測定GS的最適 pH值依序為7.4、8.2和8.2;而最佳反應溫度依序為55、60和60℃。在g- glutamyltransfer assay中,GS活性會受L-methionine-DL-sulfoximine 的抑制。以g-glutamylsynthetic與biosynthetic assays分別測得對ATP 與glutamate的apparent Michaelis常數(Km)為 0.43 / 4.52 mM 和 9.09 / 20 mM。而以g-glutamyltransfer assay分別為ADP、glutamine和 NH2OH所測得的apparent Km依序為0.03、20.83和5.26 mM;以g- glutamylsynthetic assay為NH2OH所測得的apparent Km是0.11 mM。對 NH4Cl的Km以Biosynthetic assay測定發現有兩個數值,當反應基質中NH4 Cl低於和高於7.5 mM時,其Km分別為0.8和54.64 mM。
Glutamine synthetase (GS) plays a key role in nitrogen assimilation in filamentous N2-fixing cyanobacterium Anabaena CH1. The enzyme was purified by four-step procedure involving ammonium-sulfate fractionation , diethylaminoethyl (DEAE)- Sepharose CL-6B chromatography , Sephacryl S-200 chromatography , and affinity chromatography on Matrex Gel Red A. This purification scheme resulted in a 35-fold and 19%recovery of the initial specific and total activities.The native GS had a approximate molecular mass of 619 kDa as estimated by non- denaturing PAGE, and interpolation of the Ferguson plots. It was composed of tweleve identical subunits of molecular mass 53.4 kDa.The pH optimum values were 7.4, 8.2 and 8.2 for the g- glutamyltransfer, g-glutamylsynthetic and biosynthetic assays. The temperature optimum of activity were 55, 60 and 60℃for the g-glutamyltransfer, g-glutamylsynthetic and biosynthetic activities, respectively. g-glutamyltransfer activity was inhibited by L-methionine-DL-sulfoximine.Apparent Michaelis constants (Km) for ATP and glutamate were 0.43 and 9.09 mM in g- glutamylsynthetic assay, and 4.52 and 20 mM in biosynthetic assay. That for ADP, glutamine and NH2OH were 0.03, 20.83 and 5.26 mM in g-glutamyltransfer assay, 0.11 mM for NH2OH in g- glutamylsynthetic assay. The GS of Anabaena CH1 exhibited two Km values which were 0.8 and 54.64 mM in the presence of ammonium concentrations lower and higher than 7.5 mM.
URI: http://hdl.handle.net/11455/20894
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