GS,GOGAT,GDH等酶活性测定最终版.doc

提取方法2：Zhang CF, Peng SB, Peng XX, Chavez AQ,Bennett J. Response of glutamine synthetase isoforms to nitrogen sources in rice Oryza Sativa.L roots.Plant Sci,1997,125:163-170Rhodes D, Rendo GA, Stewart GR. The control of glutamine synthetase level in Lemna minor L. Planta, 1975, 125:201-211The reaction mixture (3.0 ml) consistedof sodium ketoglutarate (10_mol), ammonium chloride(10_mol), calcium chloride (10_mol), NADH (2_mol), TrisHClbuffer (100_mol, pH 8.2) and enzyme 0.1 ml.Following a 30 min incubation period at 30C in 20mol MgSO4, 80mol -ketoglutarate, 6mol hydroxylamine, 8mol ATP and enzyme extract, the enzymatic reaction was terminated by the addition of 1.5 ml of a solution containing 0.37mol/l FeCl3, 0.2 mol/l TCA and 0.37 mol/l HCl and the amount of glutamyl hydroxamate determined at 540 nm.Lea P J, Blackwell R D, Chen F L. Enzymes of primary metabolism. In: Harborne J B. Methods in Plant Biochemistry. Vol.3. New York: Academic Press, 1990. pp 260-273the reaction mixture (3 ml) consisted of 5mol -ketoglutarate, 1mol potassium chloride, 48.75mol TrisHCl buffer (pH 7.6), 0.6mol NADH, 0.3 ml enzyme and 8mol L-glutamine300mol TrisHCl buffer (pH 8.0), 600mol ammonium chloride, 3mol calcium chloride, 0.6mol NADH, and 0.1 ml enzyme. The change of absorption value was detected at 340 nm in 3 min,Singh RD, Srivastava H S. Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen. Physiol. Plant, 1986, 66:413-416Loulakakis KA, Roubelakis-Angelakis KA. Intracellular localization and properties of NADHglutamate dehydrogenase form Vitis vinifera L.: Purification and characterization of the major leaf isoenzyme. Journal of Experimental Botany, 1990, 41:1223-1230EP酶活性的测定高玲, 叶茂炳 张荣铣“ 徐朗莱, 小麦旗叶老化期间的内肤酶植物生理学报,1998,24（2）183-188硝酸还原酶活性的测定：仪器：离心机；分光光度计；冰箱；研钵；试管等试剂：亚硝酸钠；Na2HPO412H2O；NaH2PO42H2O；磺胺；浓盐酸；萘基乙烯胺；KNO3；K2HPO43H2O；半胱氨酸；EDTA；NADH（辅酶）李合生主编，植物生理生化实验原理和技术，高等教育出版社，2000,125-127Li H-S(李合生). Experimental Principle and Technique for Plant Physiology and Biochemistry (植物生理生化实验原理和技术).Beijing: Higher Education Press, 2000. pp 125-127 (in Chinese)关于您问的“磷酸缓冲液里该加多少EDTA和半胱氨酸，均没有说明”在此书的P126页写的很全面，步骤也非常详细，请查阅， 如：提取缓冲液。0.1211g半胱氨酸、0.0372g EDTA 溶于100ml0.025mol/L pH8.7的磷酸缓冲液中。另外我们做此实验用的去离子水。孙老师：您好！首先非常感谢您的帮助！前一段时间认真的研究了您给我发的关于氮代谢关键酶测定的方法。其中尚有一些不太明白的地方，恳请您给予解读。 1、您给我了两种GS合成酶活性的测定方法，是不是这两种方法都可以测定？ 2、方法一（Lea，1990）相对简单，但不知“-谷氨酰基异羟肟酸”的标准曲线如何制作？ 再次感谢您的帮助。 祝好 罗宏海罗老师： 您好！这几天我有事没有及时看邮件，关于您提出的问题现解释一下，方法1和方法2我军做过比较试验，结果有一点差异，只要你选择一种方法即可，此外只是方法1做实验过程中可以组合配成混合液，原理一样的，即谷氨