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摘要: 甘油醛-3-磷酸脱氢酶(GAPDH)是糖酵解中的一个关键酶，在NAD+/NADP+作为辅助因子的作用下，催化甘油醛-3-磷酸形成1,3-二磷酸甘油酸。本实验首先构建了GAPDH的表达载体，并且进行了大批量的表达纯化，然后通过紫外分光光度计测定水稻中甘油醛-3-磷酸脱氢酶在不同处理条件下的酶活性，试验结果表明，当以NAD+/NADP+作为辅助因子时，吸光度平均值分别为0.417084、0.456855，表明在没有任何处理时水稻中GAPDH对辅助因子NADP+具有偏好性，与不经过任何处理相比，经过氧化性的H2O2处理1 h后，酶活性最低，吸光度平均值分别为0.360832、0.330731，经过氧化性的H2O2处理1 h后再经过还原性的DTT或β-ME处理后，酶活性均升高；其中以NAD+作为辅助因子时经过H2O2处理1 h后，然后再用还原剂DTT处理1h，酶的活性恢复的最好，吸光度平均值为0.469006，以NADP+作为辅助因子时经过H2O2处理1 h后，然后再用还原剂β-ME处理1h，酶的活性恢复的最好，吸光度平均值为0.449296，说明H2O2处理1h后，水稻中GAPDH以NAD+作为辅助因子对酶的活性恢复的较好；另外还发现处于还原状态下的甘油醛-3-磷酸脱氢酶具有较高的酶活性。61981
毕业论文关键词：水稻；甘油醛-3-磷酸脱氢酶；NAD+/NADP+；酶活性测定；吸光度
Study the Enzyme Activity of Glyceraldehyde-3-Phosphate Dehydrogenase in the Oryza sativa
Abstract: glyceraldehyde-3-phosphate dehydrogenase(GAPDH), the enzyme involved in the ubiquitous glycolysis, catalyze the oxidative phosphorylation of glyceraldehyde-3-phosphate(G3P) to 1,3-biphosphoglycerate(BGP) using nicotinamide adenine dinucleotide(NAD+)/nicotinamide adenine dinucleotide phosphate(NADP+) as an electron acceptor. In this study, we built the expression vector of GAPDH firstly, and expressed and purified the protein in large quantities to get GAPDH, and then GAPDH enzyme activity in Oryza sativa was tested at different treatment using ultraviolet spectrophotometer, The results showed that the enzyme activity of GAPDH using nicotinamide adenine dinucleotide(NAD+)/nicotinamide adenine dinucleotide phosphate(NADP+) as an electron acceptor with absorbance mean value of 0.417084, 0.456855, GAPDH had a preference for NADP+ without any treatment. GAPDH enzyme activity compared with mock decreased when it was treated with H2O2 for 1 h with absorbance mean value of 0.360832, 0.330731, However, GAPDH enzyme activity increased when it was treated with H2O2 for 1 h and then treated with DTT or β-ME, The highest enzyme activity of GAPDH using NAD+ as an electron acceptor was measured with the absorbance mean value of 0.469006 after it was treated with H2O2 for 1 h, and then treated with DTT 1h and the highest enzyme activity of GAPDH using NADP+ as an electron acceptor was measured with the absorbance mean value of 0.449296 after it was treated with H2O2 for 1 h, and then treated with β-ME 1h, GAPDH had a preference for NAD+ when it was treated with H2O2. Additionally, GAPDH had a good enzyme activity at the condition of reductive state.
Keywords: Oryza sativa; glyceraldehyde-3-phosphate dehydrogenase; NAD+/NADP+; enzyme activity assay; absorbance
摘要 i
Abstract i
目录 iii
1 绪论 1
1.1 甘油醛-3-磷酸脱氢酶的简介 1
1.2 甘油醛-3-磷酸脱氢酶的结构特点 1
1.3 甘油醛-3-磷酸脱氢酶的功能 2
1.3.1 糖酵解酶活性 3
1.3.2 细胞内膜成分的转运 3
1.3.3 DNA完整性的维持以及DNA端粒的保护