多巴胺醌对LDHB和ENO1酶活性的影响

发布时间：2018-03-21 01:29

本文选题：多巴胺　切入点：多巴胺醌　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:帕金森病作为一种常见的的神经退行性疾病,其发病原因尚不清楚。我们认为神经元细胞内游离的多巴胺能够氧化产生多巴胺醌,多巴胺醌能够结合并修饰蛋白,使蛋白功能丧失,导致细胞死亡。本实验用多巴胺对体外纯化的LDHB和ENO1酶蛋白进行处理,来研究多巴胺氧化产生的多巴胺醌对酶蛋白活性的影响,进而探究多巴胺醌毒性与帕金森病之间的关系。方法:体外扩增LDHB和ENO1酶蛋白基因序列,将两种蛋白基因序列分别连接进入表达载体pProEX-HTb。将重组表达质粒转化入BL21表达感受态菌中,利用IPTG(Isopropy1β-D-Thiogalactoside)诱导蛋白表达。超声破碎菌体提取蛋白,利用Ni-NTA亲和层析法对两种蛋白进行纯化。加入相应的酶底物,通过吸光度变化来检测酶生物活性。在不同条件下用多巴胺对酶蛋白进行处理,之后对样品的酶活性进行测定。将经过处理的样品进行变性聚丙烯酰胺凝胶电泳,利用考马斯染色法和NBT/甘氨酸盐显色法对蛋白样品进行分析。最后通过蛋白免疫印迹实验,分析LDHB蛋白样品中的醌结合蛋白。结果:体外扩增的LDHB和ENO1蛋白基因序列测序无错配。诱导表达纯化的两种蛋白具有生物催化活性。用相同浓度的多巴胺对LDHB和ENO1蛋白处理不同时间之后,随着处理时间的增加,LDHB和ENO1蛋白酶活性的损失都增加。经过不同DA浓度处理相同时间后,随着DA处理浓度增加,两种蛋白酶活性损失增加,蛋白酶活性下降;若在处理样品中预加入谷胱甘肽(GSH),能够阻止酶活性的损失,使蛋白保持活性。酪氨酸酶催化多巴胺氧化生成多巴胺醌,但不会生成活性氧等产物,生成的多巴胺醌降低了蛋白酶催化活性,同样预加入谷胱甘肽后,蛋白活性损失消失。考马斯染色结果显示经过多巴胺处理的蛋白样品中生成多聚物,多聚物的量与处理时间以及处理浓度成正比;NBT/甘氨酸盐染色结果表明处理样品中的多聚物,是由于多巴胺醌修饰产生的蛋白积聚。而谷胱甘肽能够阻止蛋白样品中多聚物形成。最后对LDHB蛋白进行Western Blots实验,结果表明处理的样品中的多聚物是醌结合的LDHB蛋白。结论:体外表达纯化的LDHB和ENO1蛋白具有一定的生物催化活性。纯化的LDHB和ENO1蛋白经过多巴胺处理,多巴胺氧化生成的多巴胺醌能够结合并修饰蛋白,使蛋白聚集形成多聚物,降低了蛋白酶活性。随着处理时间增加,处理的多巴胺浓度增加,这种影响越严重。
[Abstract]:Objective: as a common neurodegenerative disease, the pathogenesis of Parkinson's disease is unclear. We believe that dopamine free in neurons can oxidize dopamine quinone, and dopamine quinone binds and modifies proteins. In this study, we used dopamine to treat purified LDHB and ENO1 proteins in vitro to study the effect of dopamine quinone on the activity of enzyme protein. To explore the relationship between dopamine quinone toxicity and Parkinson's disease. Methods: LDHB and ENO1 protein gene sequences were amplified in vitro. The two protein gene sequences were ligated into the expression vector pProEX-HTb.Recombinant expression plasmid was transformed into BL21 expression receptive bacteria, and protein expression was induced by IPTG(Isopropy1 尾 -D-Thiogalactoside. Two kinds of proteins were purified by Ni-NTA affinity chromatography. The enzyme bioactivity was detected by absorbance change by adding the corresponding enzyme substrate. The enzyme protein was treated with dopamine under different conditions. After that, the enzyme activity of the samples was determined. The treated samples were analyzed by denaturing polyacrylamide gel electrophoresis, and the protein samples were analyzed by Cormas staining and NBT/ glycine salt colorimetry. Analysis of quinone binding proteins in LDHB protein samples. Results: there was no mismatch in sequencing of LDHB and ENO1 gene sequences amplified in vitro. The two proteins expressed by induction and purification had biocatalytic activities. The same concentration of dopamine was used to treat LDHB and ENO1. After ENO1 protein was treated for different time, The loss of both LDHB and ENO1 protease activity increased with the increase of treatment time. After different DA concentrations for the same time, with the increase of DA concentration, the loss of the two protease activities increased and the protease activity decreased. If glutathione glutathione glutathione GSH is added in the treated sample, it can prevent the loss of enzyme activity and keep the protein active. Tyrosinase catalyzes the oxidation of dopamine to dopamine quinone, but does not produce the products such as reactive oxygen species. The resulting dopamine quinone reduced protease catalytic activity, and the loss of protein activity disappeared after the addition of glutathione. Coomas staining showed that a polymer was formed in the dopamine-treated protein sample. The results of NBT / glycine salt staining showed that the amount of polymer was proportional to the treatment time and concentration. Glutathione prevents the formation of polymers in protein samples. Finally, the LDHB protein is tested by Western Blots. The results showed that the polymers in the treated samples were quinone-bound LDHB proteins. Conclusion: the purified LDHB and ENO1 proteins have certain biocatalytic activity. The purified LDHB and ENO1 proteins are treated with dopamine. The dopamine quinone produced by dopamine oxidation can bind and modify the protein, which makes the protein aggregate to form a polymer, which reduces the protease activity. With the increase of treatment time, the concentration of dopamine increases, and the effect becomes more serious.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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