【作者】 相丽；

【导师】 葛志强；

【作者基本信息】 天津大学 ， 生物化工， 2007， 硕士

【摘要】 在悬浮培养的东北红豆杉细胞中,Ce⁴⁺作为诱导子促进信号分子活性氧迸发从而增加紫杉醇的产量。为了探讨Ce⁴⁺作为诱导子影响细胞氧化还原酶类的结构和功能进而调控活性氧迸发的机制,本文以Ce⁴⁺和Ce³⁺作用下辣根过氧化物酶的构象改变为切入点,综合运用多种检测手段在分子水平上进行了较全面的研究,取得了以下结果:（1） Ce⁴⁺较Ce³⁺更有效的改变了辣根过氧化物酶的酶促反应动力学参数,酶的活力、最大反应速率以及酶与底物的亲和力都随着Ce⁴⁺和Ce³⁺浓度的增加而逐渐降低。（2）应用紫外分光光度计、荧光分光光度计以及电子顺磁共振仪对Ce⁴⁺和Ce³⁺诱导下的辣根过氧化物酶的三级结构进行了研究,经Ce⁴⁺和Ce³⁺处理后辣根过氧化物酶与其天然状态下的光谱特征不同,且这种改变随Ce⁴⁺和Ce³⁺浓度的增加而更加明显。虽然Ce⁴⁺和Ce³⁺所引起的辣根过氧化物酶的紫外特征吸收峰变化趋势相同,但是Ce⁴⁺引起的特征峰位移和强度的改变都强于Ce³⁺;并且Ce⁴⁺和Ce³⁺引起的色氨酸荧光发射峰变化趋势相反,g因子的值也有明显的差别。（3）通过圆二色和傅立叶红外光谱拟和数据发现,Ce⁴⁺和Ce³⁺使得辣根过氧化物酶的二级结构的相对百分含量发生改变,平衡从α螺旋向β转角和β折叠移动。这种二级结构间的转化对于Ce⁴⁺和Ce³⁺具有浓度依赖性;并且相同条件下Ce⁴⁺引起的转化程度远大于Ce³⁺。Ce⁴⁺和Ce³⁺对于辣根过氧化物酶的二级结构和三级结构所造成的影响不同可能与它们的离子半径、得失电子能力、电荷排布的不同有关。Ce⁴⁺作为诱导子促进活性氧的迸发可能与其对过氧化物酶的二级结构和三级结构的影响有关。更多还原

【Abstract】 Our previous studies have demonstrated that Ce⁴⁺ could induce ROS （reactive oxygen species） burst as a signal to promote pacilitaxel biosynthesis in suspension cultured Taxus cuspidate cells. To further understand the mechanism of effects of cerium ion on the ROS burst, we carefully studied the changes of the conformation of HRP treated by cerium ions, and we gained some results below:1. Ce⁴⁺ is much more efficient in reduced the value of Vmax, Km, and HRP activity than Ce³⁺. The extent of the reduced value increases accompanied by the increase of the concentration of cerium ions.2. This thesis used synchronous fluorescence spectrum, uv-visible spectrum and electron paramagnetic resonance （EPR） to detect the change of HRP tertiary structure. The change of florescence spectrum caused by Ce⁴⁺ and Ce³⁺ is opposite; although the current of uv-visible spectrum changes caused by cerium ions is similar, the changed caused by Ce⁴⁺ is still much evident than that caused by Ce³⁺; furthermore, g factor （gx and gy） in EPR induced by Ce⁴⁺ and Ce³⁺ was significantly different.3. Circular dichroism （CD） and infrared spectrum are used to compute the content of secondary structure. The percentage of transition from helical content and other structure toβstrands andβturns caused by Ce⁴⁺ is more than Ce³⁺. These results indicate that the different valence of cerium ion induces various changes of HRP conformation, and Ce⁴⁺ is more effective than Ce³⁺. This suggests that Ce⁴⁺ affects the burst of ROS through changing the conformation of redoxases.更多还原