摘要
用活性氧Ｈ２Ｏ２、Ｏ－·２和ＯＨ·处理苋菜部分纯化的ＰＥＰ羧化酶（ＰＥＰＣ）１５ｍｉｎ后，活性降低１０％～１７％，抑制程度与活性氧种类及其浓度有关。ＯＨ·的抑制效应较强，低浓度的Ｈ２Ｏ２和Ｏ－·２不影响ＰＥＰＣ活性。受Ｈ２Ｏ２和ＯＨ·作用后凝胶电泳谱上酶蛋白量减少，ＯＨ·引起酶蛋白明显降解。Ｈ２Ｏ２处理使酶活性染色带的面积减少２１％，并由单条带变成有３个未完全分离的活性带。３种活性氧与ＰＥＰＣ作用诱导产生不同的紫外吸收差示光谱：Ｈ２Ｏ２诱导形成２３０～２３２ｎｍ的负差示峰，ＯＨ·引起一个２５７ｎｍ的负差示峰，Ｏ－·２则诱导正差示峰，其峰位与峰值因Ｏ－·２浓度不同而异。ＯＨ·的清除剂ＤＭＳＯ可提高ＰＥＰＣ活性２９％～４８％，并相应影响紫外差光谱峰。结果表明氧化胁迫导致ＰＥＰＣ构象变化，酶蛋白氧化断裂或降解，活性下降。
Phosphoenolpyruvate carboxylase(PEPC) was partially purified from leaves of Amaranthus tricolor , then treated with active oxygen species such as H 2O 2, O -· 2 and OH· for 15 min. Under oxidative condition, PEPC activity was inhibited by 10%～17% depending on the species and concentration of active oxygen. Significant inhibition by OH· and higher concentrations of H 2O 2 and O -· 2 was found(Fig.2). The band intensity of PEPC protein on the electrophoretogram was reduced upon treatment with H 2O 2 and OH·, and there was obvious degradation of PEPC through OH· oxidation. Specific enzyme activity staining of PEPC on electrophoretic gel showed that the activity area was decreased to 79% of the control(Fig.3). Changes in ultraviolet absorption difference spectra of amaranth PEPC indicated that the conformation of PEPC was altered by the three active oxygen species tested(Figs. 5,6). H 2O 2 induced a negative differential peak at 230 nm or 232 nm. OH· induced a strong negative differential peak at 257 nm, whereas O -· 2 induced one or two positive peaks, the location or height of which varied with the O -· 2 concentration. DMSO, a hydroxyl radical scavenger, stimulated the PEPC activity by 29%～48%(Figs. 1,2), and altered the spectra of ultraviolet absorption and differential absorption spectra(Figs. 6,7). These results imply that oxidative stress leads to the modification of structure and conformation of PEPC and a decrease in activity.
基金
国家自然科学基金!(39570071)
广东省自然科学基金!(950714) 资助项目
关键词
活性氧
PEP羧化酶活性
苋菜
紫外差光谱
active oxygen, activity of phosphoenolpyruvate carboxylase, Amaranthus tricolor , ultraviolet difference spectra
