温度波动通过调控磷酸化和亚硝基化对磷酸丙糖异构酶活性的影响  

Temperature Fluctuations Affected the Activity of Triosephosphate Isomerase by Regulating Phosphorylation and Nitrosylation

作　　者：吴赛赛 王振宇[1] 摆玉蔷 侯成立[1] 饶伟丽 李欣[1] 张志胜[2] 张德权[1,2] WU Saisai;WANG Zhenyu;BAI Yuqiang;HOU Chengli;RAO Weili;LI Xin;ZHANG Zhisheng;ZHANG Dequan(Key Laboratory of Agro-products Quality&Safety in Harvest,Storage,Transportation,Management and Control,Ministry of Agriculture and Rural Affairs,Institute of Food Science and Technology,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]中国农业科学院农产品加工研究所,农业农村部农产品质量安全收贮运管控重点实验室,北京100193 [2]河北农业大学食品科技学院,河北保定071001

出　　处：《食品科学》2025年第9期139-147,共9页Food Science

基　　金：国家自然科学基金面上项目(32072245)。

摘　　要：本研究通过外源添加蛋白激酶A和S-亚硝基谷胱甘肽,分析磷酸化和亚硝基化影响磷酸丙糖异构酶活性随时间变化的规律。通过构建不同温度条件下磷酸丙糖异构酶磷酸化和亚硝基化修饰高表达模型,探究磷酸化和亚硝基化在调节磷酸丙糖异构酶活性中的作用。结果表明,4℃体外条件下,对照组样品在孵育6 h的磷酸丙糖异构酶活性、磷酸化水平和亚硝基化水平显著低于恒温+修饰组,磷酸化和亚硝基化共同作用提高了磷酸丙糖异构酶活性。与恒温+修饰组相比,幅度波动+修饰组磷酸化对酶活性的作用减弱,频率波动+修饰组亚硝基化对酶活性的作用增强。幅度波动+修饰组和频率波动+修饰组样品孵育6 h的β-折叠相对含量显著高于恒温+修饰组,无规卷曲相对含量显著低于恒温+修饰组。温度波动延缓磷酸丙糖异构酶结构的解聚。利用原子力显微镜观察发现,3个处理组孵育12 h的磷酸丙糖异构酶形态小于0 h,温度波动不改变磷酸丙糖异构酶的最终解聚形态。综上,在4℃体外孵育条件下,磷酸化水平和亚硝基化水平的提高延缓了磷酸丙糖异构酶的活性下降,温度幅度或频率波动影响磷酸化和亚硝基化对酶活性的调节作用,使其在孵育早期的活性提高,抑制其结构从有序向无序转变。In this study,triosephosphate isomerase was incubated in the presence of exogenous protein kinase A and S-nitrosoglutathione to elucidate the regulatory effects of phosphorylation and nitrosylation on the enzyme’s activity as a function of incubation time.High expression models of triosephosphate isomerase modification by phosphorylation and nitrosylation were constructed under different temperature conditions.The results showed that after 6 h incubation at 4℃,the triosephosphate isomerase activity,phosphorylation and nitrosylation levels of the control group were significantly lower than those of the constant temperature+modification group,suggesting that phosphorylation and nitrosylation worked together to improve the activity of triosephosphate isomerase.Compared with the constant temperature+modification group,the effect of phosphorylation on the activity of triosephosphate isomerase was weaker in the amplitude fluctuation+modification group,but stronger in the frequency fluctuation+modification group.After 6 h incubation,the relative content ofβ-sheet was significantly higher and that of random coil was significantly lower in both fluctuating temperature+modification groups than in the constant temperature+modification group.Atomic force microscopic(AFM)observation found that for all three treatment groups,the dimension of triosephosphate isomerase was smaller at 12 h than at 0 h,but no difference was observed among the three treatment groups.In conclusion,the increase in phosphorylation and nitrosylation levels slowed down the decline in the activity of triosephosphate isomerase in vitro at 4℃,and amplitude or frequency fluctuations of incubation temperature affected the regulatory effect of phosphorylation and nitrosylation on triosephosphate isomerase activity,leading to increased activity in the early stages of incubation and inhibiting the structural transition from order to disorder.

关 键 词：磷酸丙糖异构酶 温度波动 活性 磷酸化 亚硝基化 

分 类 号：TS251.1[轻工技术与工程—农产品加工及贮藏工程]