蛋白质磷酸化调控宰后肌肉糖原磷酸化酶同工酶活性机理

Glycogen phosphorylase regulates meat quality through glycolysis in early postmortem. Glycogen phosphorylase turns into glycogen phosphorylase A by phosphorylation and then startup glycolysis according to the classic theory. However, our recent research show that there are many glycogen phosphorylase isoenzymes, whose activity are regulated by phosphorylation, but the regulation mechanism is still unknown. In this project, activity characteristics of glycogen phosphorylase isoenzymes modified by phosphorylation will be identified in the postmortem situ model using muscle samples with different glycolysis levels. The phosphorylation sites of glycogen phosphorylase isoenzymes and their effects on activity during postmortem will be studied. Furthermore, the in situ and in vitro model will be established to explore the influence of protein kinase and alkaline phosphatase on the phosphorylation and activity of glycogen phosphorylase isoenzymes and to analyze the glycogen phosphorylase isoenzymes’ activity by regulating the content of catalytic substrates, metabolites and phosphate group donators. Glycogen phosphorylase isoenzymes with significantly different phosphorylation will be purified to analyze the fundamental differences of alpha-spiral, beta sheet and intermolecular force. The aim of this project is to reveal mechanism of glycogen phosphorylase isoforms' activity regulated by phosphorylation and improve current theory on muscle quality regulated by glycolysis.

糖原磷酸化酶通过调控畜禽宰后早期糖酵解影响肉品品质，经典的糖原磷酸化酶调控理论认为糖原磷酸化酶磷酸化修饰后形成糖原磷酸化酶A启动糖酵解，本项目组近期研究发现糖原磷酸化酶存在多种同工酶，其活性受磷酸化调控，但具体机理尚不清楚。本项目以糖酵解差异显著肌肉为试验材料，通过宰后肌肉原位模型鉴定不同磷酸化水平糖原磷酸化酶同工酶活性消长规律；确证宰后肌肉糖酵解过程糖原磷酸化酶同工酶磷酸化位点及其对活性作用；构建原位模型和离体模型，确证蛋白激酶、碱性磷酸酶对糖原磷酸化酶同工酶磷酸化和酶活的调控作用；调节催化底物、代谢产物和磷酸化基团供体浓度，分析糖原磷酸化酶同工酶活性变化；纯化不同磷酸化水平的糖原磷酸化酶同工酶，解析磷酸化修饰对同工酶α-螺旋、β-折叠、分子间作用力的影响，揭示磷酸化调控宰后肌肉糖原磷酸化酶同工酶活性机理，丰富和发展糖酵解调控肉品质理论。

糖原磷酸化酶通过调控畜禽宰后早期糖酵解影响肉品品质，经典的糖原磷酸化酶调控理论认为糖原磷酸化酶磷酸化修饰后形成糖原磷酸化酶A启动糖酵解，本项目组研究发现糖原磷酸化酶存在多种同工酶，其活性受磷酸化调控，但具体机理尚不清楚。本项目采用磷酸化蛋白质组学、磷酸化蛋白质免疫印迹、Phos-tag磷酸化标签、分子建模等技术，明确了糖酵解速率差异肉中糖原磷酸化酶磷酸化水平与酶活性的关系，确证了ATP对糖原磷酸化酶磷酸化水平和酶活性的调节作用，阐明了磷酸化修饰对糖原磷酸化酶空间结构的影响。研究结果表明，磷酸化修饰正向调节宰后肉中糖原磷酸化酶活性，ATP通过促进糖原磷酸化酶的磷酸化反应提高酶活性，同时直接抑制酶活性，磷酸化/去磷酸化修饰通过改变糖原磷酸化酶别构效应物结合部位和糖原结合域等结构域的结构而调节酶活性。