ATP含量与外源酶添加对罗非鱼肌原纤维蛋白磷酸化水平的影响

Effects of ATP content and exogenous enzyme addition on the phosphorylation level of myofibrillar protein in tilapia

蛋白质磷酸化修饰可影响肌肉品质,为探究外源酶添加对罗非鱼肌原纤维蛋白蛋白质磷酸化水平的影响,在向罗非鱼肌原纤维蛋白溶液中分别加入蛋白激酶A(protein kinase A,PKA)和碱性磷酸酶(alkaline phosphatase,AP)后进行体外孵育,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光染色测定不同时间段内的磷酸化水平变化。并通过将罗非鱼肌肉浸泡在不同浓度ATP溶液中测定肌原纤维蛋白的蛋白磷酸化水平以探究ATP对其的影响。结果显示,在0~72 h,PKA组磷酸化水平均显著高于对照组和AP组,PKA组整体磷酸化水平从0 h的0.35±0.01上升至12 h的0.37±0.01,而后下降至72 h的0.29±0.01,呈先上升后下降的趋势,其中,肌球蛋白重链磷酸化水平和肌动蛋白磷酸化水平分别从0 h的0.73±0.01、0.86±0.01下降至72 h的0.58±0.02和0.68±0.01。当孵育时间为0、4、24和48 h时,3组磷酸化水平均具有显著差异。在外源添加0.3 mol/L ATP后,结果显示肌原纤维蛋白整体磷酸化水平(0.46±0.00)显著高于对照组(0.42±0.01)。PKA可促进罗非鱼肌原纤维蛋白磷酸化修饰,AP则使其去磷酸化。研究表明,宰杀后罗非鱼肌肉中的ATP含量、PKA及AP活性水平是影响蛋白质磷酸化水平的关键因素。本研究可为探明罗非鱼品质变化机制与调控策略提供理论依据。

Protein phosphorylation modification can affect muscle quality.In order to investigate the effect of exogenous enzyme addition on the protein phosphorylation level of tilapia myofibrillar protein,tilapia myofibrillar protein solution was incubated in vitro by adding protein kinase A(PKA)and alkaline phosphatase(AP),respectively,and changes in phosphorylation level were determined by dodecyl sodium sulfate-polyacrylamide gel electrophoresis and fluorescence staining at different time intervals.In addition,the effect of adenosine triphosphate(ATP)on tilapia muscle was investigated by immersing the muscle in different concentrations of ATP solution to determine the level of myofibrillar fibrillar protein phosphorylation.The results showed that from 0 to 72 h,the phosphorylation level of PKA group was significantly higher than that of control group and AP group,and the overall phosphorylation level of PKA group increased from 0.35±0.01 at 0 h to 0.37±0.01 at 12 h,and then decreased to 0.29±0.01 at 72 h,with a general trend of increasing and then decreasing.In addition,the phosphorylation level of myosin heavy chain and actin phosphorylation level decreased from 0.73±0.01 and 0.86±0.01 at 0 h to 0.58±0.02 and 0.68±0.01 at 72 h,respectively(P<0.05).Phosphorylation levels were significantly different in all three groups when incubation time was 0,4,24 and 48 h.After addition of 0.3 mol/L exogenous ATP,the results showed that the overall phosphorylation level of myofibrillar proteins(0.46±0.001)was significantly higher than that of the control group(0.42±0.01).PKA promotes phosphorylation modification of myofibrillar proteins in tilapia,while alkaline phosphatase dephosphorylates them.It was shown that ATP content,PKA and AP activity levels in the muscle of slaughtered tilapia were key factors affecting the level of protein phosphorylation.This study may provide a theoretical basis for exploring the mechanism of quality change and regulatory strategies in tilapia.