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目的 为了筛选蛋白激酶CK2的抑制剂,观察山萘黄素对重组人蛋白激酶CK2的抑制效果及其酶动力学机制。方法 利用基因工程技术进行克隆、表达和纯化,获得重组人CK2的α′及β亚基在体外等摩尔混合构成CK2全酶,通过测定转移到CK2底物上的[γ32 P]ATP的32 P的放射性活性来检测CK2的活性。向反应体系中加入不同浓度的山萘黄素,观察其对CK2的抑制效果;通过固定酪蛋白浓度为2g·L- 1 ,ATP浓度为1 0 ,2 0 ,4 0和80 μmol·L- 1 或固定ATP的浓度为1 0 μmol·L- 1 ,改变酪蛋白浓度( 1 ,2 ,4和8g·L- 1 ) ,观察其酶动力学机制。结果 山萘黄素能显著抑制重组人蛋白激酶CK2的活性(IC50 =1 .9μmol·L- 1 )。抑制作用强于已知的CK2抑制剂白杨素、桑色素和金雀异黄素。酶动力学分析表明,山萘黄素与ATP(Ki=1 .1 μmol·L- 1 )及酪蛋白(Ki=3 .1 μmol·L- 1 )均呈非竞争性抑制CK2的活性。初步的化合物结构分析表明,2′和3位上的羟基对山萘黄素及芹黄素发挥其抑制效果产生实质性的负面影响。结论 山萘黄素是一种新的体外蛋白激酶CK2的有效抑制剂。黄酮类CK2的抑制剂可能通过不同的位点作用于CK2 ,这种作用主要取决于其羟基的数目和位置
Objective To screen inhibitors of protein kinase CK2 and observe the inhibitory effect of kaempferol on recombinant human protein kinase CK2 and its enzymatic kinetic mechanism. Methods Cloning, expression and purification were performed using genetic engineering techniques. The α’ and β subunits of recombinant human CK2 were obtained by equimolar mixing in vitro to form CK2 holoenzyme, and 32 P of [γ32 P] ATP transferred to CK2 substrate was determined. The radioactive activity was used to detect CK2 activity. Different concentrations of kaempferol were added to the reaction system to observe its inhibitory effect on CK2; the concentration of immobilized casein was 2 g·L-1, and the ATP concentration was 10, 20, 40, and 80 μmol·L-. 1 or fixed ATP concentration of 1 0 μmol·L - 1 , changing the casein concentration (1, 2, 4 and 8g·L - 1 ) and observing the mechanism of enzyme kinetics. Results Kaempferol can significantly inhibit the activity of recombinant human protein kinase CK2 (IC50 =1.9 μmol·L-1). The inhibitory effect is stronger than known CK2 inhibitors such as chrysin, mulberry and genistein. Enzymatic kinetic analysis showed that kaempferol with ATP (Ki=1.1 μmol·L-1) and casein (Ki=3.1 μmol·L-1) exhibited non-competitive inhibition of CK2 activity. Preliminary structural analysis of the compounds showed that the hydroxyl groups at the 2’ and 3 positions had a substantial negative effect on the inhibitory effects of kaempferol and apigenin. Conclusion Kaempferol is a new potent inhibitor of protein kinase CK2 in vitro. Inhibitors of flavonoids CK2 may act on CK2 through different sites, depending on the number and location of their hydroxyl groups.