目的观察槲皮素对重组人蛋白激酶CK2全酶的直接作用及其酶动力学机制.方法利用基因工程克隆、表达和纯化获得重组人蛋白激酶CK2α和β亚基,在体外等摩尔数混合构成有最大生物活性的重组CK2全酶,在不同条件下测定CK2的活性.CK2活性通过测定转移到CK2底物上的[γ-32P]ATP的[32P]放射活度来检测.结果重组人蛋白激酶CK2是一种Ca2+、cAMP和cGMP等第二信使非依赖性蛋白激酶,与天然CK2的性质一致.槲皮素对重组人CK2全酶具有很强的抑制作用,IC50为522 nmol/L,抑制作用大于CK2已知抑制剂DRB和A3.槲皮素对重组人蛋白激酶CK2的动力学研究表明:它与ATP和酪蛋白分别呈竞争性和非竞争性抑制作用.结论槲皮素是CK2有效抑制剂,该抑制作用可能是槲皮素抗肿瘤作用又一分子机制,为今后筛选更有效的CK2抑制剂提供了一种较为简便的筛选方法.

ObjectTo study the direct effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics. MethodsRecombinant human protein kinase CK2α and β subunits were cloned and expressed by gene engineering,and were purified.The two subunits were mixed at the same molarratio, thus reconstituting CK2 holoenzyme, which displayed the maximum bioactivity.The CK2 activity was assayed by detecting incorporation of 32 P of [γ-32 P] ATP into the substrate in the various condi-tions. ResultsThe recombinant human protein kinase CK2 was the second messenger ( Ca2+, cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme wereconsistent with those of native CK2. It was found that quercetin strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 522 nmol /L, which was much more effective than DRB and A3, known as CK2 special inhibitors. Kinetic studies of quercetinon recombinant human protein kinase CK2 showed:the inhibition was competitive with ATP and noncompetitive with casein.Conclusion Quercetinis a potent inhibitor of recombinant human protein kinase CK2. The inhibition may be another molecular mechanism of antitumo reffect of quercetin. This study provides a simple and rapidscreening method for the development of more effective inhibitors of recombinant human protein kinase CK2.

广东省自然科学基金(011766);广东医学院标志性成果扶持项目