槲皮素对重组人蛋白激酶CK2全酶的抑制作用及其动力学分析

Inhibitory effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics analysis

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- 32 P]ATP的 [32 P]放射活度来检测。结果 重组人蛋白激酶 CK2是一种 Ca2 +、c AMP和 c GMP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 5 2 2 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

Object To study the direct effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics. Methods Recombinant 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 molar ratio, 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 conditions. Results The recombinant human protein kinase CK2 was the second messenger (Ca 2+ , cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. It was found that quercetin strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 522 nmol/L, which was much more effective than DRB and A3, known as CK2 special inhibitors. Kinetic studies of quercetin on recombinant human protein kinase CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. Conclusion Quercetin is a potent inhibitor of recombinant human protein kinase CK2. The inhibition may be another molecular mechanism of antitumor effect of quercetin. This study provides a simple and rapid screening method for the development of more effective inhibitors of recombinant human protein kinase CK2.