AG372对重组人蛋白激酶CK2全酶的抑制动力学研究

Study on inhibition kinetics of AG372 on recombinant human protein kinase CK2 holoenzyme

目的 观察Tyrphostin中的AG372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 3 2 P]ATP或 [γ 3 2 P]GTP的 [3 2 P]放射活度来检测。结果 重组人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。AG372对重组人CK2全酶具有很强的抑制作用 ,IC50 为 6 2 7.1nmol·L-1,抑制作用远大于已知CK2抑制剂DRB和A3。AG372对重组人CK2的动力学研究表明 :它与GTP呈竞争性抑制作用 ,与酪蛋白呈非竞争性抑制作用。结论 AG372是一种很强的重组人CK2抑制剂。重组人蛋白激酶CK2可作为一种较为简便筛选和开发有效的CK2抑制剂的分子靶点。

OBJECTIVE: To study the direct effect of tyrphostin AG372 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS: The recombinant human protein kinase CK2 α and β subunits were cloned and expressed by gene engineering, and purified to homogeneous. The two subunits were mixed at the same molar ratio and reconstituted CK2 holoenzyme, which displayed the maximum biological activity. The CK2 activity was assayed by detecting the radio-activity 32P after [γ-32P]ATP or [γ-32P]GTP combined with the substrates in various conditions. RESULTS: The recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase. The characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG372 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 627.1 nmol&middotL-1, which was more effective than DRB and A3, known CK2 special inhibitors. The inhibition of AG372 on recombinant human CK2 was competitive with GTP and noncompetitive with casein. CONCLUSIONS: AG372 appeared to be an effective inhibitor of recombinant human protein kinase CK2 holoenzyme. The recombinant human protein kinase CK2 may be used as a molecular target for simple screening and development of more effective inhibitors of CK2.