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目的 为了观察TyrphostinAG114对重组人蛋白激酶CK2全酶的直接作用及其酶动力学机理。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或 [γ 32 P]GTP的 [32 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。AG114对重组人CK2全酶具有很强的抑制作用 ,IC50 为 2 0 .8μmol·L- 1,抑制强度介于已知CK2抑制剂 5 ,6 二氯 1 β 呋喃糖苯并咪唑 (DRB)和N (2 氨乙基 ) 5 氯萘 1 硫胺 (A3)之间。AG114对重组人CK2的动力学研究表明 :它与GTP呈混合竞争性抑制作用 ,与酪蛋白呈非竞争性抑制作用。结论 AG114不仅是酪氨酸蛋白激酶的抑制剂 ,而且是一种十分有效的蛋白激酶CK2的抑制剂。重组人蛋白激酶CK2可作为一种较为简便地筛选和开发有效的CK2抑制剂的分子靶点
Objective To observe the direct effect of TyrphostinAG114 on recombinant human protein kinase CK2 holoenzyme and its enzyme kinetics. Methods Recombinant human protein kinase CK2α and β subunits were cloned, expressed and purified by genetic engineering, and the recombinant CK2 holoenzyme with maximal bioactivity was prepared by equimolar mixing in vitro. The activity of CK2 was determined under different conditions. CK2 activity was measured by measuring [& lt; 32 & gt; P] radioactivity of [Gamma32P] ATP or [Gamma32P] GTP transferred to CK2 substrate. Results Recombinant human CK2 is a second messenger-independent protein kinase such as Ca2 +, cAMP and cGMP, which is consistent with the nature of native CK2. AG114 has a strong inhibitory effect on the recombinant human CK2 holoenzyme with an IC50 of 20.8 μmol·L-1 and an inhibitory strength between 5 and 6 of the known CK2 inhibitor furanose benzimidazole (DRB) and N (2 aminoethyl) 5 chloronaphthalene 1 thiamine (A3). Kinetic studies of AG114 against recombinant human CK2 showed that it exhibited mixed and competitive inhibition with GTP and exhibited non-competitive inhibition with casein. Conclusion AG114 is not only an inhibitor of tyrosine kinase, but also a very potent inhibitor of protein kinase CK2. Recombinant human protein kinase CK2 can be used as a simple and convenient molecular target for the screening and development of an effective CK2 inhibitor