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背景与目的:蛋白激酶CK2是一种高度进化保守性的丝/苏氨酸激酶,它的活性在人的多种癌症是上调的。CK2可作为一种癌基因起作用,它的靶向抑制可用于肿瘤的治疗。为了寻找特异性CK2抑制剂,本文观察Tyrphostin中的AG1109对重组人蛋白激酶CK2全酶的直接作用,以探讨其酶动力学机制。方法:利用基因工程克隆、表达和纯化而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底物上的犤γ-32P犦ATP或犤γ-32P犦GTP的犤32P犦放射活度来检测。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。AG1109对重组人CK2全酶具有很强的抑制作用,其IC50为9.7μmol/L,抑制作用稍大于已知的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)。AG1109对重组人CK2的动力学研究表明,它与GTP呈以竞争性为主的混合型抑制作用,与酪蛋白呈非竞争性抑制作用。结论:AG1109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地筛选和开发有效的CK2抑制剂的分子靶点。
BACKGROUND & OBJECTIVE: Protein kinase CK2 is a highly evolutionarily conserved serine/threonine kinase whose activity is up-regulated in many human cancers. CK2 can act as an oncogene and its targeted inhibition can be used for the treatment of tumors. In order to search for specific CK2 inhibitors, the direct action of AG1109 in Tyrphostin on the recombinant human protein kinase CK2 holoenzyme was investigated to investigate its enzymatic kinetic mechanism. Methods: Recombinant human protein kinase CK2α and β subunits were cloned, expressed and purified by genetic engineering. They were mixed in equal moles in vitro to construct recombinant CK2 holoenzyme with maximal biological activity. CK2 activity was determined under different conditions. CK2 activity was detected by measuring the 犤32P犦 radioactivity of 犤γ-32P犦ATP or 犤γ-32P犦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 natural CK2. AG1109 has a strong inhibitory effect on recombinant human CK2 holoenzyme, with an IC50 of 9.7 μmol/L, slightly more inhibitory than the known CK2 inhibitor 5,6-dichloro-1-β-furanose benzimidazole (DRB). ) and N-(2-aminoethyl)-5-chloronaphthalene-1-thiamine (A3). The kinetic study of recombinant human CK2 by AG1109 showed that it is a competitive inhibitor of GTP and non-competitive with casein. Conclusion: AG1109 is a very potent recombinant human CK2 inhibitor. Recombinant human protein kinase CK2 can be used as a molecular target for screening and developing effective CK2 inhibitors.