摘要
目的 观察Tyrphostin中的AG1112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。AG1112对重组人CK2全酶具有很强的抑制作用 ,IC50 为 6 0 μmol·L-1,抑制作用大于CK2已知抑制剂DRB和A3。AG1112对重组人CK2的动力学研究表明 :它与GTP和酪蛋白分别呈混合性和非竞争性抑制作用。结论 由于AG1112对重组人CK2显示出很强的抑制作用 。
AIM To study the direct effect of tyrphostin AG1112 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 α and βsubunits 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 incorporation of 32 P of [γ 32 P]ATP or [γ 32 P]GTP into the substrate in the various conditions. RESULTS The recombinant human CK2 was a 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. AG1112 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 6 0 μmol·L -1 , which was more effective than DRB and A3, known CK2 special inhibitors. Kinetic studies of AG1112 on recombinant human CK2 showed that the inhibition was mixed(competitive is dominant) with GTP and noncompetitive with casein. CONCLUSION The recombinant human protein kinase CK2 may be used as a molecular target for simpler screening and development of more effective inhibitors of CK2 since AG1112 showed effective in this recombinant CK2 model.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2001年第6期657-660,共4页
Chinese Pharmacological Bulletin
基金
广东省重点学科基金 ( 2 0 0 0 1)
广东省卫生厅科研基金 (A2 0 0 0 487)
广东医学院标志性成果扶持项目 (XK0 0 0 2 )
