摘要
目的 :观察TyrphostinAG34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。AG34对重组人CK2全酶具有很强的抑制作用 ,IC50 为 1.0 μmol·L 1,抑制作用远大于CK2已知抑制剂 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)。AG34对重组人CK2的动力学研究表明 :它与GTP呈现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 :AG34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 AG34 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHOD: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 to reconstitute CK2 holoenzyme which had 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. AG34 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 1.0μmol·L 1 , which was more effective than 5,6 dichloro 1 β D ribofuranosylbenzimidazole(DRB) and N (2 aminoethyl) 5 chloronaphthalene 1 sulfonamide (A3), the known CK2 special inhibitors. Kinetic studies of AG34 on recombinant human CK2 showed that the inhibition was mixed (competitive is dominant) with GTP and noncompetitive with casein.CONCLUSION:AG34 is not only an effective inhibitor of protein tyrosine kinases, but also a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 may be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.
出处
《中国现代应用药学》
CAS
CSCD
北大核心
2002年第3期185-188,共4页
Chinese Journal of Modern Applied Pharmacy
基金
广东省自然科学基金 ( 0 1176 6 )
广东省卫生厅科研基金 (A2 0 0 0 487)
湛江市科委科技计划项目资助
