氨氯吡咪对重组人蛋白激酶CK2全酶的抑制动力学

目的　了解氨氯吡咪对重组人蛋白激酶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 ,在不同条件下通过测定转移到CK2底物上的 γ 3 2 P]GTP的 3 2 P]放射活度来测定CK2的活性。结果　氨氯吡咪对重组人CK2全酶具有抑制作用 ,IC50 为 2 5 7 5 7μmol·L-1 。氨氯吡咪对重组人CK2全酶的动力学研究表明 :它与GTP呈竞争性抑制作用 ,抑制常数Ki 值为 2 36 30 μmol·L-1 ;与酪蛋白呈非竞争性抑制作用 ,抑制常数Ki 值为 1 6 3 6 3μmol·L-1 。结论　氨氯吡咪除了能抑制cAMP或ATP依赖的酶以外 ,还可以抑制以GTP为磷酸供体的蛋白激酶。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

Inhibitory kinetics of amiloride on recombinant human protein kinase CK2 holoenzyme

AIM To study the direct effect of amiloride on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 α and β subunits were cloned,expressed and purified by gene engineering. The two subunits were mixed at the same molar ratio to form CK2 holoenzyme,which displayed the maximum biological activity. CK2 activity was assayed by detecting incorporation of 32 P of γ 32 P]GTP into the substrate in various conditions. RESULTS Amiloride inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 257 57 μmol·L -1 . Kinetic studies of amiloride on recombinant human CK2 showed that the inhibition was competitive with GTP with the K i of 236 30 μmol·L -1 ,and noncompetitive with casein with the K i of 163 63 μmol·L -1 . CONCLUSION Amiloride inhibits the activity of cAMP or ATP dependent enzymes as well as the kinase with GTP as phosphate donor,and the recombinant human protein kinase CK2 may be used as a molecular target for simple screening and development of effective inhibitors of CK2.