Inhibitory kinetics of amiloride on recombinant human protein kinase CK2 holoenzyme / 中国药理学通报

ABSTRACT

AIM To study the direct effect of amiloride on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 ? and ? 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 [? 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 ?mol?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 ?mol?L -1 ,and noncompetitive with casein with the K i of 163 63 ?mol?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.