3,6-二羟黄酮对重组人蛋白激酶CK2全酶的抑制作用及其动力学分析

 目的观察3,6二羟黄酮对重组人蛋白激酶CK2全酶的直接作用及其酶动力学机制,寻找CK2的抑制剂。方法以重组人CK2全酶为分子靶点,检测不同浓度3,6二羟黄酮对CK2活性的影响,并通过酶的动力学分析其抑制作用机制。CK2活性通过测定转移到CK2底物上的[γ³²P]ATP的[³²P]放射活度来检测。结果重组人CK2与天然CK2的性质完全一致。3,6二羟黄酮对重组人CK2全酶具有较强的抑制作用,IC₅₀为1393μmol·L^-1;进一步分析,它与ATP呈竞争性抑制CK2的活性,抑制常数K_i值为10.67μmol·L^-1;与酪蛋白呈以非竞争性为主的混合性抑制CK2的活性,抑制常数K_i值为17.34μmol·L^-1。结论3,6二羟黄酮是一种重组人蛋白激酶CK2的抑制剂。重组人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。

Inhibitory effect and its kinetic analysis of 3,6-dihydroxyflavone on recombinant human protein kinase CK2 holoenzyme

OBJECTIVE To find the inhibitor of protein kinase CK2 by investigating the direct effect of 3,6-dihydroxyflavone on holoenzyme and its kinetics. METHODS The effects of a series of concentrations of 3,6-dihydroxyflavones on recombinant human protein kinase CK2 holoenzyme were investigated. The mechanism of inhibition was analyzed by its kinetics.The CK2 activity was assayed by detecting incorporation of ³²P of [γ-³²P] ATP into the substrate under various conditions.RESULTS The characteristic and function of the reconstituted holoenzyme were consistent with those of native CK2. 3,6-dihydroxyflavone strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 (IC₅₀=13.93 μmol·L^-1). Kinetic studies showed that 3,6-dihydroxyflavone inhibitied CK2 competitively with ATP (K_i 10.67 μmol·L^-1 ) and noncompetitively dominantly with casein (K_i 17.34 μmol·L^-1).CONCLUSIONS The recombinant human protein kinase CK2 may be used as a molecular target for simple screening and development of more effective inhibitors of CK2. 3,6-dihydroxyflavone is an effective inhibitor of CK2 holoenzyme.