人早幼粒白血病HL-60细胞蛋白激酶CK2的研究

Study on protein kinase CK2 from HL 60 cells

目的：研究人早幼粒白血病ＨＬ－６０细胞蛋白激酶ＣＫ２的性质。方法：经二次ＤＥ－５２离子交换纤维素柱，一次肝素－Ｓｅｐｈａｒｏｓｅ４８亲和层析柱纯化后的ＣＫ２（纯化了１３９倍，回收率为１１．８％），以［γ－３２Ｐ］ＡＴＰ和［γ－３２Ｐ］ＧＴＰ为磷酸供体，以去磷酸化酪蛋白为底物，对其性质进行研究。结果：Ｃａ２＋、ｃＡＭＰ、ｃＧＭＰ等第二信使对ＨＬ－６０细胞的ＣＫ２活性无显著影响。肝素抑制ＣＫ２活性，而精胺激活ＣＫ２，肝素与精胺对ＣＫ２的作用在一定浓度范围内呈剂量依赖性。对ＣＫ２的动力学研究测得其米氏常数Ｋｍ（ＧＴＰ）为３４．０μｍｏｌ／Ｌ。结论：ＣＫ２是一种不依赖于Ｃａ２＋、ｃＡＭＰ、ｃＧＭＰ等第二信使的蛋白激酶，肝素是其抑制剂，精胺是其激活剂。

Objective: To study the characterization of CK2 from HL 60 cells.Methods:CK2 was purified from HL 60 cells by double chromatography of DE 52 and a heparin sepharose 4B affinity column(the procedure resulted in a 139 fold purification of the kinase activity with a yield of 11.8%).CK2 activity was detected by using[γ 32 P]ATP or[γ 32 P]GTP as phosphoryl donor and dephosphated casein as substrate.Results:CK2 activity was not affected by Ca 2+ ,cAMP and cGMP.Hepar in inhibited CK2 activity with low concentrations and spermine stimulated CK2 activity effectively.Using dephosphated casein(2 g/L)as substrate,the Km of CK2 for GTP was 34. 0 μ mol/L.Conclusion:CK2 from HL 60 cells is a kinase independent of the second messengers such as Ca 2+ ,cAMP and cGMP.Heparin is the inhibitor of CK2,and spermine is the activator of CK2.