摘要
目的：纯化人早幼粒白血病(human promyelocytic leukemia)HL-60细胞的蛋白激酶CK2，观察蛋白激酶CK2在HL-60细胞胞膜、胞浆和胞核中的活性，并寻找HL-60 细胞中蛋白激酶CK2的天然底物。方法：采用二次离子交换纤维素DE-52柱和Hp-Sepharose 4B亲和层析柱分离纯化蛋白激酶CK2；采用蔗糖梯度离心法分离HL-60细胞的胞膜、胞浆和胞核；蛋白激酶CK2活性以掺入到CK2底物上的[γ-32P]GTP的32P放射活度来检测；以[γ-32P]GTP为磷酸供体，以精胺为激活剂，以肝素为抑制剂，用放射自显影的方法检测底物蛋白磷酸化。结果：经3次层析后，以[Arg]3-Ala-Asp-Ser-[Asp]5为底物测得蛋白激酶CK2纯化了139倍，回收率11.8％；以去磷酸酪蛋白为底物测得蛋白激酶CK2在HL-60细胞的胞浆和胞核中的活力分别为(354±47) U/mg蛋白和(353±30) U/mg蛋白；以[Arg]3-Ala-Asp-Ser-[Asp]5为底物测得蛋白激酶CK2在HL-60细胞的胞浆和胞核中的活力分别为(60±4) U/mg蛋白和(66±9) U/mg蛋白，HL-60细胞胞浆中59 kDa、54 kDa、33 kDa、28 kDa和22 kDa的蛋白,以及胞核中77 kDa和51 kDa 的蛋白质可被蛋白激酶CK2磷酸化。结论：HL-60细胞中有高活性的蛋白激酶CK2存在；它主要位于HL-60细胞的胞浆和胞核中；蛋白激酶CK2的β亚基有自磷酸化作用；
Objective：This study was designed to purify protein kinase CK2 in human promyelocytic leukemia HL-60 cells and to investigate the activity of protein kinase CK2 on the membrane, the cytoplasm and the nuclei of the cells and find the natural substrates of CK2 in HL-60 cells. Methods：Using double chromatography of DE-52 and a heparin-Sepharose 4B affinity column to purify CK2; Using sucrose gradient centrifugation to separate the membrane, the cytoplasm and the nuclei of HL-60 cells. The activity of CK2 was assayed by detecting radioactivity of 32P of [γ-32P]GTP which was incorporated into the substrate. Using [γ-32P] GTP as the phosphoryl donor, spermine as the activator and heparin as the inhibitor, the phosphorylated substrates were visualized by autoradiography of dried SDS-polyacrylamide gel. Results：Using [Arg]3-Ala-Asp-Ser-[Asp]5 as the substrate, the purify procedure resulted in a 139-fold purification of the kinase with the yield of 11.8％; Using dephosphated casein as the substrate, the activity of CK2 on the cytoplasm and the nuclei of HL-60 cells were (354±47) U/mg protein and (353±30) U/mg protein, respectively; Using [Arg]3-Ala-Asp-Ser-[Asp]5 as the substrate, the activity of CK2 on the cytoplasm and the nuclei of HL-60 cells were (60±4) U/mg protein and (66±9) U/mg protein, respectively. The 59 kDa, 54 kDa, 33 kDa, 28 kDa, and 22 kDa proteins in the cytoplasm and 77 kDa, 51 kDa proteins in the nuclei were phosphorylated by CK2. Conclusions：There is high activity of protein kinase CK2 in HL-60 cells. Protein kinase CK2 localized chiefly on the cytoplasm and the nuclei of HL-60 cell. The βsubunit of CK2 was autophosphorylated. The 59 kDa, 54 kDa, 33 kDa, 22 kDa proteins in cytoplasm and 77 kDa, 51kDa proteins in nuclei may be the natural substrates of CK2.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2001年第7期734-737,共4页
Chinese Journal of Cancer
基金
广东省自然科学基金项目(940586)
广东医学院青年基金项目(XQ9803)
