Watanabe F, Teraoka H, Iijima S, Mimori T, Tsukada K
Department of Pathological Biochemistry, Tokyo Medical and Dental University, Japan.
Biochim Biophys Acta. 1994 Sep 8;1223(2):255-60. doi: 10.1016/0167-4889(94)90234-8.
A double-stranded DNA-dependent protein serine/threonine kinase (DNA-PK) was purified from a nuclear extract of Raji Burkitt's lymphoma cells by a three-step column-chromatographic procedure. The main silver-stained band visualized after SDS/PAGE corresponded to an autophosphorylated polypeptide of about 350-kDa that represents the catalytic component. The existence of Ku DNA-binding protein as a regulatory component in the purified enzyme was revealed by Western blot/enzyme immunoassay and direct inhibition test with anti-Ku sera from the autoimmune patients. The DNA-PK catalyzed phosphorylation of synthetic peptides corresponding to Myc and RB proteins in a DNA-dependent manner, indicating that DNA-PK may recognize a second core-sequence motif Pro-Ser/Thr- in addition to the putative consensus sequences of -Ser/Thr-Gln. The level of enzyme activity was significantly higher in DMSO-induced G0/G1-arrested Raji cells as well as in the cells after release from DMSO than in the log-phase cells.
通过三步柱色谱法从Raji伯基特淋巴瘤细胞的核提取物中纯化出一种双链DNA依赖性蛋白丝氨酸/苏氨酸激酶（DNA-PK）。SDS/PAGE后可见的主要银染条带对应于一条约350 kDa的自磷酸化多肽，它代表催化成分。通过蛋白质印迹/酶免疫测定以及来自自身免疫患者的抗Ku血清的直接抑制试验，揭示了Ku DNA结合蛋白作为纯化酶中的调节成分的存在。DNA-PK以DNA依赖性方式催化对应于Myc和RB蛋白的合成肽的磷酸化，表明DNA-PK除了假定的共有序列-Ser/Thr-Gln之外，还可能识别第二个核心序列基序Pro-Ser/Thr-。在二甲基亚砜（DMSO）诱导的G0/G1期停滞的Raji细胞以及从DMSO释放后的细胞中，酶活性水平显著高于对数期细胞。
