Prinos P, Slack C, Lasko D D
Department of Medicine, McGill University, Montreal, Quebec, Canada.
J Cell Biochem. 1995 May;58(1):115-31. doi: 10.1002/jcb.240580114.
Proteins that catalyze 5' phosphorylation of an oligodeoxyribonucleotide substrate can be fractionated by polymin P treatment of whole cell extracts of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, Polymin P-precipitable polynucleotide kinase (PP-PNK), was further purified with a 1,500-fold increase of specific activity compared to the crude Polymin P pellet fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3' monophosphate was not a substrate. PP-PNK preparations exhibited an apparent KM of 52 microM for ATP and 8 microM for oligo dT25. The enzyme preparation displayed no detectable 3' phosphatase or cyclic 2',3' phosphohydrolase activities. The sedimentation coefficient of the PP-PNK activity was 3.8S as determined by sucrose density gradient analysis; the Stokes radius was 45 A, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimum in the neutral to alkaline range in several buffer systems and is distinct from the DNA kinase with an acidic pH optimum previously described in calf thymus.
能够催化寡聚脱氧核糖核苷酸底物5'磷酸化的蛋白质，可以通过用聚胺P处理小牛胸腺全细胞提取物来进行分级分离。在Q-琼脂糖上进行阴离子交换色谱分析显示，聚胺P上清液组分中有三个可分离的活性峰，聚胺P沉淀组分中有一个活性峰。后一种活性，即聚胺P可沉淀的多核苷酸激酶（PP-PNK），经过进一步纯化后，比粗聚胺P沉淀组分的比活性提高了1500倍。寡核苷酸、一个去磷酸化的2.9 kb EcoRI片段和聚（A）可被该酶制剂磷酸化，但胸苷3'单磷酸不是底物。PP-PNK制剂对ATP的表观KM为52 microM，对寡聚dT25的表观KM为8 microM。该酶制剂未显示出可检测到的3'磷酸酶或环2'，3'磷酸水解酶活性。通过蔗糖密度梯度分析测定，PP-PNK活性的沉降系数为3.8S；斯托克斯半径为45 Å，估计分子量为72 kDa。在几种缓冲系统中，该酶的最适pH在中性至碱性范围内，与先前在小牛胸腺中描述的最适pH为酸性的DNA激酶不同。
