Izuta S, Kohsaka M, Saneyoshi M
Nucleic Acids Symp Ser. 1985(16):241-4.
DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.
通过以下纯化步骤，已从发育中的樱鳟（Oncorhynchus masou）睾丸中纯化出2750倍的DNA聚合酶α2-引发酶：分级提取、磷酸纤维素（第一次）、硫酸铵分级分离、DEAE-纤维素、磷酸纤维素（第二次）、羟基磷灰石和单链DNA-纤维素柱色谱。该酶的最终制剂的比活性为107,000单位/毫克蛋白质（以活化的鲑鱼精子DNA作为模板引物）。在该纯化过程的所有阶段，DNA引发酶活性（依赖rGTP将标记的dGMP掺入聚（dC）中或依赖rNTP将dNMP掺入M13单链DNA中）与DNA聚合酶α活性紧密相关。通过使用以M13 DNA为模板的依赖rNTP的DNA合成，研究了六种3'-脱氧核糖核苷酸对DNA引发酶活性的抑制作用。抑制常数（Ki）大于依赖DNA的RNA聚合酶I和II的抑制常数。然而，Ki/Km值非常接近。
