Youdale T, MacManus J P, Whitfield J F
Can J Biochem. 1982 Apr;60(4):463-70. doi: 10.1139/o82-054.
Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 X 10(-4) for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS-polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.
首次从再生大鼠肝脏中大量纯化出哺乳动物核糖核苷酸还原酶的两个不同亚基L1和L2。它们通过dATP-琼脂糖亲和色谱法分离。与dATP-琼脂糖结合的亚基L1用50 mM ATP洗脱，并通过分子排阻高压液相色谱（HPLC）纯化至同质（如十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳所示）。该亚基的表观相对分子质量（Mr）为45 000，对CDP的Km为0.9×10⁻⁴。不与dATP-琼脂糖结合的亚基L2通过pH 5.2沉淀，然后在CM-葡聚糖凝胶、分子排阻HPLC和DEAE-纤维素上进行色谱法纯化。该亚基含铁，通过HPLC分子排阻色谱法测定其表观Mr为120 000，但在SDS-聚丙烯酰胺凝胶电泳上显示两条带（Mr 75 000和Mr 47 000）。L1和L2单独都没有任何酶活性，但结合时它们将CDP还原为dCDP。
