Drocourt J L, Thang D C, Thang M N
Eur J Biochem. 1978 Jan 16;82(2):355-62. doi: 10.1111/j.1432-1033.1978.tb12030.x.
Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.
天然的大肠杆菌多核苷酸磷酸化酶能够保留在蓝色葡聚糖-琼脂糖凝胶上。结合的酶不能被其单核苷酸底物（如ADP、UDP、CDP、GDP和IDP）置换，但很容易被其多聚底物洗脱。在相同条件下，结合在蓝色葡聚糖-琼脂糖凝胶上的乳酸脱氢酶不会被聚肌苷酸洗脱，但可被NADH特异性置换。另一方面，已知已失去多核苷酸结合位点的胰蛋白酶处理过的多核苷酸磷酸化酶，不会与亲和柱结合。天然的多核苷酸磷酸化酶也能紧密结合到聚尿苷酸-琼脂糖上，只有在高盐浓度下才会从其上被置换下来。胰蛋白酶处理过的酶根本不会结合在聚肌苷酸-琼脂糖上。此外，连接在蓝色葡聚糖-琼脂糖凝胶上的天然酶仍具有活性，这表明核苷二磷酸能够自由进入活性中心。综合这些结果表明，染料配体不会插入到单核苷酸结合位点上，而是表明它结合在多核苷酸结合区域。本文讨论了这项研究的意义以及蓝色葡聚糖-琼脂糖凝胶亲和色谱法在其他对核酸有亲和力的蛋白质上的应用。
