Osborne W R
J Biol Chem. 1980 Aug 10;255(15):7089-92.
As part of a study of the immune defect related to the absence of purine nucleoside phosphorylase, the substrate analogue 6-hydroxy-9-p-aminobenzylpurine was synthesized. This inosine analogue was a competitive inhibitor with an inhibition constant of about 200 muM. Using trichloro-s-triazine, the inhibitor was coupled to Sepharose, producing an efficient, reusable biospecific affinity gel. The gel was used to purify purine nucleoside phosphorylase from human red cells with an 85% yield and a specific activity of 95 mumol/min/mg. The molecular weight of native purine nucleoside phosphorylase was estimated as 90,400 using high pressure liquid chromatography. A subunit molecular weight of 31,600 was established using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, a trimeric structure for the native enzyme is indicated, which is in accordance with the evidence derived from genetic studies.
作为对与嘌呤核苷磷酸化酶缺失相关的免疫缺陷研究的一部分，合成了底物类似物6-羟基-9-对氨基苄基嘌呤。这种肌苷类似物是一种竞争性抑制剂，抑制常数约为200μM。使用三氯-s-三嗪，将抑制剂与琼脂糖偶联，制备出一种高效、可重复使用的生物特异性亲和凝胶。该凝胶用于从人红细胞中纯化嘌呤核苷磷酸化酶，产率为85%，比活性为95μmol/分钟/毫克。使用高压液相色谱法估计天然嘌呤核苷磷酸化酶的分子量为90400。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳确定亚基分子量为31600。因此，表明天然酶具有三聚体结构，这与遗传研究得出的证据一致。
