Wilson J M, Baugher B W, Mattes P M, Daddona P E, Kelley W N
J Clin Invest. 1982 Mar;69(3):706-15. doi: 10.1172/jci110499.
We have explored the possibility of using cultured lymphoblasts from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a source of cells for the isolation and characterization of mutant forms of the enzyme. HPRT from lymphoblasts derived from six male patients of five unrelated HPRT-deficient families was highly purified and characterized with regard to: (a) level of immunoreactive protein, (b) absolute specific activity, (c) isoelectric point, (d) migration during nondenaturing polyacrylamide gel electrophoresis, and (e) apparent subunit molecular weight. There experiments were performed on small quantities of lymphoblasts using several micromethods involving protein blot analysis of crude extracts as well as isolation and characterization of enzyme labeled in culture with radioactive amino acids. The lymphoblast enzymes from four of the patients exhibited structural and functional abnormalities that were similar to the recently described abnormalities found with the highly purified erythrocyte enzymes from these same patients. In addition, a previously undescribed HPRT variant was isolated and characterized from lymphoblasts derived from two male siblings. This unique variant has been called HPRT Ann Arbor. We conclude that lymphoblastoid cell lines can be used as a source of cells for the detection, isolation, and characterization of structural variants of human HPRT.
我们探讨了利用次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HPRT）缺乏症患者的培养淋巴细胞作为细胞来源，用于分离和鉴定该酶突变形式的可能性。对来自五个无亲缘关系的HPRT缺乏症家族的六名男性患者的淋巴细胞中的HPRT进行了高度纯化，并在以下方面进行了表征：（a）免疫反应性蛋白水平，（b）绝对比活性，（c）等电点，（d）非变性聚丙烯酰胺凝胶电泳中的迁移情况，以及（e）表观亚基分子量。使用了几种微量方法对少量淋巴细胞进行了这些实验，这些方法包括对粗提物进行蛋白质印迹分析，以及对在培养中用放射性氨基酸标记的酶进行分离和表征。四名患者的淋巴细胞酶表现出结构和功能异常，这些异常与最近在这些患者的高度纯化红细胞酶中发现的异常相似。此外，从两名男性同胞的淋巴细胞中分离并鉴定出一种先前未描述的HPRT变体。这种独特的变体被称为HPRT安阿伯。我们得出结论，淋巴母细胞系可作为检测、分离和鉴定人HPRT结构变体的细胞来源。
