Human hypoxanthine-guanine phosphoribosyltransferase. Demonstration of structural variants in lymphoblastoid cells derived from patients with a deficiency of the enzyme.
Human hypoxanthine-guanine phosphoribosyltransferase. Demonstration of structural variants in lymphoblastoid cells derived from patients with a deficiency of the enzyme.
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DOI：
10.1172/jci110499
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发表时间：
1982-03
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影响因子：
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通讯作者：
J. Wilson;B. W. Baugher;P. Mattes;P. Daddona;W. Kelley
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文献类型：
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作者：
J. Wilson;B. W. Baugher;P. Mattes;P. Daddona;W. Kelley
我们已经探索了使用培养的淋巴母细胞的次黄嘌呤鸟嘌呤磷酸核糖转移酶（HPRT）的缺乏症患者作为细胞的来源的突变形式的酶的分离和表征的可能性。来自5个不相关HPRT缺陷家族的6名男性患者的淋巴母细胞的HPRT被高度纯化，并在以下方面进行表征：（a）免疫反应蛋白水平，（B）绝对比活性，（c）等电点，（d）非变性聚丙烯酰胺凝胶电泳期间的迁移，和（e）表观亚基分子量。使用几种微量方法对少量淋巴母细胞进行实验，包括粗提物的蛋白质印迹分析以及用放射性氨基酸标记的培养物中的酶的分离和表征。四名患者的淋巴母细胞酶表现出结构和功能异常，这与最近描述的高度纯化的红细胞酶从这些相同的患者中发现的异常相似。此外，以前未描述的HPRT变异体分离和来自两个男性同胞的淋巴母细胞的特点。这种独特的变种被称为HPRT安阿伯。我们的结论是，淋巴母细胞系可用作检测，分离和表征人类HPRT结构变异的细胞来源。
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.