Dick J E, Wright J A
J Cell Physiol. 1980 Oct;105(1):63-72. doi: 10.1002/jcp.1041050109.
There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. THe antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki value of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony-forming ability. Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.
关于人体组织中核糖核苷酸还原酶活性的研究非常少。在本报告中，我们描述了一种快速便捷的方法，用于测定正常人二倍体成纤维细胞中嘌呤和嘧啶核糖核苷酸的还原情况，并使用该方法研究这些细胞中该活性的一些一般特性。对ADP和CDP还原酶进行了表征，研究其对正效应物ATP和dGTP、负效应物dATP以及还原剂二硫苏糖醇的反应。ADP和CDP的表观Km值分别确定为0.1 mM和0.04 mM。抗肿瘤药物羟基脲以非竞争性方式抑制嘌呤和嘧啶还原酶，ADP和CDP的Ki值分别为0.40 mM和0.41 mM。这些Ki估计值比一些永久细胞系报道的值高约四到五倍。对羟基脲细胞毒性作用的研究表明，抑制酶活性的药物浓度与集落形成能力下降之间存在密切相关性。显然，在少量正常人二倍体细胞中研究核糖核苷酸还原的能力将对遗传和生化研究有用。
