Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.
Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.
复制标题
快速检测转导细胞中大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖转移酶活性。
DOI：
10.1093/nar/13.8.2921
复制
发表时间：
1985
影响因子：
14.9
通讯作者：
Berg,P
中科院分区：
文献类型：
--
作者：
Chu,G;Berg,P
用大肠杆菌基因 Ecogpt 转导的培养哺乳动物细胞会合成细菌酶黄嘌呤-鸟嘌呤磷酸核糖转移酶 (XGPT) (1)。本文描述了通过薄层色谱将14 C-黄嘌呤(X)转化为14 C-黄嘌呤单磷酸(XMP)和14 C-黄嘌呤核苷(XR)来测量粗细胞提取物中XGPT活性的方法。该方法快速、易于使用、灵敏且在较宽的 XGPT 活性范围内呈线性，可用于检测瞬时转染或稳定转化 Ecogpt 的细胞中的 XGPT。在我们的研究中，我们发现人类细胞系 (XP2OS) 将黄嘌呤转化为 XMP。这种活性可能是由次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HGPT）变体催化的，因为低活性很容易被次黄嘌呤抑制。 X 向 XMP 的低水平转化可以解释为什么某些细胞系在含有霉酚酸和 X 的培养基中未被杀死。
Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of14C-xanthine (X) to14C-xanthine monophosphate (XMP) and14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP2OS) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.