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用于检测转导细胞中大肠杆菌黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶活性的快速测定法。

Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.

作者信息

Chu G, Berg P

出版信息

Nucleic Acids Res. 1985 Apr 25;13(8):2921-30. doi: 10.1093/nar/13.8.2921.

DOI:10.1093/nar/13.8.2921
PMID:3889850
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC341204/
Abstract

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 of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-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 (XP20S) 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.

摘要

用大肠杆菌基因Ecogpt转导的培养哺乳动物细胞可合成细菌酶黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶（XGPT）（1）。本文描述了一种通过薄层层析跟踪14C - 黄嘌呤（X）转化为14C - 黄嘌呤单磷酸（XMP）和14C - 黄苷（XR）来测量粗细胞提取物中XGPT活性的方法。该方法快速、易用、灵敏，在广泛的XGPT活性范围内呈线性，并且已用于检测用Ecogpt瞬时转染或稳定转化的细胞中的XGPT。在我们的研究过程中，我们发现一种人类细胞系（XP20S）可将黄嘌呤转化为XMP。这种活性可能由一种变异的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶（HGPT）催化，因为低活性很容易被次黄嘌呤抑制。X向XMP的低水平转化可能解释了为什么一些细胞系在含有霉酚酸和X的培养基中未被杀死。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/183ea58e90b7/nar00302-0253-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/f1576e8ce9ef/nar00302-0249-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/8d6306f22ae6/nar00302-0250-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/addb0a58f661/nar00302-0251-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/183ea58e90b7/nar00302-0253-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/f1576e8ce9ef/nar00302-0249-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/8d6306f22ae6/nar00302-0250-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/addb0a58f661/nar00302-0251-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db69/341204/183ea58e90b7/nar00302-0253-a.jpg

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本文引用的文献

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Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072-6. doi: 10.1073/pnas.78.4.2072.
2
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J Biol Chem. 1983 Jun 25;258(12):7469-75.
3
Mol Cell Biol. 1988 Feb;8(2):893-902. doi: 10.1128/mcb.8.2.893-902.1988.
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J Bacteriol. 1988 Jun;170(6):2796-801. doi: 10.1128/jb.170.6.2796-2801.1988.
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