Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.
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.
D000818:Animals; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008722:Methods; D051379:Mice; D010430:Pentosyltransferases / Q000032:analysis*; D012263:Ribonucleosides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D014161:Transduction, Genetic; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism
D012263:Ribonucleosides; D012265:Ribonucleotides; D014970:Xanthines; D019820:Xanthine; C011141:xanthosine monophosphate; C005893:xanthosine; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase