Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt.
The enzyme xanthine-guanine phosphoribosyltransferase from Escherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a Km value of 2.5, 42 and 182 microM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a Km value of 38.5 microM for PRib-PP with guanine as second substrate and of 100 microM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010957:Plasmids
D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase