Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt |
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Authors: | Surinder S. Deo Romesh Saini Roswell S. Coles Raghbir S. Athwal |
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Affiliation: | 1. Department of Microbiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 100 Bergen Street, Newark, NJ 07103, U.S.A. |
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Abstract: | The enzyme xanthine-guanine phosphoribosyltransferase from scherichia 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 μM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a Km value of 38.5 μM for PRib-PP with guanine as second substrate and of 100 μM 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. |
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Keywords: | Xanthine-guanine phosphoribosyltransferase Thioguanine inhibition Substrate specificity (Plasmid pSV2gpt) |
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