Deo S S, Tseng W C, Saini R, Coles R S, Athwal R S
Biochim Biophys Acta. 1985 May 8;839(3):233-9. doi: 10.1016/0304-4165(85)90003-0.
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.
通过单步 GMP-琼脂糖亲和层析，从携带质粒 pSV2gpt 的大肠杆菌细胞中纯化出黄嘌呤-鸟嘌呤磷酸核糖转移酶，纯化倍数达 30 倍，接近均一状态。该酶对底物鸟嘌呤、黄嘌呤和次黄嘌呤的 Km 值分别为 2.5、42 和 182 μM，其中鸟嘌呤是最优先的底物。当以鸟嘌呤作为第二底物时，该酶对 PRib-PP 的 Km 值为 38.5 μM；当以黄嘌呤作为第二底物时，Km 值为 100 μM。它受到 6-硫鸟嘌呤、GMP 的显著抑制，也受到其他一些嘌呤类似物的较弱抑制。已发现硫鸟嘌呤是最有效的抑制剂。黄嘌呤-鸟嘌呤磷酸核糖转移酶的亚基分子量测定为 19000。在非变性聚丙烯酰胺凝胶电泳凝胶上进行的原位活性测定表明，第二种大肠杆菌磷酸核糖转移酶优先使用次黄嘌呤而非鸟嘌呤作为底物，且不使用黄嘌呤。
