Dovey H F, McKerrow J H, Aldritt S M, Wang C C
J Biol Chem. 1986 Jan 15;261(2):944-8.
Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.
次黄嘌呤磷酸核糖基转移酶和鸟嘌呤磷酸核糖基转移酶活性对于曼氏血吸虫童虫中鸟嘌呤核苷酸的供应至关重要。在曼氏血吸虫成虫的粗提物中，这两种活性在尺寸排阻色谱、离子交换色谱和聚焦色谱中共同洗脱，并且对热处理表现出相似的稳定性，这表明它们与一种酶蛋白——次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶相关联。该酶已通过在85℃下进行热处理以及聚焦色谱结合在表观pI为5.27±0.15时洗脱的方法进行了纯化。对天然酶进行孔径梯度电泳，随后进行活性染色，结果显示酶的分子量为105,000。无论使用次黄嘌呤还是鸟嘌呤作为底物，活性染色模式均保持不变，这进一步支持了单一蛋白——次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶的存在。对纯化后的蛋白进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳产生一条单一蛋白带，亚基分子量估计为64,000，这表明天然酶是一种二聚体。初步动力学研究表明，纯化后的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶与鸟嘌呤反应的速率是与次黄嘌呤反应速率的两倍，但它根本不作用于黄嘌呤。将全长小鼠神经母细胞瘤次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶cDNA克隆pHPT5和含有大肠杆菌黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶基因的质粒pSV2 - gpt用作曼氏血吸虫DNA消化物Southern印迹的探针，在相对宽松的条件下未发现明显的杂交信号。针对人红细胞次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶和大肠杆菌黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶制备的多克隆抗体在曼氏血吸虫蛋白提取物的酶联免疫吸附测定中进行了检测，未发现可检测到的交叉反应蛋白。因此，曼氏血吸虫次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶可能与哺乳动物次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶或细菌黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶具有相当有限的同源性，并且可能是抗血吸虫化疗药物设计的一个有吸引力的靶点。
