McIvor R S, Wohlhueter R M, Plagemann P P
J Cell Physiol. 1985 Mar;122(3):397-404. doi: 10.1002/jcp.1041220309.
Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330-fold from Novikoff rat hepatoma cells by ammonium sulfate precipitation, DEAE-Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 microM, KmRib-1-P = 88 microM, KmUrd = 16 micron, and KmPi = 130 microM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 microM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose-1-phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N-(phosphonacetyl)-L-aspartate ("PALA"), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-1-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.
在六种不同的哺乳动物细胞系的超声提取物中检测到尿苷磷酸化酶活性，并且该酶与尿苷激酶一起，提供了一条通过尿苷将尿嘧啶转化为UMP的途径。在八种不同的哺乳动物细胞系中均未检测到尿嘧啶磷酸核糖基转移酶活性。通过硫酸铵沉淀、DEAE-葡聚糖凝胶色谱、羟基磷灰石色谱和葡聚糖凝胶G-200分级分离，从诺维科夫大鼠肝癌细胞中纯化尿苷磷酸化酶，纯化倍数达5330倍。通过凝胶过滤法测得该酶的分子量约为45,000。在共底物浓度饱和的情况下，针对所有四种底物分析纯化后酶的动力学，得出参数KmUra = 360 μM、KmRib-1-P = 88 μM、KmUrd = 16 μM和KmPi = 130 μM。然而，在完整细胞中，尿苷的磷酸解反应的表观Km为231 μM。用0.5 mM肌苷处理的诺维科夫细胞表现出尿嘧啶摄取速率增加，这与细胞内核糖-1-磷酸的增加成正比。然而，在嘧啶从头合成被吡唑呋林或N-（膦酰乙酰基）-L-天冬氨酸（“PALA”）阻断的细胞中，即使肌苷增强了摄取，尿嘧啶的摄取仍不足以支持细胞增殖。这些观察结果与该酶的动力学特征一致，并提供证据表明核糖-1-磷酸的细胞内水平在尿苷磷酸化酶介导的尿嘧啶摄取中起限速作用。
