Voytek P
J Biol Chem. 1975 May 25;250(10):3660-5.
Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process.
等电聚焦被用作胸苷磷酸化酶分离的最后一步，发现该酶的等电点为4.1。分析型丙烯酰胺凝胶电泳显示，纯化的酶制剂含有一条主要的蛋白带，该带对胸苷磷酸化酶活性呈阳性染色，通常还有一条迁移速度较快的次要无活性带。单独或在敏化剂存在下，胸苷磷酸化酶在253.7nm紫外线照射下失活，遵循一级失活动力学。在pH 5和7.4时，酶的失活速率相同，添加各种嘧啶碱基和核苷可提高两个pH值下的失活速率，但在pH 5时提高幅度更大。失活速率与胸苷或胸腺嘧啶浓度的线性图相同。在7.8mM胸苷或胸腺嘧啶存在下，在pH 5和7.4时，胸苷磷酸化酶的光失活分别增加了11倍和4.4倍。随着5-碘-2'-脱氧尿苷或5-碘尿嘧啶浓度增加，得到抛物线曲线。5.2mM的5-碘尿嘧啶使胸苷磷酸化酶的光失活速率在pH 5时增加212倍，在pH 7.4时增加100倍。然而，5.0mM的5-碘-2'-脱氧尿苷仅使酶的光失活增加83倍（pH 5）和21倍（pH 7.4）。5-溴-2'-脱氧尿苷或5-溴尿嘧啶在使酶敏化方面都不如碘类似物有效。5-碘尿嘧啶或5-碘-2'-脱氧尿苷与胸腺嘧啶的组合导致的失活速率高于各化合物的加和失活速率，而任何一种碘类似物与胸苷的组合导致的失活速率较低。增加磷酸盐或氯化钠的浓度会降低单独的胸苷磷酸化酶的光失活速率，并保护该酶免受不同碱基和核苷引起的敏化作用。在有或没有5-碘尿嘧啶或5-碘-2'-脱氧尿苷存在的情况下进行照射，胸苷磷酸化酶中伯氨基数量没有明显的定量变化。在Sephadex G-150上对辐照后的酶进行检测表明，形成了一种更大的蛋白质物种，并且5-碘尿嘧啶促进了这一过程。
