Purification, activity, and expression levels of two uridine-cytidine kinase isoforms in neuroblastoma cell lines

Nucleosides, Nucleotides and Nucleic Acids Pub Date : 2016-12-01 DOI:10.1080/15257770.2015.1124998

R. Meinsma, A. V. van Kuilenburg

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引用次数: 8

Abstract

ABSTRACT Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme. The UCK1 antibody showed no cross reactivity against UCK2. Our data showed that UCK1 and 2 are both expressed in several neuroblastoma cell lines, including four MYCN single copy cell lines and five MYCN amplified cell lines, with the exception that UCK1 was not expressed in SJNB8. These results indicate that UCK2 in neuroblastoma might be used as a selective target for chemotherapy using UCK2-dependent pyrimidine analogues.

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两种尿苷-胞苷激酶亚型在神经母细胞瘤细胞系中的纯化、活性和表达水平

尿嘧啶-胞苷激酶(UCK)催化尿嘧啶、胞苷和几种嘧啶核糖核苷类似物的磷酸化。我们在大肠杆菌中过表达和纯化了人类UCK的两种已知亚型，制备了针对UCK1的特异性抗体，并对UCK1和2的动力学特性进行了表征。与纯化的重组UCK1酶相比，用尿苷和胞苷纯化的重组UCK2酶的Vmax分别高出22倍和8倍。与纯化的重组UCK2酶相比，尿苷和胞苷的UCK1的Km分别高出39倍和40倍。UCK1抗体与UCK2无交叉反应性。我们的数据显示，UCK1和2在几种神经母细胞瘤细胞系中均有表达，包括4种MYCN单拷贝细胞系和5种MYCN扩增细胞系，但UCK1在sjb8中不表达。这些结果表明，神经母细胞瘤中的UCK2可能作为使用UCK2依赖性嘧啶类似物进行化疗的选择性靶点。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Nucleosides, Nucleotides and Nucleic Acids

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