Navarro J M, Olmo N, Turnay J, López-Conejo M T, Lizarbe M A
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Mol Cell Biochem. 1998 Oct;187(1-2):121-31. doi: 10.1023/a:1006808232059.
Differences on 5'-nucleotidase activity in intact Rugli and BCS-TC2 cells (rat glioblastoma and human colon adenocarcinoma cell lines, respectively) are not due to differences in the characteristics of the ectoenzyme. A membrane-bound 5'-nucleotidase from BCS-TC2 cells has been purified to homogeneity with a high specific activity (130 U/mg), yielding a single 72-kDa band on SDS-PAGE. It is a metalloenzyme and, after inhibition by EDTA, its activity can be partially restored by divalent cations. The hydrolysis of the nucleosides 5'-monophosphate used as substrate follows Michaelis-Menten kinetics; ADP and concanavalin A are competitive and non-competitive inhibitors of the AMPase activity, respectively. This ecto-5'-nucleotidase is a high-mannose glycoprotein; deglycosylation converts the 72-kDa into a 59-kDa protein with a concomitant activity loss. The enzyme purified from BCS-TC2 cells shows similar characteristics from that previously isolated from Rugli cells; differences between them are mainly due to glycosylation. Polyclonal antibodies against 5'-nucleotidase from BCS-TC2 cells also show cross-reactivity with the enzyme from Rugli cells. When the ectoenzyme activity is measured in cells in culture, Rugli cells present a higher activity than BCS-TC2 cells however, they express very low amounts of ecto-5'-nucleotidase. Our results also show a reduction in protein level and enzyme activity associated with a decrease in the differentiation degree and an increase in tumorigenicity of human colon adenocarcinoma BCS-TC2 sublines.
完整的鲁格利细胞和BCS - TC2细胞（分别为大鼠胶质母细胞瘤和人结肠腺癌细胞系）中5'-核苷酸酶活性的差异并非由于胞外酶特性的差异所致。从BCS - TC2细胞中纯化出的一种膜结合5'-核苷酸酶，其纯度达到同质性，具有高比活性（130 U/mg），在SDS - PAGE上呈现出一条单一的72 kDa条带。它是一种金属酶，在被EDTA抑制后，其二价阳离子可部分恢复其活性。用作底物的核苷5'-单磷酸的水解遵循米氏动力学；ADP和伴刀豆球蛋白A分别是AMP酶活性的竞争性和非竞争性抑制剂。这种胞外5'-核苷酸酶是一种高甘露糖糖蛋白；去糖基化将72 kDa的蛋白转化为59 kDa的蛋白，同时活性丧失。从BCS - TC2细胞中纯化的酶与先前从鲁格利细胞中分离出的酶具有相似的特性；它们之间的差异主要归因于糖基化。针对BCS - TC2细胞5'-核苷酸酶的多克隆抗体也与鲁格利细胞中的该酶显示出交叉反应性。当在培养细胞中测量胞外酶活性时，鲁格利细胞的活性高于BCS - TC2细胞，然而，它们表达的胞外5'-核苷酸酶量非常低。我们的结果还表明，人结肠腺癌BCS - TC2亚系的蛋白质水平和酶活性降低与分化程度降低和致瘤性增加相关。
