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Ecto-5′-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells

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Abstract

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.

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Authors and Affiliations

  1. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain

    Juana M. Navarro, Nieves Olmo, Javier Turnay, M. Teresa López-Conejo & M. Antonia Lizarbe

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  1. Juana M. Navarro
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  2. Nieves Olmo
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  3. Javier Turnay
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  4. M. Teresa López-Conejo
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  5. M. Antonia Lizarbe
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Navarro, J.M., Olmo, N., Turnay, J. et al. Ecto-5′-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells. Mol Cell Biochem 187, 121–131 (1998). https://doi.org/10.1023/A:1006808232059

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