Ecto-5'-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells.
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.
Navarro
J M
JM
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Olmo
N
N
Turnay
J
J
López-Conejo
M T
MT
Lizarbe
M A
MA
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000230:Adenocarcinoma / Q000201:enzymology*; D000906:Antibodies; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002413:Cations, Divalent; D006023:Glycoproteins; D037102:Lectins; D008667:Metalloproteins; D009711:Nucleotides; D004492:Edetic Acid; D015720:5'-Nucleotidase