In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an
5'-nucleotidase in Walker 256 cells. For the
ATP,
ADP and
AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for
divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of
ATP and
ADP hydrolysis was observed in the presence of high concentrations of
sodium azide and 0.5 mM of
Gadolinium chloride. These activities were insensitive to
ATPase,
adenylate kinase and
alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of
inorganic phosphate min(-1) mg of protein(-1) for
ATP,
ADP and
AMP, respectively. Using RT-PCR analysis we identified the
mRNA of two members of the ecto-
nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a
5'-nucleotidase. The presence of NTPDases and
5'-nucleotidase enzymes in Walker 256
tumor cells may be important to regulate the ratio
adenine nucleotides/
adenine nucleoside extracellularly, therefore motivating
tumor growth.