Neuro-2a (N2a)
neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine
polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound
BODIPY FL
guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM.
Diadenosine pentaphosphate,
diadenosine tetraphosphate,
diadenosine triphosphate, and the
nucleoside monophosphate
AMP behaved as an inhibitor of
BODIPY FL
guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide
pyrophosphatase/
phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of
divalent cations, being strongly inhibited by
EDTA and activated by Zn(2+)
ions. Both NPP1 and NPP3
isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3
protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main
isozyme involved in the extracellular degradation of dinucleotides in these cells, this
enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine
polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide
pyrophosphatase/
phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound
BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells differentiate into a neuronal-like phenotype. Activity assays in differentiated cells showed that the ApnA-hydrolytic activity largely depends on the NPP1
isozyme.