Kinetic and biochemical characterization of an ecto-nucleotide pyrophosphatase/phosphodiesterase (EC 3.1.4.1) in cells cultured from submandibular salivary glands of rats

The participation of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in the nucleotide hydrolysis by salivary gland cells of rats was evaluated using p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate for this enzyme. We investigated the biochemical characteristics of this ectoenzyme in cells cultured from submandibular salivary glands of rats. Primary cell cultures demonstrated ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activities, which could be observed by extracellular hydrolysis of p-Nph-5′-TMP and other biochemical characteristics such as dependence of metal ions, dependence of pH alkaline and inactivation by a metal ion chelator. The Km value for the hydrolysis of p-Nph-5′-TMP was 280.7 ± 34.2 μM (mean ± S.D., n = 4) and Vmax was 721.31 ± 225 nmol p-nitrophenol/min/mg (mean ± S.D., n = 4). We suggest that E-NPP is co-localized with an ecto-ATP diphosphohydrolase/ecto-NTPDase and an ecto-5′-nucleotidase, since these enzymes probably act under different conditions. It may be postulated that the physiological role for these ecto-enzymes is to terminate the action of the co-transmitter ATP, generating adenosine.