ABSTRACT
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 microM (mean+/-S.D., n=4) and Vmax was 721.31+/-225nmol 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.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Nucleotides/metabolism , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Submandibular Gland/enzymology , Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Analysis of Variance , Animals , Cells, Cultured , Enzyme Activation , Hydrolysis , Nucleotides/chemistry , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/pharmacokinetics , Pyrophosphatases/chemistry , Pyrophosphatases/pharmacokinetics , Rats , Submandibular Gland/metabolismABSTRACT
The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.
