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An Ecto-Nucleotide Pyrophosphatase Is One of the Main Enzymes Involved in the Extracellular Metabolism of ATP in Rat C6 Glioma
Authors:Bert Grobben  Katelijne Anciaux  Dirk Roymans  Cristiana Stefan  Mathieu Bollen  Eddy L Esmans  Herman Slegers
Institution:Department of Biochemistry, Cellular Biochemistry, University of Antwerp, Universitaire Instelling Antwerpen, Antwerpen-Wilrijk, and Department of Chemistry, Nucleoside Research, University of Antwerp, Universitair Centrum Antwerpen, Antwerp, and; Department of Biochemistry, Catholic University of Leuven, Leuven, Belgium
Abstract:Abstract : The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the α-and γ-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for ~50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 μ M abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a K m for ATP of 17 ± 5 μ M . The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.
Keywords:Glial cells  ATP hydrolysis  Nucleotide pyrophosphatase  Nucleoside diphosphate kinase (NDPK/nm23
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