Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ.
During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.
Martín-Satué
Mireia
M
Laboratori de Neurobiologia Cellular i Molecular, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, Institut d'Investigacions Biomèdiques de Bellvitge-Universitat de Barcelona, Spain. martinsatue@ub.edu
Torrejón-Escribano
Benjamín
B
Felipe
Antonio
A
de Aranda
Inmaculada Gómez
IG
Elías
Marc
M
Marsal
Jordi
J
Blasi
Juan
J
Solsona
Carles
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004557:Electric Organ / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D014101:Torpedo
D018076:DNA, Complementary; D011755:Pyrophosphatases