title: Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes.
abstract: We purified to homogeneity and characterized NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. SDS/PAGE and immunoblotting with antibodies specific to these enzymes revealed a molecular mass estimated at 72 kDa for NTPDase1 and 66 for NTPDase2. Both enzymes exhibited kinetic properties typical for all members of the NTPDase family, e.g. low substrate specificity for tri- and diphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. The calculated Km value for NTPDase1 in respect to ATP as a substrate (97 microm) was three times lower in comparison to analogous values for NTPDase2 (270 microm). Additionally, NTPDase1 had a three times higher Kcat/Km coefficient than NTPDase2 (860 and 833 micromol product.s(-1), respectively). We have also demonstrated that in spite of differences in the affinity of ATP for both hydrolases, these enzymes have similar molecular activity. Taken together, these results indicate that NTPDase1 would terminate P2 receptor-mediated signal transmission whereas activity of NTPDase2 may contribute to decreasing high (toxic) concentrations of ATP and/or to production of another signal molecule, ADP.
authors.affiliation: Department of Biochemistry, Institute of General and Molecular Biology, N. Copernicus University, Toruń, Poland.
authors.forename: Filip
authors.initials: F
authors.lastname: Kukulski
authors.forename: Michal
authors.initials: M
authors.lastname: Komoszyński
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000302:isolation & purification* / Q000378:metabolism; D001081:Apyrase; D002540:Cerebral Cortex / Q000378:metabolism*; D007700:Kinetics; D013552:Swine / Q000378:metabolism; D013569:Synapses / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen