Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

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 t...

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Veröffentlicht in:European journal of biochemistry 2003-08, Vol.270 (16), p.3447-3454
Hauptverfasser: Kukulski, Filip, Komoszyński, Michal
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Komoszyński, Michal
description 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 μm) was three times lower in comparison to analogous values for NTPDase2 (270 μm). Additionally, NTPDase1 had a three times higher Kcat/Km coefficient than NTPDase2 (860 and 833 μmol 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.
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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 μm) was three times lower in comparison to analogous values for NTPDase2 (270 μm). Additionally, NTPDase1 had a three times higher Kcat/Km coefficient than NTPDase2 (860 and 833 μmol 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. 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subjects Adenosine Triphosphatases - isolation & purification
Adenosine Triphosphatases - metabolism
Animals
Antigens, CD - isolation & purification
Antigens, CD - metabolism
Apyrase
central nervous system
Cerebral Cortex - metabolism
ecto‐nucleoside triphosphate disphosphohydrolase
extracellular purines
Kinetics
P2 receptors
signal transmission
Swine - metabolism
Synapses - metabolism
title Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes
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