tailieunhanh - Báo cáo khoa học: 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 typical for all members of the NTPDase family, . low substrate specificity for tri-anddiphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. | Eur. J. Biochem. 270 3447-3454 2003 FEBS 2003 doi Purification and characterization of NTPDasel ecto-apyrase and NTPDase2 ecto-ATPase from porcine brain cortex synaptosomes Filip Kukulski and Michal Komoszynski Department of Biochemistry Institute of General and Molecular Biology N. Copernicus University Torun Poland 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 . 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 M was three times lower in comparison to analogous values for NTPDase2 270 M . Additionally NTPDase1 had a three times higher Kcat Km coefficient than NTPDase2 860 and 833 Ltmol 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. Keywords central nervous system extracellular purines P2 receptors signal transmission ecto-nucleoside triphosphate disphosphohydrolase. Extracellular ATP and ADP as well as UTP and UDP participate in biological signaling particularly neurotransmission processes in the central nervous system CNS by activating nucleotide P2 receptors 1 2 . Nucleotide mediated signal transmission is terminated by hydrolysis of pyrophosphate bonds present in the agonist structure 3 4 . In the CNS .