Apyrases are active against both di- and triphosphate nucleotides (NDPs and NTPs) and hydrolyse NTPs to nucleotide monophosphates (NMPs) in two distinct successive phosphate-releasing steps, with NDPs as intermediates. They differ from ATPases, which specifically hydrolyse ATP, by hydrolysing both ATP and ADP. The eukaryotic enzymes requires Ca2+, but Mg2+ can substitute. Most of the ecto-ATPases that occur on the cell surface and hydrolyse extracellular nucleotides belong to this enzyme family.
Apyrases are active against both di- and triphosphate nucleotides (NDPs and NTPs) and hydrolyse NTPs to nucleotide monophosphates (NMPs) in two distinct successive phosphate-releasing steps, with NDPs as intermediates. They differ from ATPases, which specifically hydrolyse ATP, by hydrolysing both ATP and ADP. The eukaryotic enzymes requires Ca2+, but Mg2+ can substitute. Most of the ecto-ATPases that occur on the cell surface and hydrolyse extracellular nucleotides belong to this enzyme family.
chicken NTPDase8 is not susceptible to substrate inactivation or agents that cause membrane perturbation, but its soluble mutant, lacking C- and N-termini, is susceptible to inhibition. This inhibition of the mutant can be abolished by mutant enzyme-crosslinking on membranes with glutaraldehyde, the ATPase activities of glutaraldehyde-treated chicken NTPDase8 ECD preincubated with ATP, ADP, and phosphate are respectively 95%, 80%, and 89% of the control
chicken NTPDase8 is not susceptible to substrate inactivation or agents that cause membrane perturbation, but its soluble mutant, lacking C- and N-termini, is susceptible to inhibition. This inhibition of the mutant can be abolished by mutant enzyme-crosslinking on membranes with glutaraldehyde, the ATPase activities of glutaraldehyde-treated chicken NTPDase8 ECD preincubated with ATP, ADP, and phosphate are respectively 95%, 80%, and 89% of the control
the pH optimum of MgATPase of the wild-type full-length chicken NTPDase8 is in the neutral pH range while the pH optimum of the mutant MgADPase activity is more acidic at about pH 6.0
activity range, the activity of the full-length enzyme increases with temperature up to 60°C, whereas the ATPase activity of the mutant truncated NTPDase8 ECD decreases at temperatures higher than 25°C even when assayed for 1 min
NTPDase8 is a cell surface ectonucleotidase with a large extracellular domain containing the active site and is anchored to the membrane by two transmembrane domains at the N- and C-termini
NTPDase8 is a cell surface ectonucleotidase with a large extracellular domain containing the active site and is anchored to the membrane by two transmembrane domains at the N- and C-termini
a soluble truncated mutant NTPDase8, lacking the extracellular domain, shows 85% reduced activity compared to the full-length membrane-bound enzyme. Also activity of the soluble chicken NTPDase8 decreases with time in a temperature-dependent manner as a result of inactivation by ATP, ADP, and phosphate, in contrast to the wild-type full-length enzyme
NTPDase8 is a cell surface ectonucleotidase with a large extracellular domain containing the active site and is anchored to the membrane by two transmembrane domains at the N- and C-termini
NTPDase8 is a cell surface ectonucleotidase with a large extracellular domain containing the active site and is anchored to the membrane by two transmembrane domains at the N- and C-termini
construction of a hu-ck ACR1,5 chimera in which the extracellular domain of human NTPDase2 is anchored to the membrane by the two transmembrane domains of the chicken NTPDase8. The hu-ck ACR1,5 chimera is the first chimeric NTPDase reported that shows a resistance to membrane perturbation and substrate inactivation. The strength of interaction of the respective transmembrane domain pairs of the human NTPDase2 and chicken NTPDase8 determine their different responses to membrane perturbation and substrate. The chimeric mutants all show highly reduced ATPase activities, overview. Catalysis at the active site in the extracellular domain of the hu-ck ACR1,5 chimera is no longer negatively affected by membrane perturbation in the lipid bilayer by detergent and temperature
construction of a hu-ck ACR1,5 chimera in which the extracellular domain of human NTPDase2 is anchored to the membrane by the two transmembrane domains of the chicken NTPDase8. The hu-ck ACR1,5 chimera is the first chimeric NTPDase reported that shows a resistance to membrane perturbation and substrate inactivation. The strength of interaction of the respective transmembrane domain pairs of the human NTPDase2 and chicken NTPDase8 determine their different responses to membrane perturbation and substrate. The chimeric mutants all show highly reduced ATPase activities, overview. Catalysis at the active site in the extracellular domain of the hu-ck ACR1,5 chimera is no longer negatively affected by membrane perturbation in the lipid bilayer by detergent and temperature
generation of a soluble truncated mutant NTPDase8 by removal of amino acids 1-28 (containing TMD1) and 464-493 (containing TMD2), the mutant shows 85% reduced activity compared to the full-length membrane-bound enzyme. Generation of chimeric mutant Ck-hu TMD1, encoding a protein in which the N-terminus (aa 1-28) of the chicken NTPDase8 is substituted with the corresponding region (aa 1-29) of the human NTPDase2, which includes its TMD1, and of chimeric mutant Ck-hu TMD2, encoding a protein in which the C-terminus (aa 465-493) of the chicken NTPDase8 is substituted with the corresponding region (aa 461-495) of the human NTPDase2, which includes its TMD2
generation of a soluble truncated mutant NTPDase8 by removal of amino acids 1-28 (containing TMD1) and 464-493 (containing TMD2), the mutant shows 85% reduced activity compared to the full-length membrane-bound enzyme. Generation of chimeric mutant Ck-hu TMD1, encoding a protein in which the N-terminus (aa 1-28) of the chicken NTPDase8 is substituted with the corresponding region (aa 1-29) of the human NTPDase2, which includes its TMD1, and of chimeric mutant Ck-hu TMD2, encoding a protein in which the C-terminus (aa 465-493) of the chicken NTPDase8 is substituted with the corresponding region (aa 461-495) of the human NTPDase2, which includes its TMD2
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant NTPDase8s from HEK293 cell plasma membranes by ammonium sulfate fractionation and nickel affinity column chromatography