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Updated: Feb 23, 2026
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Characterization of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) activity in mouse
Dhébora Albuquerque Dias1,2, Bruna de Barros Penteado1,2, Lucas Derbocio Dos Santos1,2
1Laboratório de Bioquímica Geral e de Microrganismos, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
Abstract:
This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+ . A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.
