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Updated: May 18, 2026
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions
M C Ribeiro1, M S Costa-Alves, M Wengert
1Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Background:
The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.
Methods:
We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-(32)P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.
Results:
This activity was linear with time up to 20min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5mM MgCl(2) was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN(3)) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN(3). The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2±1.2nmol Pixmg(-1)xmin(-1) and K(0.5) of 0.07±0.01mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60min of ischemia.
Conclusion:
Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.
General Significance:
This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.
