Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes

Biochim Biophys Acta. 2000 Jun 1;1466(1-2):234-44. doi: 10.1016/s0005-2736(00)00165-6.

Abstract

Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / antagonists & inhibitors
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / metabolism*
  • Animals
  • Binding, Competitive
  • Catalysis
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Dinucleoside Phosphates / pharmacology*
  • Enzyme Inhibitors / pharmacology*
  • Hydrolysis
  • Liver / enzymology
  • Male
  • Pyridoxal Phosphate / analogs & derivatives*
  • Pyridoxal Phosphate / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Substrate Specificity
  • Suramin / pharmacology*
  • Triazines / pharmacology*

Substances

  • Dinucleoside Phosphates
  • Enzyme Inhibitors
  • Triazines
  • pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid
  • diadenosine tetraphosphate
  • Cibacron Blue F 3GA
  • Pyridoxal Phosphate
  • Suramin
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • ectoATPase