Steady-state binding of [3H]ATP to rat liver plasma membranes and competition by various purinergic agonists and antagonists

摘要: Abstract Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using 3H-labelled ATP as radioligand under complete inhibition ecto-ATPase activity by excess EDTA. Binding [3H]ATP to the is saturable, reversible and apparently involves one population specific binding with Kd about 90 nM capacity (Bmax) 15 pmol/mg protein. A broad spectrum purinergic agonists antagonists examined potential inhibitors measured binding. The displacement studies showed following rank order inhibitory potency for [3H]ATP-binding (pIC50 values in parentheses): ATPγS (7.49)>2-MeSATP (7.18)>ATP (6.91)>ADPβS (6.64)≥ADP (6.56)≫RB2 (6.14)≫suramin (5.40)≫Ap4A (4.57)>α,β-MeATP (4.19)≥β,γ-MeATP (3.97). AMP, adenosine, Ap5A, PPADS, β-glycerophosphate well non-adenine nucleoside triphosphates GTP, UTP CTP did not exert any effect at concentration ranges 10−6–10−4 M. In ascertain whether its analogues are capable interacting same domain, 2-MeSATP ADP were treated alternative ligands that could compete unlabelled sites. 2-fold increase value ATP-receptor interaction observed presence (60 nM) or (250 without modulation Bmax value, confirming effects these compounds competitive nature. These demonstrate able interact a single domain membranes, which may be identified ligand-binding component P2 purinoceptors P2Y1 subtype.