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

摘要

Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using ~3H-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity be excess EDTA. Binding of [~3H]ATP to the membranes is saturable, reversible and apparently involves one population of specific binding sites with K_d of about 90 nM and binding capacity (B_(max)) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhibitors of the measured binding. The displacement studies showed the following rank order of inhibitory potency for [~3H]ATP-binding sites (pIC_(50) 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) Ap_4A (4.57) > α,β-MeATP (4.19) ≥ β,γ-MeATP(3.97). AMP, adenosine, Ap_5A, PPADS, β-glycerophosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10~(-6)-10~(-4) M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-MeSATP and ADP were treated as alternative ligands that could compete with unlabelled ATP for its binding sites. A 2-fold increase of K_d value for ATP-receptor interaction was observed in the presence of 2-MeSATP (60 nM) or ADP (250 nM) without any modulation of B_(max) value, confirming that inhibitory effects of these compounds are competitive in nature. These studies demonstrate that ATP and its analogues are able to interact with a single binding domain on liver plasma membranes, which may be identified as ligand-binding component of P2 purinoceptors of the P2Y_1 subtype.