[Interaction of native 5'-ATP with rat liver cell membranes and adipose tissue]

Studies have been carried out to evaluate specific binding and enzymatic hydrolysis of native 5'-ATP by rat liver and adipose tissue plasma membranes. Addition of [14C]ATP to membranes resulted in sequential nucleotide hydrolysis occurring via stepwise enzymatic reactions: ATP-->ADP-->AMP-->adenosine, thus indicating a high ecto-phosphatase activity of the plasma membranes under study. The ATP-binding kinetic characteristics were studied using a high-speed filtration technique under complete inhibition of membrane nucleotidases by excess EDTA (40 mM). Comparative analysis of [3H]ATP displacement by other nucleotides (ADP, AMP, GTP, GMP) revealed that only ADP was in equimolar competition with ATP for its binding sites. Adenosine and beta-glycerophosphate did not exert any significant effect on [3H]ATP binding to the membranes. Graphical representation of [3H]ATP binding data in a Scatchard plot showed a linear dependence, indicating the existence of a single ATP-binding site with Kd about 50-60 nM and Bmax of 5-7 pmol/mg protein. It is suggested that these sites represent a nucleotide-binding component of P2 purine receptors and are tightly coupled with the catalytic site of membrane ecto-ATPase.