CHARACTERIZATION OF [H-3] AMP BINDING TO RAT ADIPOSE PLASMA-MEMBRANESAND ITS SUBSTRATE-SPECIFICITY

Studies were carried out to evaluate the binding of [H-3]AMP to rat ad ipose plasma membranes and ro study the substrate specifity of this pr ocess. The [H-3]AMP binding to membranes was studied by high-speed fil tration under conditions of virtually complete inhibition of 5'-nucleo tidase acivity by EDTA/Na. The Scatchard plot revealed the existence o f a single class of AMP-binding sites of the membrane surface with K-d equal to 2.14 +/- 0.210 mu M acid binding capacity (B-max) of 26.0 +/ - 0.68 pmoles per mg protein, The addition of ATP (12.5 mu M) or ADP ( 3.5 mu M) to the incubation medium resulted in the two-fold increase i n K-d value, whereas in the presence of adenosine (400 mu M) and its p harmacological antagonist theophylline (200 mu M), the number of AMP-b inding sites decreased, Therefore ATP and ADP but not adenosine compet e with AMP for the same nucleotide-binding site, It is suggested that the observed [H-3]AMP binding may be primarily caused by the AMP inter action with specific substrate-binding active center of the membrane e ctoenzyme 5'-nucleotidase.