Characterization of [3H]AMP binding to rat adipose plasma membranes and its substrate specificity

We evaluated the binding of [3H]AMP to rat adipose plasma membranes and studied the substrate specificity of the process. The [3H]AMP binding was investigated by high-speed filtration under conditions of virtually complete inhibition of the 5'-nucleotidase activity by EDTA/Na. The Scatchard plot revealed the existence of a single class of AMP-binding sites on the membrane surface with Kd of 2.14 +/- 0.210 microM and the binding capacity (Bmax) of 26.0 +/- 0.68 pmol per mg protein. Addition of ATP (12.5 microM) or ADP (3.5 microM) to the incubation medium resulted in a two-fold increase of Kd, whereas in the presence of adenosine (400 microM) or its pharmacological antagonist theophylline (200 microM), the number of AMP-binding sites decreased. Therefore, ATP and ADP but not adenosine compete with AMP for the same nucleotide-binding site. It is suggested that the observed [3H]AMP binding may be primarily caused by the interaction of AMP with a specific substrate-binding active centre of the membrane ectoenzyme 5'-nucleotidase.