Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A(2)s

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an elec trical shock and fractionated with C-4 followed by C-18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted lase r desorption ionization time-of-flight mass spectrometry to be 15 347 and 1 5 404 Da, respectively. The inhibitory activity was also measured against o ther membrane receptors, such as the A2 adenosine receptor, muscarinic acet ylcholine receptor and capsaicin receptor. Ligand binding to the A2 and mus carinic receptors was also severely inhibited by these proteins. However, t hey did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor bindin g occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A(2) from various organisms. The phospholipase A(2) activity of both proteins was tested using Dole's assay technique. Both pr oteins showed phospholipase A(2) activity, and therefore, they were designa ted as PLA(2)-R1 and PLA(2)-R2, respectively. In addition, their ligand-bin ding inhibitory activity depended on their phospholipase A(2) activity. Thi s is the first finding that the frog secretes a phospholipase A(2) similar to that of snake venoms, which posess inhibitory activity against the adeno sine A1, adenosine A2 and muscarinic receptors.