Figure 1.

Isolation and characterization of potential BRS-3 ligand from toad skin. A) Elution profile of toad skin extract on C18HCE reverse-phase HPLC column (4.6 × 150 mm, 5 μm) and activities of 48 fractions (1 min) tested for their abilities to induce intracellular Ca²⁺ mobilization in HEK293 cells expressing BRS-3 receptor. Signal is expressed as ratio of increment obtained by BRS-3–transfected HEK293 cells vs. HEK293 wild-type cells. Elution was performed with linear gradient from 5 to 15% CH₃CN in 30 min, then from 15T to 95% CH₃CN in 10 min. Flow rate was 1 ml/min. Fraction 46 (arrow) was most active. B) PGB2 purification and structure elucidation. Inset shows structure of PGB2 and its response in BRS-3/HEK monitored by Ca²⁺ mobilization. Error bars represent sem of triplicate measurements for each point.