Physicochemical characterization and solubilization of endothelin receptors

[125I]Endothelin-1 (ET-1) bound to specific endothelin receptors (ET-R) with high affinity (Kd = 0.4-1 nM) and dissociated very slowly from ET-R at 37 degrees C (K-1 = 2.4 x 10(-3)/h). The binding of ET-1 was reduced in acidic (pH = 4) and alkaline (pH > 8.5) medium but was stable at near neutral pH (pH 6.5-7.5). Covalent affinity labeling of ET-R with [125I] endothelins (ET-1,2,3) demonstrated that ET-1 and ET-2 bound specifically to three proteins with approximate mol masses of 75, 52, and 34 kDa, whereas [125I]ET-3 bound mainly to a protein with a mol mass of 34 kDa. The binding of [125I]ET-1 to the three mol-mass species was effectively displaced with ET-1 and ET-2. However, ET-3 displaced the binding to the 34-kDa band, only. ET-R were solubilized, in active endothelin-binding forms, with 1% digitonin and 0.1% cholate. Solubilized ET-R sedimented on sucrose density gradients (SDG) containing 0.1% CHAPS, 10% glycerol, and 0.4 M KCl, as 7-8S complexes. Binding of ET-1, ET-3, vasoactive intestinal constrictor (VIC) or sarafotoxin (SRTX-6b) to the solubilized ET-R, and subsequent analysis on SDG, demonstrated similar sedimentation characteristics, suggesting that these peptides are bound to similar receptors.