KINETIC ANALYSES DEMONSTRATE THAT THE EQUILIBRIUM ASSUMPTION DOES NOT APPLY TO [I-125] ENDOTHELIN-1 BINDING DATA

The kinetics of [I-125]Endothelin-1 ([I-125]ET-1) binding were studied using membranes from rat heart, rat lung, rat brain, and porcine vascular smooth muscle at 37-degrees-C in 0.05M Tris-HCl buffer (pH=7.4). The dissociation half-life (t1/2, diss.) for bound [I-125]ET-1 was in excess of 30 hours for each tissue studied. Equilibrium-time requirements for proper Scatchard analysis of [I-125]ET-1 were also far in excess of 30 hours for each tissue. These data suggest that determination of dissociation constants, K(d), and receptor concentrations, B(max), by conventional Scatchard analysis is not feasible with [I-125]ET-1. Kinetic analyses may provide a more accurate means for determining [I-125-ET-1] binding characteristics including K(d) and B(max).