A rapid method for the measurement of prostaglandin receptors

A method is presented which provides for a simple and rapid determination of prostaglandin receptors in large numbers of tissue samples. Binding of prostaglandin F2 alpha, to preparations of microsomal membranes obtained from the ovaries of PMSG/hCG-treated rats served as the model system for this study. After incubating the membranes with [3H]-PGF2 alpha +/- graded concentrations of PGF2 alpha for 90 min at 22 degrees C, dextran-coated charcoal was added to adsorb the free PGF2 alpha during a 10 minute period. Centrifugation of the mixture for 3 minutes atop a 2.2M sucrose cushion resulted in the charcoal pelleting to the bottom while the [3H]PGF2 alpha-membrane complex remained at the interface. There was no further significant diffusion of PGF2 alpha after centrifugation, enabling large numbers of samples to be handled within a short period of time. The binding data revealed both high and low affinity receptors: Kd = 5.8 nM and 77 nM with binding capacities of 360 and 4000 fmole/mg protein. Both PGE1 and PGF1 alpha partially displaced [3H]PGF2 alpha from the binding sites whereas PGA2 was without effect. Binding equilibrium was reached within 90--140 min. The results are compatible with those of investigators using other techniques.