Receptor binding of selectively labeled (Tyr-10) and (Tyr-13)-mono-125I-glucagons and competition by homologous 127I-labeled isomers.

Two monoiodinated derivatives of glucagon were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The purified (Tyr-10) and (Tyr-13)-mono-125I-labeled glucagon isomers were characterized and studied with respect to their binding to the receptors of isolated intact rat hepatocytes. The extent of steady-state binding to cellular receptor sites differed for the two labeled glucagon tracers at 37 degrees C as well as at 15 degrees C with (Tyr-10)-mono-125I-glucagon displaying higher receptor binding. The apparent equilibrium constants, Kd,app at 37 degrees C are 3.6 +/- 0.4 nM (mean +/- S.E. of three independent experiments) for the tyrosine-13-labeled tracer and 5.9 +/- 0.6 nM for the tyrosine-10-labeled glucagon with native glucagon as competitor. Since the observed Kd in the competition assay is a function of the true Kd values of the monoiodinated radioactive glucagon isomers and native glucagon, the dissociation constants were also measured with chemically identical tracer and competitor. Under these conditions, we obtained Kd values of 1.3 +/- 0.2 nM for the tyrosine-10-labeled analog and 2.0 +/- 0.2 nM for the tyrosine-13-labeled glucagon isomers confirming the higher receptor binding affinity of (Try-10)-mono-125I-glucagon. All competition curves fit the mathematical expression for a model of non-cooperative binding to a single class of receptors.