Binding of tyrosine-A-14(125I)-monoiodoinsulin to human erythrocytes.
We have studied the method for the determination of human erythrocyte insulin receptor concentrations using tyrosine-A-14-monoiodoinsulin as the labelled ligand with increasing amounts of unlabelled insulin in a saturation assay. An overnight incubation at 0-+4 degrees C was found to give the highest receptor concentrations and highest affinities for the ligand. Insulin receptor concentrations were found to be very low and lower in erythrocytes from normal females than from normal male subjects (7.0 +/- 1.9 and 10.5 +/- 1.6 receptors per erythrocyte, respectively). Our results suggest that an initially homogenous class of insulin binding sites upon exposure to insulin can appear in different forms with different binding affinities for the ligand. This was deduced from the changes in the forms of Scatchard plots when the saturation assay was performed at different times at low temperature. Variability in the forms of Scatchard plots (linear to biphasic or vice versa) in samples obtained on different occasions from the same subjects also support this view. The apparent dissociation constants (mean values) were 150 and 550 pmol/l for the linear components of the biphasic plots and 300 pmol/l for the linear plot. These values lie well within the normal plasma concentrations of insulin. Addition of spermine to the incubation mixture was shown to further accentuate the high affinity part of the Scatchard plot. The high and low affinity forms of the receptor could provide an ideal means to rapidly regulate the response to insulin.
D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000097:blood; D007457:Iodine Radioisotopes; D008297:Male; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000032:analysis*; D012737:Sex Factors; D013096:Spermine / Q000097:blood
D007328:Insulin; D007457:Iodine Radioisotopes; D013096:Spermine; C029491:insulin, 3-iodo-Tyr(A14)-; D011972:Receptor, Insulin