REgulation of insulin binding to isolated hepatocytes: correction for bound hormone fragments linearizes Scatchard plots.
Fragments of 125I-labeled insulin (125I-insulin) are rapidly produced after the initial cell binding process. After association of 125I-insulin with hepatocytes, hormone fragments remain bound to cells. At 23 degrees C, approximately 20% of the label bound at steady state was soluble in trichloroacetic acid. Correction of saturation experiments for the presence of bound trichloroacetic acid-soluble insulin fragments decreased the number and increased the affinity of 125I-insulin-binding sites. Label extracted from cell pellets recovered from saturation experiments was characterized by gel filtration; 59%, 55%, 40%, and 36% of the bound label was from intact hormone after recovery from incubation mixtures containing 0.18, 0.60, 4.6, and 7.5 nM applied 125I-insulin, respectively. At high applied 125I-insulin concentrations, the hormone predominantly interacted with lower affinity degradation systems. When binding data were corrected to assay for undegraded 125I-insulin only, curvilinear Scatchard plots were linearized. The insulin receptor is therefore not composed of heterogeneous or negatively cooperative sites. It is necessary to correct for retained fragments of 125I-insulin in order to define mechanisms through which hormone binding and cellular response may be regulated.
D000818:Animals; D001667:Binding, Competitive; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D010446:Peptide Fragments; D011972:Receptor, Insulin