Characterization of insulin-binding to the hepatoma cell line H35

We have studied the characteristics of insulin-binding to its receptor in H35 hepatoma cells. Insulin-binding was time, temperature and pH dependent. Optimum pH was 8.0-8.2. Binding at 21 degrees C reached a steady state after 90 min of incubation at a level of 30.0 +/- 2.6% per mg protein. Pre-incubation of the cells with unlabelled exogenous insulin resulted in a decrease of insulin-binding which was time and concentration dependent. Pre-incubation with 10 micrograms/ml for 24 h resulted in a decrease to 35-40% of initial binding. Scatchard plots of the binding data were curvilinear in control as well as in down regulated cells. Analysis of the Scatchard plots revealed that decrease of insulin-binding to down regulated cells was due to a decrease of insulin-binding sites, while affinity constants did not change.