▲ EXPERIMENTAL FIGURE 13-4 Binding assays for cell-surface receptors can determine the Kd for high-affinity ligands and the number of receptors per cell. Shown here are data for insulin-specific receptors on the surface of liver cells. A suspension of cells is incubated for 1 hour at 4° C with increasing concentrations of 125I-labeled insulin; the low temperature is used to prevent endocytosis of the cell-surface receptors. The cells are separated from unbound insulin, usually by centrifugation, and the amount of radioactivity bound to them is measured. The total binding curve A represents insulin specifically bound to high-affinity receptors as well as insulin nonspecifically bound with low affinity to other molecules on the cell surface. The contribution of nonspecific binding to total binding is determined by repeating the binding assay in the presence of a 100-fold excess of unlabeled insulin, which saturates all the specific high-affinity sites. In this case, all the labeled insulin binds to nonspecific sites, yielding curve C. The specific binding curve B is calculated as the difference between curves A and C. From curve B, the Kd for insulin binding (=1.4 X 10"8 M, or 14 nM) and the number of receptor molecules per cell (=33,000) can be determined. [Adapted from A. Ciechanover et al., 1983, Cell 32:267.]

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