Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-1 00. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak 11) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak 11). Both peaks were glycoproteins. At 4°C peak 1 showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak I1 had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak I1 an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4°C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak 11).Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with M , 400000, 365000, 320000, 290000, and 245000 under non-reducing conditions. For peak I1 two major receptor bands with M , 210000 and 115000 were found. The peak I1 receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with M , 130000. The reductive generation of the peak I1 receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak I1 receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.

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The binding of the 1251-labelled insulin-like growth factors I and I1 (lZ5I-IGF I and 1251-IGF 11) to the highmolecular-mass binding protein of human serum was characterized. With diluted human serum both growth factors showed optimal specific binding at 4°C and pH 5-6. When 0.1% Triton X-100 was present in the incubation buffer an increase in the affinity of the IGF-binding protein was induced, which produced an enhanced binding of IGF I and IGF 11. Competition experiments with various peptide hormones revealed that the native IGF-binding protein complex binds both the IGF I and IGF I1 with high specificity. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF I and IGF I1 respectively, indicating that in human serum only a single class of non-interacting binding sites is present. At optimal binding conditions the dissociation constants were determined to be 0.28 x Human serum was gel-filtered on Sepharose CL-6B at neutral pH and the eluate was assayed for binding activity with both IGF I and IGF 11. One peak with an apparent molecular mass of 175 kDa and a Stokes radius of 4.8 nm was determined for both growth factors. Thus, our data suggest that human serum contains one class of high-molecular-mass binding protein with comparable binding characteristics for IGF I and IGF 11.M for IGF I binding and 0.66 x M for IGF 11.The insulin-like growth factors (IGFs) are a family of polypeptide hormones that have close structural and functional homologies with insulin. They elicit classical insulin effects on all target tissues of insulin and show potent in vitro growth-promoting activities for many different cultured cells (reviewed by [l, 21). Unlike insulin and other peptide hormones, the IGFs circulate in serum and other body fluids associated with specific binding proteins [3 -51. Two forms of IGF-binding proteins with apparent molecular masses of about 150 -200 kDa (175-kDa binding protein) and 40 kDa (40-kDa binding protein) have been described. In normal and acromegalic serum the 175-kDa binding protein has been found to be more abundant [6 -81, while in fetal serum, amniotic fluid and cerebrospinal fluid the 40-kDa binding protein predominates [9 -111.Biosynthesis of the 175-kDa binding protein is known to be regulated by the growth hormone. In human serum it is associated with the majority of circulating IGF [12-141. Under acid conditions, the native IGF is released from the 175-kDa protein while the binding protein itself dissociates into the acid-stable IGF-binding subunit (53 kDa) and at least one acid-labile subunit, which appears not to be directly involved in the binding of IGF [6, 15, 161. However, attempts to reconstitute the 175-kDa binding protein from its subunits dissociated under acid conditions have not been successful [6]. Therefore most investigators have used the acid-stripped binding protein subunit to characterize the ligand-binding properties after the removal of endogenous IGFs by gel filtration chromatography under acid pH conditions. In this study...

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