The biochemical characterization of detergent-solubilized insulin-like growth factor II receptors from rat placenta

A membrane preparation, the R3, obtained by differential centrifugation of rat placental homogenates is enriched in receptors that bind insulin-like growth factor II (IGF-II) preferentially and with avidity (Daughaday, W.H., Mariz, I.K., and Trivedi, B. (1981) J. Clin. Endocrinol. Metab. 53, 282-288). When this preparation was incubated with 2% (w/v) octyl-beta-D-glucopyranoside for 60 min at 0-4 degrees C, 60% of the membrane protein was solubilized without loss of binding activity. The 125I-IGF-II binding properties of the detergent-solubilized receptors were found to be similar to those of the membrane-associated receptor. The rate constants for association, ka, and dissociation, kd, and equilibrium dissociation constant, KD, were 8.5 X 10(8) M-1 min-1, 7.5 X 10(-3) min-1, and 1.3 nM for the detergent-solubilized receptors and 5.3 X 10(8) M-1 min-1, 4.2 X 10(-3) min-1, and 0.6 nM for the membrane receptors. Gel chromatography on Sephacryl S-300 concentrated the solubilized receptors into a major peak of binding activity with a Stokes radius of 7.2 nm; a second peak of less specific binding had a Stokes radius of 4.3 nm. The receptors in the major peak bound 125I-IGF-II with a KD of 0.6 nM; the total binding capacity, Ro, was 21.6 pmol mg of protein-1 compared to 1.6 pmol mg of protein-1 for the membrane-associated receptor. Centrifugation of the receptors on 5-20% (w/v) gradients of sucrose in H2O or D2O disclosed a heterogeneous pattern of receptor distribution. When they were labeled with 125I-IGF-II prior to centrifugation, a major form of the receptor with a sedimentation constant, S20,w, of 9.9 X 10(13) s and other, possibly smaller, forms of the receptor were observed. However, only the 9.9 s20,w form of the receptor was observed if it was labeled with 125I-IGF-II subsequent to centrifugation. Based on these hydrodynamic measurements and a partial specific volume of 0.72 cm3/g, the IGF-II receptor was calculated to have a Mr of 290,000 and frictional ratio, f/fo, of 1.6. This value for the Mr is similar to the mass of 220,000 or 250,000 Dal determined by cross-linking 125I-IGF-II to the membrane- or detergent-solubilized receptors with disuccimidyl suberate and separating the complex by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels in the absence or presence of dithiothreitol, respectively.