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Insulin-like growth factor II receptors. Molecular radius and molecular weight determination using quantitative
Abstract:
High resolution quantitative polyacrylamide gel electrophoresis was employed under nondenaturing conditions to calculate a molecular weight, Mr, for rat placental membrane receptors that bind insulin-like growth factor II (IGF-II). An n-octylglucoside-soluble extract of receptors that had been enriched 20-fold during Sephacryl S-300 gel chromatography and designated peak 1 (Perdue, J. F., Chan, J. K., Thibault, C., Radaj, P., Mills, B., and Daughaday, W. H. (1983) J. Biol. Chem. 258, 7800-7811) was incubated with 125I-IGF-II in the presence or absence of an excess of unlabeled IGF-II and electrophoresed in glass tubes containing highly cross-linked polyacrylamide gels at concentrations ranging from 5 to 12% (w/v). A specifically labeled peak of IGF-II binding activity was identified by freezing, slicing, and counting the gels. The proteins eluted from the region of the gel when cross-linked to 125I-IGF-II with disuccinimidyl suberate and electrophoresed in the presence of sodium dodecyl sulfate have the same molecular weight, Mr, as the previously described IGF-II receptor. From the slope of a plot of log of the relative mobility, RF, at each of 6 to 7 gel concentrations for the 125I-IGF-II-receptor and for each of seven standard proteins (Ferguson-Hedrick plot), a retardation coefficient, KR, was determined. Using a reference curve constructed from a plot of square root KR versus the molecular radius, R, of the standard proteins, the IGF-II receptor was estimated to have a R of 4.13 nm and a calculated Mr of 250,000. Thus, quantitative procedures that separate native proteins based on their size and charge have provided information which is in good agreement with the results of studies of the IGF-II-receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and supports the premise that it is a monomeric single chained glycoprotein, constrained by intradisulfide bonds and with a mass of 250 kDa.