Adipocyte insulin-binding species: The size and subunit composition of the larger binding species

Several investigators have reported that there are both large and small insulinbinding proteins in plasma membranes; the larger protein demonstrates nonlinear Scatchard binding, and the smaller protein has linear binding. We now present evidence that the larger insulin-binding species consists of four proteins of different sizes. Rat epididymal adipocyte plasma membranes were prebound with ¹²⁵I-insulin and then exposed to 1 mM disuccinimidyl suberate for 15 min at 2°C. The membranes were solubilized in 0.1% Triton X-100 and applied to a Sepharose 6B column. Peaks of radioactivity from the column were dialyzed, lyophilized, and analyzed by dodecyl-sulphate gel electrophoresis (5%, 100/1; mono/bisacrylamide). Autoradiograms of the gels were scanned with a densitometer. The Sepharose chromatogram revealed four radioactive peaks: peak 1 at column void volume; peak 2, K_av = 0.27; peak 3, K_av = 0.77; and peak 4, K_av = 1.09. Dodecyl sulphate electrophoresis of fractions in peak 2 demonstrated four bands on autoradiography; peak 1 did not enter the gel and peaks 3 and 4 ran with the dye front. Molecular weight estimates of the four insulin-binding species in peak 2 were 600, 500, 420, and 350 K. On dithiothreitol reduction each insulin-binding species yielded subunits of M_r ? 135 and 18 K. The three largest binding species demonstrated an additional 45-K dalton protein on dithiothreitol reduction, and the 500-K and 420-K binding species also yielded a 49-K dalton protein. These results suggest that the large insulin-binding protein in rat epididymal adipocytes contains several insulin-binding species, and that these insulin-binding species differ in the number of and the type of subunits they contain. In addition, it may be postulated that the nonlinear Scatchard binding associated with the larger binding protein is a consequence of the heterogeneity of the insulin-binding species in this Sepharose peak.