Isolation of adipocyte plasma membrane antigens by immunoaffinity chromatography. Insulinomimetic antibodies do not bind directly to the insulin receptor or the glucose transport system.

Antiserum against rat adipocyte intrinsic plasma membrane proteins raised in rabbits has previously been shown to mimic insulin action on rat adipocyte glucose transport activity and lipolysis (Pillion, D. J., and Czech, M. P. (1978) J. Biol. Chem 253, 3761-3764). In the present studies, extraction of lipids from adipocyte membranes previously treated with dimethylmaleic anhydride to remove extrinsic proteins resulted in no loss of antigenic activity indicating that a lipid or glycolipid is not a major antigenic determinant inthe intrinsic membrane protein preparation. Periodate treatment of adipocyte membranes abolished subsequent antigen-antibody interaction. Immobilized wheat germ agglutinin or concanavalin A adsorbed all of the antigenic components of detergent-solubilized adipocyte intrinsic membrane proteins and these membrane antigens were released upon incubation with N-acetylglucosamine or a-methyl-D-mannoside, respectively. The purified immunoglobulin fraction of antiserum against intrinsic membrane proteins was immobilized by reaction with CNBr-Sepharose to form an immunoaffinity column, and cholate-solubilized intrinsic membrane proteins were passed through the column. The 94,000 dalton glycoprotein fraction present in the original sample was adsorbed by the column, whereas cytochalasin B-sensitive D-glucose transport activity could be reconstituted from the material which did pass through the column. Elution of the column with 5 M Nal in order to release bound antigens produced a material enriched in the 94,000 dalton glycoprotein as well as several other proteins. ‘261-Insulin was cross-linked to its receptor on the adipocyte plasma membrane as previously described (Pilch, P. F., and Czech, M. P.