Identification of photoaffinity labeled insulin receptor proteins by linear polyacrylamide gradient gel electrophoresis under non-denaturing conditions

Abstract

Photoaffmity probes have been used to selectively label functional components in a number of membrane systems [l-lo]. To identi’fy the labeled molecules, membranes were subsequently solubilized with sodium dodecyl sulfate (SDS) and analyzed by SDSpolyacrylamide gel electrophoresis. A major disadvantage of this technique is that non-covalent interactions among proteins are abolished, precluding the detection of proteins whose functional state depends on a complex subunit structure. On the other hand, it has been shown that a number of membrane proteins can be extracted from the membrane under non denaturing conditions, thus affording their functional integrity [ 111. This observation has prompted us to examine the possibility of performing the photolabeling reaction in the presence of non-ionic detergent. In this communication we report the covalent linking of a photosensitive, radioactive insulin analogue to proteins previously solubilized from porcine liver membranes. Electrophoresis of the reaction mixture on linear polyacrylamide gradient gels showed that proteins migrating at positions corresponding to an MW of 300 000 and 600 000, respectively, were specifically labeled, suggesting these proteins to be the non-denatured binding components of the insulin receptor.