A ONE-STEP PROCEDURE FOR BIOTINYLATION AND CHEMICAL CROSS-LINKING OF LYMPHOCYTE SURFACE AND INTRACELLULAR MEMBRANE-ASSOCIATED MOLECULES

The use of cell surface biotinylation has become a popular alternative to radioiodination in studies involving labeling of cell surface mole cules. The possibility of using biotinylation and chemical cross-linki ng to label and covalently link associated molecules in T lymphocytes was explored using immunoprecipitation, SDS-PAGE analysis, and protein detection by enhanced chemiluminescence (ECL). Reduced and nonreduced SDS-PAGE analysis of CD45 and Thy-1 mAb immunoprecipitates from 1% Tr iton X-100 lysates of murine thymocytes surface biotinylated in the pr esence of the homobifunctional cross-linker DTSSP [3,3'-dithio-bis (su lfosuccinimidylpropionate), 0.2 mg/ml] revealed that the surface molec ules CD45 and Thy-1 can be biotinylated and chemically linked, and is consistent with a previous report using cell surface radioiodination. Also, CD45 and CD3 mAb immunoprecipitates from 1% digitonin lysates of the murine T cell clone D10 and of the human leukemic T cell line Jur kat, biotinylated after permeabilization of the cells with lysolecithi n (20-25 mu g/ml), revealed several additional coprecipitating molecul es of 16, 32, 34, 56, 60 and 80 kDa not detected in immunoprecipitates from lysates of surface-biotinylated cells. The failure to detect the se molecules in lysates of surface-biotinylated cells suggests that th ese molecules biotinylate poorly by cell surface labeling, or are loca lized intracellularly. The intracellular localization of some of these molecules is supported by the fact that the 56-kDa molecule seen in C D45 mAb immunoprecipitates from lysates of Jurkat cells biotinylated i n the presence of lysolecithin could be identified as the src-like tyr osine kinase p56(lck) by immunoblotting with p56(lck) Abs. The results show, therefore, that certain associated cell surface and intracellul ar proteins can be biotinylation and chemically linked using a single- step procedure. We expect that the technique will further broaden the use of biotinylation and nonradioactive protein detection, particularl y in studies characterizing the molecular associations of cell surface receptors with other molecules, either on the cell surface or inside the cell. (C) 1995 Academic Press, Inc.