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 molecules. The possibility of using biotinylation and chemical cross-linking 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% Triton X-100 lysates of murine thymocytes surface biotinylated in the presence of the homobifunctional cross-linker DTSSP [3,3'-dithio-bis(sulfosuccinimidylpropionate), 0.2 mg/ml] revealed that the surface molecules 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 Jurkat, biotinylated after permeabilization of the cells with lysolecithin (20-25 micrograms/ml), revealed several additional coprecipitating molecules of 16, 32, 34, 56, 60 and 80 kDa not detected in immunoprecipitates from lysates of surface-biotinylated cells. The failure to detect these molecules in lysates of surface-biotinylated cells suggests that these molecules biotinylate poorly by cell surface labeling, or are localized intracellularly. The intracellular localization of some of these molecules is supported by the fact that the 56-kDa molecule seen in CD45 mAb immunoprecipitates from lysates of Jurkat cells biotinylated in the presence of lysolecithin could be identified as the src-like tyrosine kinase p56lck by immunoblotting with p56lck Abs.(ABSTRACT TRUNCATED AT 250 WORDS)
Altin
J G
JG
Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra City, Australian Capital Territory.
Pagler
E B
EB
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005260:Female; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D051379:Mice; D008808:Mice, Inbred CBA; D010539:Permeability; D013601:T-Lymphocytes / Q000378:metabolism*
D017252:CD3 Complex; D003432:Cross-Linking Reagents; D008244:Lysophosphatidylcholines; D001710:Biotin; D017493:Leukocyte Common Antigens