The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes.
N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000378:metabolism; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000187:drug effects; D002352:Carrier Proteins / Q000032:analysis; D002999:Clone Cells / Q000276:immunology; D003208:Concanavalin A / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D008766:Methylmannosides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D002352:Carrier Proteins; D008766:Methylmannosides; D003208:Concanavalin A; D014107:Tosyllysine Chloromethyl Ketone