The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes.

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Abstract
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.