Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes.

We describe the purification of a T cell specific serine proteinase derived from a cloned murine cytolytic T lymphocyte line. Analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mol. wt of approximately 60 kd under non-reducing conditions and of approximately 30 kd under reducing conditions. The proteinase cleaves the model peptide substrate H-D-Pro-Phe-Arg-NA, at the 4 nitroanilide (NA) group with high efficiency. Much lower or no activity of the enzyme is found against synthetic peptide substrates carrying other amino acid (AA) sequences at position P2, P3 adjacent to L-arginine or against substrates in which AA other than L-arginine are bound to the NA group. Optimal pH for the cleavage of H-D-Pro-Phe-Arg-NA is in the range of 8.0-8.5. The enzyme is strongly inhibited by inhibitors of serine proteinases, diisopropylfluorophosphate, phenylmethane-sulfonyl fluoride, m-aminobenzamidine, aprotinin, and leupeptin but not by inhibitors of either thiol-, metallo- or carboxyl-proteinases. We propose the designation TSP-1 (T-cell derived serine proteinase 1) for this enzyme. TSP-1 has the capacity to stimulate B lymphocytes for proliferation in the absence of antigen.