Activation of uPA by tryptase ϵ. A single-chain pro-uPA was incubated in buffer alone (left lane) or buffer containing pro–tryptase ϵ, active wild-type tryptase ϵ, or its inactive C90A/D92A mutant (lanes 2, 3, and 4, respectively). After a 2-hour incubation at 37°C, the treated samples were subjected to SDS-PAGE under reducing conditions; the resulting gels were stained with Coomassie blue. N-terminal amino acid sequence analysis also was performed on the indicated approximately 33-kDa product detected in the third lane (arrow). (B) Aliquots of the resulting reactions were evaluated for the presence of an active protease that cleaves H-Glu-Gly-Arg-pNA. As noted (▴), tryptase ϵ treatment of pro-uPA resulted in the generation of a mature enzyme that readily cleaved the uPA-sensitive substrate. (C) Time-dependent activation of pro-uPA with tryptase ϵ is shown in the left 4 lanes. In lane 5, tryptase ϵ was added to a 20-fold excess of pro-uPA after incubation with plasma (2% final volume) for 30 minutes at room temperature. Lane 6 on the far right is the control, depicting the proteins in plasma before tryptase ϵ treatment. In a second experiment (data not shown), purified, recombinant tryptase ϵ again quickly activated pro-uPA even in the presence of plasma proteins. (D) pro-tPA and plasminogen were incubated in the absence (-) or presence (+) of active tryptase ϵ. After a 2-hour incubation at 37°C, the resulting reaction products were subjected to SDS-PAGE under reducing conditions. The resulting gel was stained with Coomassie blue. The top and bottom arrows point to plasminogen and pro-tPA, respectively. As noted, tryptase ϵ was unable to activate either zymogen under the tested conditions.