Fig. 4.

Trypsinogen activity is activated by proteolytic cleavage. (A and B) A full-length mouse trypsinogen5 cDNA from the mouse pancreas was cloned into pcDNA3 (Invitrogen) and expressed in 293T cells. The indicated amounts of cell lysates (2–8 μL of 5 μg/μL lysates) were mixed with a trypsin-specific substrate (BioVision) in the presence or absence of added enteropeptidase. Tryptic activity was monitored by the amount of released pNA, measuring spectrophotometric units (A₄₀₅). (C and D) The effects of Spink3 were examined by adding cell lysates expressing Spink3, a major intrinsic trypsin inhibitor in mouse pancreas, to lysates expressing trypsinogen5 (C) or mouse Prss1 (D). (E) The DNA sequence encoding the activation peptide in the trypsinogen5 expression vector was replaced with sequences encoding a PACE cleavage site (-RTKR-) so that tryptic activity is activated by ubiquitously expressed PACE protease. (F) 293FT cells transfected with PACE-trypsinogen5 or control vector were stained with FITC-labeled annexin V to detect apoptosis.