THE HUNT FOR THE MYSTERIOUS ENZYME Y

Background and Aims: Enzyme Y was described as an undefined proteolytic activity derived from purified human cationic tryspinogen, which is capable of rapid degradation of human cationic and anionic trypsinogens to inert products. It was proposed that enzyme Y acts as a protective factor in the pathogenesis of pancreatitis (Rinderknecht et al. 1988. A possible zymogen self-destruct mechanism preventing pancreatic autodigestion. Int J Pancreatol 3, 33-44). The aim of the present study was to investigate whether enzyme Y is generated from cationic trypsinogen or it is a protease contaminating the cationic trypsinogen preparation. Methods: Native human trypsinogens were purified from pancreatic juice with anion-exchange and ecotin-affinity chromatography. Recombinant trypsinogens were expressed in Escherichia coli or HEK 293T cells. Trypsinogen degradation was measured with activity assays and SDS-PAGE. Results: Incubation of purified native cationic trypsinogen at 37 °C in the presence of 0-0.1 mM calcium concentrations resulted in full degradation of the zymogen within 60 min, without a significant release of active trypsin. In striking contrast, under the same conditions recombinant cationic trypsinogen suffered minimal degradation and significant autoactivation was observed. Pancreatic secretory trypsin inhibitor (PSTI) added at the beginning of the incubation completely prevented trypsinogen degradation, whereas it had no protective effect when added at 5 min. Calcium in 1-10 mM concentrations completely inhibited trypsinogen degradation. Conclusions: The results demonstrate that enzyme Y is not derived from cationic trypsinogen, but appears to be a contaminating protease in native cationic trypsinogen preparations. The time-dependent effect of PSTI on enzyme Y activity suggests that trypsin is required to activate enzyme Y from a zymogen form, but active enzyme Y itself is not PSTI-sensitive. Consequently, it is very likely that enzyme Y is identical to one or more of the known pancreatic chymotrypsins or elastases. Currently, experiments are underway to generate recombinant forms of these enzymes and test their ability to degrade trypsinogens.