GUINEA-PIG LUNG TRYPTASE - LOCALIZATION TO MAST-CELLS AND CHARACTERIZATION OF THE PARTIALLY PURIFIED ENZYME

Tryptase (EC 3.4.21.59), the major secretory product of human mast cel ls, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of all ergic disease. However, there is little information available on the p roperties, or even the presence, of this protease in commonly used spe cies of laboratory animals. We, here, report the demonstration and cha racterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to al-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appea red to be present in all mast cells staining with Alcian Blue. A trypt ic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose c hromatography, and gel filtration. This enzyme was found to be multime ric with a subunit of 38 kDa and a native molecular mass of 860 +/- 10 0 kDa. Inhibitor studies identified it as a serine protease. Like huma n tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N -(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride), but n ot by al-proteinase inhibitor, soybean trypsin inhibitor, or antithrom bin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and huma n enzymes were seen in activity toward a panel of 10 tryptic p-nitroan ilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar K -m but a 5-fold lower k(cat) than human tryptase, and with L-Pyr-Gly-A rg-pNA the guinea pig enzyme had a 10-fold lower K-m and a 30% greater k(cat) than its human counterpart. Heparin stabilised guinea pig tryp tase, but did not alter its kinetic parameters as it did with human tr yptase, decreasing the K-m towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guin ea pigs suggests that this species may be an appropriate model to inve stigate the actions of tryptase in vivo, provided cognizance is taken of the differences that do exist.