Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme.
Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-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 appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.
McEuen
A R
AR
Immunopharmacology Group, Southampton General Hospital, UK.
He
S
S
Brander
M L
ML
Walls
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D053818:Chymases; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D053802:Tryptases
D011480:Protease Inhibitors; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases