Tryptase from human pulmonary mast cells. Purification and characterization.
Tryptase, the predominant neutral protease in human mast cell secretory granules, was purified to homogeneity from dissociated and concentrated pulmonary mast cells by sequential chromatography on Dowex 1-X2, DEAE-Sephadex, and heparin-agarose. Purified tryptase gave a single stained protein band on polyacrylamide gels after electrophoresis at pH 4.3 in the presence of 4 M urea. The enzyme has an apparent molecular weight of 120,000 to 140,000 by gel filtration chromatography. Electrophoresis of purified tryptase under denaturing conditions revealed subunits with molecular weights of 37,000 and 35,000 in a molar ratio of 1:1, consistent with a tetrameric subunit structure for the holoenzyme of Mr = 144,000. Both subunits bind [3H]diisopropyl fluorophosphate as assessed by the correspondence of radioactivity with the two stained protein bands in a polyacrylamide gel after electrophoresis of purified tryptase under denaturing conditions, indicating that all four subunits of the holoenzyme may have active site capacity. Purified tryptase has a specific activity for tosyl-L-arginine methyl ester of 97 units/mg (1 unit = 1 mumol of substrate cleaved/min at 22 degrees C). Human pulmonary mast cells contain tosyl-L-arginine methyl ester-esterase at levels more than 100-fold higher than those of human neutrophils, eosinophils, and monocytes. One million mast cells contain about 1.1 units, or 6 to 19 micrograms of tryptase, and have the capacity to contribute dominant levels of this enzyme at tissue sites of mast cell degranulation.
D000596:Amino Acids / Q000032:analysis; D003594:Cytoplasmic Granules / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D008407:Mast Cells / Q000201:enzymology*; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000302:isolation & purification*
D000596:Amino Acids; D007531:Isoflurophate; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase