Purification and partial characterization of an alpha-chymotrypsin-like protease of rat peritoneal mast cells.

An alpha-chymotrypsin-like enzyme was isolated from mast cells of the rat peritoneal cavity by extraction with 0.8 M potassium phosphate, 2 per cent protamine sulfate followed by affinity chromatography on hen ovoinhibitor-agarose and adsorption on barium sulfate. This procedure yielded over 9 mg of protease from the peritoneal lavage fluid of 100 rats, equivalent to 44 per cent of the initial activity. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical isoelectric focusing, and amino-terminal sequence analysis. The protease contains no covalently bound carbohydrate and has a molecular weight of approximately 26,000. The enzyme molecule is a single polypeptide chain with an amino-terminal sequence homologous to that of the B chain of bovine alpha-chymotrypsin. The kinetic parameters, Km and kcat, for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester were determined at pH 8.0 and 25 degrees C as 1.1 X 10(-3) M and 84 sec-1, respectively. The value of the second-order rate constant for inactivation of mast cell protease by diisopropylphosphofluoridate was 300 times lower than for bovine alpha-chymotrypsin.