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% 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% 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 MW of .apprx. 26,000. The enzyme molecule is a simple polypeptide chain with an amino-terminal sequence homologous to that of the B chain of bovine .alpha.-chymotrypsin. The kinetic parameters, Km and kcat [catalytic rate constant], for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester were determined at pH 8.0 and 25.degree. C as 1.1 .times. 10-3 M and 84 s-1, respectively. The value of the 2nd-order rate constant for inactivation of mast cell protease by diisopropylphosphofluoridate was 300 times lower than for bovine .alpha.-chymotrypsin.