Purification and characterization of the chymotrypsin-like enzyme of the bovine granulocyte

A chymotrypsin-like enzyme (EC 3.4.21.20) was isolated from bovine granulocytes, and purified 14-fold by affinity chromatography on 4-phenylbutylamine Affi-gel. The molecular weights of the isoenzymes were estimated as 16 000, 19 300 and 24 000 by SDS-polyacrylamide gel electrophoresis. A Michaelis constant of 2 mM and a catalytic constant of 34.6 s⁻¹ were determined with Bz-Tyr-OEt. The esterolytic activity of the enzyme could be inhibited both by PMSF and by TPCK. It was also inhibited by chymostatin ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.11 μ}\text{g/ml}$) and by the cytosol inhibitor of the bovine granulocyte ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{′ = 1 μ}\text{M}$). The chymotrypsin-like enzyme of the bovine granulocyte shares a number of the kinetic properties common to the chymotrypsin-like enzyme of the human granulocyte. The two granulocytes showed nearly identical chymotrypsin-like enzyme activities per cell.