Hydrolysis of a chymotrypsin substrate and of naphthol AS-D chloroacetate by human leukocyte granules.

Abstract

The subcellular distribution has been investigated of two esterases from human neutrophil granulocytes obtained from patients with chronic myelocytic leukemia. One esterase hydrolyzed the typical chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester, the other hydrolyzed naphthol AS-D chloroacetate, a substance which is used in cytochemistry for the demonstration of esterase activity. Both enzymes could be recovered almost exclusively in the granule fraction and were optimally active at about pH 7.4. After lysis of the granules by hypoosmotic shock 73% of the naphthol AS-D chloroacetate esterase activity could be dissolved in buffered 0.15M sodium chloride (pH 7.0) in contrast to only 17% of the N-acetyl-L-tyrosine ethyl esterase activity. The soluble lysate contained 59% of the total granule protein. It was subjected to acrylamide-gel electrophoresis at acid pH and multiple bands of naphthol AS-D chloroacetate esterase activity were demonstrated on the gels. Acrylamide-gel electrophoresis of the insoluble proteins and the soluble lysate of the granule fraction was carried out after dissolution of the proteins in phenol-acetic acid-urea and the resulting electrophoretic patterns were compared. Hydrolysis of naphthol AS-D chloroacetate by the granule fraction was compared to hydrolysis of the same substrate effected by the pure proteases chymotrypsin, trypsin, and pancreatopeptidase E.