A rapid method for purification of human granulocyte cationic neutral proteases: purification and characterization of human granulocyte chymotrypsin-like enzyme.
A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001769:Blood; D002417:Cattle; D002918:Chymotrypsin / Q000097:blood* / Q000302:isolation & purification; D003594:Cytoplasmic Granules / Q000201:enzymology; D004355:Drug Stability; D006098:Granulocytes / Q000201:enzymology*; D006801:Humans; D007659:Ketones / Q000494:pharmacology; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D009928:Organ Specificity; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood* / Q000302:isolation & purification; D013045:Species Specificity; D013696:Temperature; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D000596:Amino Acids; D007659:Ketones; D000515:alpha 1-Antitrypsin; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D010196:Pancreatic Elastase