Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases.
We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D053818:Chymases; D004283:Dog Diseases / Q000201:enzymology*; D004285:Dogs; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D012515:Mast-Cell Sarcoma / Q000201:enzymology / Q000662:veterinary*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000097:blood*; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D012878:Skin Neoplasms / Q000201:enzymology / Q000662:veterinary*
D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D053818:Chymases