Purification and N-terminal amino-acid sequence analysis of rat polymorphonuclear leukocyte cathepsin G.
Cathepsin G was purified by single-step cation-exchange chromatography from rat polymorphonuclear leukocytes, obtained from the peritoneal cavity after induction of a mild peritonitis. The 26 N-terminal amino acids were determined and showed 73% identity to those of human cathepsin G. Total amino-acid composition demonstrated a high degree of basic amino acids in accordance with its high affinity for the cationic-exchange gel medium. The protein was found to be a glycoprotein with a glucosamine content of 7.4% of the calculated Mr28,900. On SDS/polyacrylamide-gel electrophoresis the protein showed a Mr of 28,400. It migrated as two bands in a gradient SDS/polyacrylamide-gel indicating isoforms. The pH optimum for the proteinase was determined to be 8.0-8.5 using Suc-Ala-Ala-Pro-Phe-Nan as substrate (Suc = 3-carboxypropionyl; Nan = 4-nitroanilide). Km and Kcat/Km values for Suc-Ala-Ala-Pro-Phe-Nan were 0.86mM and 280M-1S-1 and for Suc-Phe-Leu-Phe-Nan 0.24mM and 3600M-1S-1, respectively.
Björk
P
P
Department of Surgical Pathophysiology, University of Lund, Malmö General Hospital.
Ohlsson
K
K
D000595:Amino Acid Sequence; D000818:Animals; D056649:Cathepsin G; D002403:Cathepsins / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry*; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases
D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534097:Ctsg protein, rat