RELEASE OF DOG POLYMORPHONUCLEAR LEUKOCYTE CATHEPSIN-G, NORMALLY AND IN ENDOTOXIN AND PANCREATITIC SHOCK - ISOLATION AND PARTIAL CHARACTERIZATION OF DOG POLYMORPHONUCLEAR LEUKOCYTE CATHEPSIN-G

Dog polymorphonuclear leukocyte cathepsin G was isolated from a granule extract using a two-step procedure including affinity chromatography on a Trasylol-Sepharose gel and ion-exchange chromatography on a CM 52 column. 22 of the first 24 N-terminal amino acids were determined and showed 83% and 71% identity to those of human and rat cathepsin G, respectively. Total amino-acid composition demonstrated the basic nature of the protein. In an SDS/polyacrylamide-gel electrophoresis the protein showed an M(r) of 29 400 compared to the M(r) of 26 800 calculated from the total amino-acid composition. The enzyme was shown to form complexes with alpha-1-alpha-2-macroglobulin and alpha-1-proteinase inhibitor. A specific enzyme-linked immunosorbent assay was developed for the determination of cathepsin G/alpha-1-proteinase inhibitor complex in dog plasma and tissue fluids. The mean concentration of cathepsin G in normal dog plasma was determined to be 38-mu-g/l, measured as cathepsin G/alpha-1-proteinase inhibitor complex. When active dog cathepsin G was added to normal dog plasma in vitro, approximately 56% could be measured by the assay. Slow intravenous infusion of a lethal dose of endotoxin in dogs was followed by a marked drop in white blood cell count and thrombocytes and a simultaneous rapid increase in plasma cathepsin G concentration, reaching a maximum level of 150-mu-g/l. Bile-induced experimental pancreatitis in dogs was accompanied by successive increase in cathepsin G levels in plasma as well as in peritoneal exudates, reaching a maximum level of about 300-mu-g/l in plasma and 18 mg/l in the exudates during the late stages of disease.