Preliminary data on isolation of an elastase-like proteinase and its inhibitor from ovine neutrophil granulocytes.
Ovine polymorphonuclear leukocytes (PMNs) were purified by counterflow centrifugation of the buffy coat. The resulting cell preparation was gently sonicated and ultracentrifuged for separation of cytosol and cell organelles. The proteolytic activity of the isolated enzyme was determined with the specific substrate MeO-Suc-Ala-Ala-Pro-Val-Nan (methoxysuccinyl-[L-alanyl]2-L-prolyl-L-valine p-nitroanilide). Elastase-like activity was identified in the insoluble (particular) fraction, while an inhibitor of elastase activity was found in the supernatant (cytosolic fraction). Further purification of the elastase-like enzyme was achieved by cation exchange chromatography (SP-Sephadex C-25 and CM-Sephadex C-25) and gel permeation chromatography (Bio-Gel P-30). Purification of the inhibitor from cytosol was accomplished using anion exchange chromatography (DE32 diethylaminoethyl cellulose) and two gel permeation steps (Sephadex G-75 and TSK-G 2000 SW - HPLC column). Degrees of purity in all separation steps were controlled with SDS-polyacrylamide gel electrophoresis. The molecular mass of the elastase-like enzyme is in a range of 25 to 27 kDa. The isoelectric point is between 8.0 and 9.0. Three isoenzymes were found. The optimal activity lies at pH 8.0. The molecular mass of the inhibitor is about 40 kDa.
Junger
W
W
Ludwig-Boltzmann-Institut für experimentelle Traumatologie, Wien.
Hallström
S
S
Redl
H
H
Schlag
G
G
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003594:Cytoplasmic Granules / Q000032:analysis; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000032:analysis* / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000032:analysis*; D011480:Protease Inhibitors / Q000032:analysis*; D012756:Sheep
D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase