Isolation and characterization of porcine leukocyte elastase. Leukocyte elastase-inhibitor complexes in porcine blood, II.
Porcine leukocyte elastase was purified from granulocytes by chelating chromatography on copper chelate Sepharose and by ion exchange chromatography on CM-Sepharose. Thus an enzyme preparation with a specific activity (substrate: MeOSuc(Ala)2ProValNan) of 89.3 U/mg protein was obtained. Dodecyl sulphate gel electrophoresis revealed one protein band corresponding to a molecular mass of 27 kDa. The amino acid composition was determined and isoleucine was identified as the only N-terminal amino acid residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 2000 1 . mol-1 . min-1. The dissociation constants, Ki, of the complexes of porcine leukocyte elastase with various inhibitors were calculated. The kinetic constants for the elastase-catalysed hydrolysis of MeOSuc(Ala)2ProValNan, Suc(Ala)2ValNan and Suc(Ala)3Nan were determined, as well as the kinetic constants of the inactivation of leukocyte elastase by active site mapping reagents. Detergents such as Triton X-100, Tween 20 and Brij 35, as well as porcine serum albumin, activated the porcine leukocyte elastase preparation.
D000818:Animals; D004355:Drug Stability; D007962:Leukocytes / Q000201:enzymology*; D046911:Macromolecular Substances; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015843:Serpins; D013552:Swine; D013816:Thermodynamics
D046911:Macromolecular Substances; D011480:Protease Inhibitors; D011506:Proteins; D015843:Serpins; D010196:Pancreatic Elastase