cathepsin-g and 4-nitroaniline

Pseudomonas aeruginosa, which may cause severe lung infections, secretes a metalloelastase that may interfere with the assay of neutrophil elastase and cathepsin G in lung secretions. Using nuclear magnetic resonance spectroscopy, we have shown that P. aeruginosa elastase (PsE) cleaves succinyl-Ala3-p-nitroanilide between the first and the second alanine residue, rendering this substrate inefficient for the assay of neutrophil elastase. The cleavage occurs with a kcat/Km of 2.4 X 10(3) M-1 s-1, a value eightfold higher than the kcat/Km for the chromogenic cleavage of succinyl-Ala3-p-nitroanilide by neutrophil elastase. P. aeruginosa elastase also cleaves the elastase substrate succinyl-Ala3-Val-p-nitroanilide between the second and the third alanine residue and the cathepsin G substrate succinyl-Ala2-Pro-Phe-p-nitroanilide at the Pro-Phe linkage. By contrast, methoxysuccinyl-Ala2-Pro-Val-p-nitroanilide, another elastase substrate, is not hydrolyzed by the bacterial enzyme. Our data indicate that synthetic substrates should be used with caution to assay elastase and cathepsin G in lung secretions or other biologic fluids in which metalloproteinases may be present.

Topics: Amino Acid Sequence; Aniline Compounds; Cathepsin G; Cathepsins; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Pancreatic Elastase; Pseudomonas aeruginosa; Serine Endopeptidases; Substrate Specificity

Rat is a likely test animal for determining the efficacy of proteinase inhibitor drugs directed toward human leukocyte elastase and cathepsin G. We therefore sought to assess and compare relevant properties of both human and rat leukocyte elastase and cathepsin G. Some differences between the pairs of proteinases from the two species were found, however both pairs of enzymes displayed comparable specificity toward various natural (plant and animal) proteinase inhibitors and also toward specific peptide substrates and a serine proteinase-specific reagent. Such overlapping specificity implies similarity of reactive center topography and sequence homology around the extended substrate/inhibitor binding regions of these proteinases. This apparent homology leads us to conclude that a pharmacologically effective inhibitor of leukocyte proteinases in the rat would probably also be effective in man.

Topics: Aniline Compounds; Animals; Binding Sites; Cathepsin G; Cathepsins; Chromogenic Compounds; Humans; Hydrogen-Ion Concentration; Kinetics; Leukocytes; Male; Nitrophenols; Pancreatic Elastase; Rats; Serine Endopeptidases; Species Specificity; Substrate Specificity