A Rapid Procedure for the Large Scale Purification of Elastase and Cathepsin G from Human Sputum

Martodam, Raymond R.; Baugh, Robert J.; Twumasi, D Y; Liener, Irvin E.

doi:10.1080/00327487908061669

Cited by 95 publications

(46 citation statements)

References 18 publications

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“…Human NE was purified from purulent sputum as described by Martodam et al (1979). The batch used in the present work was 9000 active, as judged by active-site titration (Powers et al, 1984) done with acetyl-Ala-2-AzaAla-pnitrophenyl ester (Enzyme System Product, Livermore, CA, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

Boudier

Bieth

1994

Biochemical Journal

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N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-l s-'. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass = 2.6 x 10' M-1 s-', dissociation rate constant kdiSs = 2.9 x10-3 s-s and equilibrium dissociation constant Ki =

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Section: Methodsmentioning

confidence: 99%

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

Boudier

Bieth

1994

Biochemical Journal

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“…Human leukocyte elastase (HLE) was isolated from purulent sputum (8) and active site-titrated using acetyl-Ala 2 -AzaAla-p-nitrophenyl ester (9) from Enzyme System Products (Livermore, CA). Porcine trypsin and bovine chymotrypsin were purchased from Biosys (Compiègne, France) and active site-titrated using p-nitrophenyl p-guanidinobenzoate (10) and N-trans-cinnamoylimidazole (11) from Sigma, respectively.…”

Section: Enzymesmentioning

confidence: 99%

Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet

Kellenberger

Boudier

Bermúdez

et al. 1995

Journal of Biological Chemistry

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Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel ␤-sheet were studied. Their inhibitory effect on two serine proteases (bovine ␣-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong ␣-chymotrypsin inhibitor (K i ‫؍‬ 0.2 nM) and interacts with leukocyte elastase with a K i of 0.12 M. The other two peptides, PMP-D2 and HI, interact only weakly with ␣-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both ␣-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a K i of 3 nM.In the last decade, naturally occurring serine protease inhibitors (1) have been the focus of many studies, mainly for two reasons: first, the target proteins control functions in a variety of fundamental proteolytic processes in humans and mammals (blood clotting, digestion, inflammation, fibrinolysis), in invertebrates such as insects (immune system, digestion, protection against their predators) or worms (protection against their host), and plants (protection against insect attack); second, low molecular weight inhibitors of serine proteases have been attractive tools for studying the general aspects of protein conformation and protein-protein interactions (2). In the present study, we report the inhibitory properties of three homologous peptides (primary and tertiary structures) toward ␣-chymotrypsin, trypsin, and human leukocyte elastase.We have previously isolated two peptides, PMP-C and PMP-D2, from the brain and the fat body of the insect Locusta migratoria (3). These peptides are composed of 36 and 35 residues, respectively, and are cross-linked by three disulfide bonds. The Thr-9 of PMP-C has an uncommon O-glycosidic linkage to a single fucose moiety. There is 40% strict identity between PMP-C and PMP-D2 with conservation of the Cys positions (3). Moreover, they are located on the same peptidic precursor, and, by Northern blot analysis, it has been shown that the gene encoding this precursor is mainly transcribed in the fat body (4). In this paper, we describe the isolation and characterization of HI, a novel locust peptide.Because the isolation from insect extracts is time-consuming and yields only small amounts of peptides, we have prepared, at a reasonable scale (5-10 mg), by solid-phase synthesis, PMP-D2 (5) and PMP-C with and without the fucose moiety. 1Although they are small peptides with a high disulfide content, no sequence similarities could be found when comparing them with small toxins or small protease inhibitors. However, the milligram quantities of PMP-D2 obtained by solid-phase synthesi...

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“…FPM treatment by human leucocyte elastase HLE was isolated and purified from purulent expectorated sputum (Martodam et al, 1979) and stored at -20°C until used. For analysing the response of the FPM to the leucocyte protease, 100 pl of HLE 250 pg/ml in MEM-HEPES was introduced through the upper hole of the Swinnex filters and deposited over the epithelial surface of nine different FPM preparations.…”

Section: Adherence Systemmentioning

confidence: 99%

Adherence of Pseudomonas aeruginosa to respiratory epithelium and the effect of leucocyte elastase

Plotkowski

Beck²,

Tournier³

et al. 1989

Journal of Medical Microbiology

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Summary. The tracheobronchial secretions from patients with cystic fibrosis often contain high amounts of free proteases. To evaluate whether human leucocyte elastase (HLE) can favour the persistence of bacterial airways infection, we exposed the frog palate mucosa to HLE and then to radiolabelled Pseudomonas aeruginosa and followed the sequence of events by scanning electronmicroscopy. In response to HLE there was a marked outpouring of mucus and a desquamation of the epithelium. P. aeruginosa was shown to adhere to recently secreted granules of mucus and to the exposed submucosal underlying connective tissues. For the eight different bacterial strains studied, a significative adherence to HLE-injured mucosa was observed only in strains that possessed internal haemagglutinating activity. Neither the presence of fimbriae, nor of the mucoid exopolysaccharide, nor of the bacterial surface haemagglutinating activity could be related to adherence of P. aeruginosa to the injured mucosa. These results support the hypothesis that HLE enhances bacterial infection of the respiratory mucosa both by inducing mucus hypersecretion and by exposing receptors to the microbial adhesins. It is also suggested that P. aeruginosa internal lectins may be implicated in adherence to host tissues.

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A Rapid Procedure for the Large Scale Purification of Elastase and Cathepsin G from Human Sputum

Cited by 95 publications

References 18 publications

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet

Adherence of Pseudomonas aeruginosa to respiratory epithelium and the effect of leucocyte elastase

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