A colorimetric method for measuring the esterolytic activity of elastase

Bieth, Joseph G.; Meyer, Jean-François

doi:10.1016/0003-2697(73)90459-4

Cited by 25 publications

(6 citation statements)

References 7 publications

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“…The purified cathepsin G migrated as a single broad band on 7.5% polyacrylamide disc gels run at pH 4 (24) and as a single band ofapparent Mr 30,000 on NaDodSO4/7.5% polyacrylamide-slab gels. It readily hydrolyzed the chymotrypsin substrate N-acetyl-DL-phenylalanine ,3naphthyl ester (25) but was free of HLE as judged by polyacrylamide gel electrophoresis and by nearly complete lack of activity against the synthetic HLE substrate succinyl-Lalanyl-L-alanyl-L-alanyl-p-nitroanilide (26). Lactoferrin was purified from human breast milk (27) and was fully saturated with iron.…”

mentioning

confidence: 99%

Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.

Campbell¹

1982

Proc. Natl. Acad. Sci. U.S.A.

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Interactions between polymorphonuclear neutrophils and mononuclear phagocytes are potentially of great importance in a variety of inflammatory processes. As part of a continuing effort to elucidate the physiologic importance of human alveolar macrophage receptor-mediated binding of neutrophil Oleukocyte) elastase, I have studied the binding of leukocyte elastase and two other neutrophil granule glycoproteins, cathepsin G and lactoferrin, to human alveolar macrophages. Saturable binding of all three ligands at 0°C was observed, with equilibrium dissociation constants of 4.0 x 10-7, 2.0 x 10-7, and 1.7 X 10-6 M, respectively. All bound to a similar number (54-73 X 106) of sites per cell. Binding of all three ligands was inhibited by the polysaccharide fucoidin, and extensive cross-inhibition of their binding to macrophages was observed. The results indicate that alveolar macrophages possess a relatively low-affinity, high-volume receptor for a family of neutrophil granule glycoproteins, which would be ideally suited for clearing released neutrophil granule contents from the extracellular space in inflamed tissues.Human polymorphonuclear neutrophils (PMN) release human leukocyte elastase (HLE) and other granule constituents in response to a variety of stimuli (1-6). Removal of HLE and other proteinases from the extracellular space depends upon receptor-mediated clearance by the reticuloendothelial system (7-18). Alveolar macrophages are capable of binding and internalizing HLE by two mechanisms: (i) binding of HLE-a2 macroglobulin complexes (14)(15)(16)(17)(18), which requires that the HLE first be bound to this circulating proteinase inhibitor; and (ii) binding of HLE without a requirement for previous interaction with a proteinase inhibitor (18). I have recently reviewed these two pathways for proteinase metabolism and their possible importance to lung defenses against proteolytic injury (18).Because another PMN granule glycoprotein, lactoferrin, also binds to a specific macrophage receptor (19-21), I hypothesized that HLE and lactoferrin bind to the same macrophage receptor and that additional PMN granule glycoproteins might also bind to macrophages. The present work provides evidence to support that hypothesis. These findings are important to an understanding of PMN-macrophage interactions at inflammatory foci and suggest that cross-inhibition of macrophage binding of PMN granule glycoproteins in vivo may produce effects that would not be expected from in vitro studies utilizing single purified proteins. METHODS Reagents and Proteins. Fucoidin was obtained from K & K.Versilube F50 is a product of Harwick (Akron, OH). Succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide was purchased from Bachem (Torrance, CA). Na'25I (11-17 mCi/,ug; 1 Ci = 3.7 x 100 becquerels) was obtained from Amersham. Trasylol was a gift from Bayer (Wuppertal, Federal Republic of Germany). Phenylmethylsulfonyl fluoride (PhMeSO2F), N-acetyl-DLphenylalanine -naphthyl ester, human transferrin, porcine pancreatic trypsin, and bovine ser...

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confidence: 99%

Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.

Campbell¹

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…These data also permit estimation of the capability of macrophages to bind the HLE released by neutrophils in response to physiologic stimuli. Published data (38) show that 5 x 106 neutrophils release 22 ,Ag of HLE (78 x 106 molecules/cell) in response to an immunologic stimulus in vitro. Based upon our estimated total number of binding sites, each macrophage has the capacity to bind the HLE released by eight neutrophils.…”

Section: Discussionmentioning

confidence: 99%

“…To inactivate HLE, the enzyme was exposed to the serine protease inhibitor PMSF, essentially as described by Polgar and Bender (21). PMSF completely inactivated the HLE, as measured by inability to hydrolyze SLAPN (22), even after prolonged incubation. The inactive HLE was dialyzed against PBS to remove unreacted PMSF.…”

Section: Methodsmentioning

confidence: 97%

Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro.

Campbell¹,

White²,

Senior³

et al. 1979

J. Clin. Invest.

110

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“…This substrate is regarded as highly specific for elastase [ 191. Hydrolysis of Ac-Ala,-OMe was measured calorimetrically [20] at pH 7.4. Derivatives of tri-alanine chloromethyl ketones were synthesized by Dr K. Bankowski (Institute of Fundamental Problems of Chemistry, Warsaw University).…”

Section: Methodsmentioning

confidence: 99%

Inactivation of some pancreatic and leucocyte elastases by peptide chloromethyl ketones and alkyl isocyanates

Ardelt¹

1976

FEBS Letters

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A colorimetric method for measuring the esterolytic activity of elastase

Cited by 25 publications

References 7 publications

Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.

Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.

Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro.

Inactivation of some pancreatic and leucocyte elastases by peptide chloromethyl ketones and alkyl isocyanates

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