Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (kcat/Km) in the 105M-1·s-1 range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02—0.7pg of cathepsin G/cell (n = 15) at their surface. This means that about 104 purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.
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September 2002
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Research Article|
September 15 2002
Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates
Sylvie ATTUCCI;
Sylvie ATTUCCI
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Brice KORKMAZ;
Brice KORKMAZ
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Luiz JULIANO;
Luiz JULIANO
†Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil
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Eric HAZOUARD;
Eric HAZOUARD
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Catherine GIRARDIN;
Catherine GIRARDIN
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Michèle BRILLARD-BOURDET;
Michèle BRILLARD-BOURDET
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Sophie RÉHAULT;
Sophie RÉHAULT
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Philippe ANTHONIOZ;
Philippe ANTHONIOZ
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
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Francis GAUTHIER
Francis GAUTHIER
1
∗INSERM EMI-U 00-10 ‘'Protéases et Vectorisation'’, Laboratory of Enzymology and Protein Chemistry, University François Rabelais, 2bis Bd Tonnellé, 37032 TOURS Cedex, France,
1To whom correspondence should be addressed (e-mail gauthier@univ-tours.fr).
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Publisher: Portland Press Ltd
Received:
February 25 2002
Revision Received:
May 30 2002
Accepted:
June 28 2002
Accepted Manuscript online:
June 28 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 965–970.
Article history
Received:
February 25 2002
Revision Received:
May 30 2002
Accepted:
June 28 2002
Accepted Manuscript online:
June 28 2002
Citation
Sylvie ATTUCCI, Brice KORKMAZ, Luiz JULIANO, Eric HAZOUARD, Catherine GIRARDIN, Michèle BRILLARD-BOURDET, Sophie RÉHAULT, Philippe ANTHONIOZ, Francis GAUTHIER; Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates. Biochem J 15 September 2002; 366 (3): 965–970. doi: https://doi.org/10.1042/bj20020321
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