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Characterisation of peroxidasin activity in isolated extracellular matrix and direct detection of hypobromous acid formation.
Bathish, Boushra; Turner, Rufus; Paumann-Page, Martina; Kettle, Anthony J; Winterbourn, Christine C.
Afiliação
  • Bathish B; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
  • Turner R; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
  • Paumann-Page M; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
  • Kettle AJ; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
  • Winterbourn CC; Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand. Electronic address: Christine.winterbourn@otago.ac.nz.
Arch Biochem Biophys ; 646: 120-127, 2018 05 15.
Article em En | MEDLINE | ID: mdl-29626421
ABSTRACT
Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 µM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 µM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bromatos / Brometos / Proteínas da Matriz Extracelular / Peroxidase / Matriz Extracelular / Peróxido de Hidrogênio Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Revista: Arch Biochem Biophys Ano de publicação: 2018 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bromatos / Brometos / Proteínas da Matriz Extracelular / Peroxidase / Matriz Extracelular / Peróxido de Hidrogênio Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Revista: Arch Biochem Biophys Ano de publicação: 2018 Tipo de documento: Article