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Titolo:
Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence
Autore:
Forghani, F; Ouellet, M; Keen, S; Percival, MD; Tagari, P;
Indirizzi:
Merck Frosst Ctr Therapeut Res, Pointe Claire, PQ H9R 4P8, Canada Merck Frosst Ctr Therapeut Res Pointe Claire PQ Canada H9R 4P8 P8, Canada
Titolo Testata:
ANALYTICAL BIOCHEMISTRY
fascicolo: 2, volume: 264, anno: 1998,
pagine: 216 - 221
SICI:
0003-2697(19981115)264:2<216:AOPGSI>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
SELECTIVE-INHIBITION; HUMAN CYCLOOXYGENASE-2; H SYNTHASE; ARACHIDONIC-ACID; PURIFICATION; MECHANISM; STOMACH; POTENT; COX-2; ASSAY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Tagari, P Merck Frosst Ctr Therapeut Res, POB 1005, Pointe Claire, PQ H9R 4P8, Canada Merck Frosst Ctr Therapeut Res POB 1005 Pointe Claire PQ CanadaH9R 4P8
Citazione:
F. Forghani et al., "Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence", ANALYT BIOC, 264(2), 1998, pp. 216-221

Abstract

The inducible form of the heme-protein prostaglandin G/H synthase (PGHS-2 or COX-2) has been established as a pivotal enzyme in the cascade of eventsleading to inflammation, hyperalgesia, and pyresis and represents a major therapeutic target in inflammatory disease. Accordingly, we have exploited the heme-catalyzed hydroperoxidase activity of recombinant hCOX-2 to generate luminescence in the presence of luminol, or a cyclic naphthalene hydrazide, and the substrate arachidonic acid. Arachidonate-induced luminescence was shown to be an index of real-time catalytic activity and demonstrated the turnover inactivation of the enzyme. Luminol luminescence was proportional to hCOX-2 concentration and gave accurate K-m determinations for arachidonate. Inhibition of hCOX-2 activity, measured by luminescence, by a varietyof selective (for COX-2) and nonselective inhibitors showed rank orders ofpotency similar to those observed with other in vitro and whole cell methods using the recombinant protein. The sensitivity of the luminescence assayalso allowed determination of inhibitor potency at substrate concentrations below K-m, distinguishing competitive inhibitors such as ibuprofen from time-dependent inhibitors such as DuP-697. Finally the use of higher quantum-yielding luminol analogues allowed measurement of cyclooxygenase activity at extremely low substrate and protein concentrations, enabling a variety of novel assay formats. (C) 1998 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 04:47:49