Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Mevkh, Alevtina T.; Sud’ina, Galina F.; Golub, N B; Варфоломеев, С. Д.

doi:10.1016/0003-2697(85)90444-0

Cited by 28 publications

(18 citation statements)

References 12 publications

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“…The preparation of a microsomal cell extract broadly followed the method of Percival et al (1994). The fluorogenic COX-2 assay is based on the paper of Mevkh et al (1985). Due to the slow, tight-binding nature of the diarylheterocycles, the dissociation of inhibitor from the pseudoirreversible end complex was analyzed by monitoring the recovery in peroxidase activity over the initial pseudolinear phase of the progress plot because cyclooxygenase self-inactivates with time as it turns over arachidonic acid (AA) (Wu et al, 2003).…”

Section: Enzyme Kineticsmentioning

confidence: 99%

The Cyclooxygenase-2 Inhibitor GW406381X [2-(4-Ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] Is Effective in Animal Models of Neuropathic Pain and Central Sensitization

Bingham¹,

Beswick²,

Bountra³

et al. 2004

J Pharmacol Exp Ther

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The pathogenic form of the cyclooxygenase (COX) enzyme, COX-2, is also constitutively present in the spinal cord and has been implicated in chronic pain states in rat and man. A number of COX-2 inhibitors, including celecoxib and rofecoxib, are already used in man for the treatment of inflammatory pain. Preclinically, the dual-acting COX-2 inhibitor, GW406381X tion injury model and reversed thermal hyperalgesia in the mouse partial ligation model, both models of neuropathic pain. GW406381X, was also effective in a rat model of capsaicininduced central sensitization, when given intrathecally (ED 50 ϭ 0.07 g) and after chronic but not acute oral dosing. Celecoxib and rofecoxib had no effect in this model. Several hypotheses have been proposed to try to explain these differences in efficacy, including central nervous system penetration, enzyme kinetics, and potency. The novel finding of effectiveness of GW406381X in these models of neuropathic pain/central sensitization, in addition to activity in inflammatory pain models and together with its central efficacy, suggests dual activity of GW406381X compared with celecoxib and rofecoxib, which may translate into greater efficacy in a broader spectrum of pain states in the clinic.The role of prostaglandins, formed through the action of the two isoforms of the cyclooxygenase (COX)

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Section: Enzyme Kineticsmentioning

confidence: 99%

The Cyclooxygenase-2 Inhibitor GW406381X [2-(4-Ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] Is Effective in Animal Models of Neuropathic Pain and Central Sensitization

Bingham¹,

Beswick²,

Bountra³

et al. 2004

J Pharmacol Exp Ther

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“…The isolation and purification of PGHS-1 from ram seminal vesicles was performed according to previously published literature methods (23).…”

Section: Isolation and Purification Of Pghs-1 Enzyme From Ram Seminalmentioning

confidence: 99%

“…The tissue sections were subjected to a series of purification steps equivalent to those used when isolating PGHS from sheep seminal vesicles, except that the buffers contained protease inhibitors (23).…”

Section: Purification Of Atherosclerotic Tissue Obtained From Endartementioning

confidence: 99%

Tyrosine nitration in prostaglandin H2 synthase

Deeb

Resnick

Mittar

et al. 2002

Journal of Lipid Research

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In this study, we investigated the effects of various nitrogen oxide (NO x ) species on the extent of prostaglandin H 2 synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO x species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-( Nmethyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO x species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and Ͻ 1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1.Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration. -Deeb, R.

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“…In addition protohemin IX with both carboxyl groups substituted lost the cofactor ability while the modification of one carboxyl did not influence the enzyme activity [12].…”

Section: Introductionmentioning

confidence: 91%

Prostaglandin H synthase

et al. 1993

Self Cite

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Prostaglandin H synthase (PGHS) as apo-PGHS, holo-PGHS, and holo-PGHS, inactivated in the course of catalysis was studied using chemical modification with dietbyl pyrocarbonate (DEPC). The exhausted reaction with DEPC corresponded to the modification of 7 histidine residues in apo-PGHS and 4 in holo-PGHS. All 18 histidine residues became accessible for modification with DEPC in the enzyme, inactivated in the course of catalysis. The velocities of tryptic cleavage of all the three forms into two fragments were fairly different but independent of modification. Based on the results we hypothesize fast and dramatic changes in the protein structure in the course of the substrate conversion.Prostaglandin H synthase; Diethyl pyrocarbonate; Inactivation in the course of the reaction; Chemical modification

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Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Cited by 28 publications

References 12 publications

The Cyclooxygenase-2 Inhibitor GW406381X [2-(4-Ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] Is Effective in Animal Models of Neuropathic Pain and Central Sensitization

The Cyclooxygenase-2 Inhibitor GW406381X [2-(4-Ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-pyrazolo[1,5-b]pyridazine] Is Effective in Animal Models of Neuropathic Pain and Central Sensitization

Tyrosine nitration in prostaglandin H2 synthase

Prostaglandin H synthase

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