Purification and characterization of sheep platelet cyclo-oxygenase Acetylation by aspirin prevents haemin binding to the enzyme

Boopathy, Rathanam; Balasubramanian, A.S.

doi:10.1042/bj2390371

Cited by 21 publications

(4 citation statements)

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“…It thus expresses two activities: a cycloxygenase (dioxygenase) activity which converts arachidonic acid into prostaglandin G2 (PGG2), and a peroxidase activity which reduces PGG2 into prostaglandin H2 (PGH2) (eq 1) (Ohki et al, 1979;Hemler & Lands, 1980;Kulmacz et al, 1985;Markey et al, 1987). It can be released upon extraction in the presence of detergents and purified from bovine (Miyamoto et al, 1976;Hemler etal., 1976) and sheep seminal vesicles (Van der Ouderaa et al, 1977;Roth et al, 1981;Jahnke et al, 1986; Kulmacz & Lands, 1987;Marnett et al, 1984) and from sheep platelets (Boopathy & Balasubramanian, 1986).…”

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confidence: 99%

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: Formation of iron(III)-.sigma.-phenyl complexes

Mahy¹,

Gaspard²,

Mansuy³

1993

Biochemistry

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Phenylhydrazones of various aromatic and aliphatic aldehydes or ketones act as good substrates of the dioxygenase reaction of prostaglandin synthase (PGHS). Corresponding alpha-azo hydroperoxides are formed as intermediates with maximum initial rates of O2 consumption between 8 and 230 mol (mol of PGHS)-1 s-1 for benzophenone and hexanal phenylhydrazone, respectively. The Km values for these reactions vary from 100 to 300 microM. These alpha-azo hydroperoxides are then converted to the corresponding alpha-azo alcohols by the peroxidase reaction of PGHS. During such oxidations of phenylhydrazones by PGHS, a new complex of this hemeprotein characterized by peaks at 438 and 556 nm is formed. This complex was obtained both by direct reaction of PGHS Fe(III) with phenyldiazene and by reaction of PGHS Fe(III) with phenylhydrazine in the presence of O2. By analogy to results previously reported for hemoglobin, myoglobin, catalase, and cytochrome P450, this species should be a sigma-phenyl PGHS FeIII-Ph complex. The PGHS FeIII-Ph complex should derive from an oxidation of the intermediate alpha-azo alcohol by PGHS Fe(III), cleavage of the resulting alkoxy radical with formation of a ketone (or aldehyde) and Ph*, and combination of PGHS Fe(II) with Ph*. Such an oxidation of alpha-azo alcohols by lipoxygenase-FeIII with formation of Ph* was reported previously. The formation of Ph* and of PGHS FeIII-Ph is likely the cause of the inhibitory effects previously reported for arylhydrazones toward PGHS.

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confidence: 99%

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: Formation of iron(III)-.sigma.-phenyl complexes

Mahy¹,

Gaspard²,

Mansuy³

1993

Biochemistry

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“…^XjH synthase (PGHS) catalyses the first two steps of the biosynthesis of prostaglandins, thromboxanes, and prostacyclin (Hemler et al, 1976;Miyamoto et al, 1976; Van Der Ouderaa et al, 1977). It first acts as a dioxygenase (cyclooxygenase) which oxidizes arachidonic acid to prostaglandin G2 (PGG2) and then as a peroxidase which reduces the hydroperoxide PGG2 to prostaglandin H2 (PGH2) (Hamberg et al, 1974;Ohki et al, 1979) to apparent electrophoretic homogeneity from bovine (Miyamoto et al, 1976) and sheep (Hemler et al, 1976;Marnett et al, 1984;Nastainczyk et al, 1984;Roth et al, 1981;Van Der Ouderaa et al, 1977) vesicular glands and sheep platelets (Boopathy & Balasubramanian, 1986), and it has been characterized as a homodimer of two 70-kDa polypeptide subunits (Boopathy & Balasubramanian, 1986;Hemler et al, 1976; Marnett et al, 1984; Miyamoto et al, 1976; Nastainczyk et al, 1984; Roth et al, 1981;Van Der Ouderaa et al, 1977).…”

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confidence: 99%

Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines

Mahy

Mansuy²

1991

Biochemistry

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Various N-alkylhydroxylamines such as N-hydroxyamphetamine react with prostaglandin synthase (PGHS) from sheep seminal vesicles, with the formation of new complexes characterized by a Soret peak around 421 nm. These complexes are very stable toward O2 or dithionite but are destroyed upon oxidation by Fe(CN)6K3 with regeneration of starting PGHS-FeIII. Their spectral characteristics, chemical properties, and routes of formation (either by direct oxidation of RNHOH or by in situ reduction of RNO2 in the presence of dithionite) are very similar to those previously reported for nitrosoalkane complexes of hemoglobin-, myoglobin-, and cytochrome P-450-FeII. Their FeII-N(O)R structure was completely confirmed in the case of N-hydroxyamphetamine, both by extraction of the heme complex by butanone and by identification to authentic protoporphyrin IX-FeII-N(O)-amphetamine, and by insertion of this authentic complex into apoPGHS. Phenylhydroxylamine also reacts with PGHS-FeIII to give a PGHS-FeII-N(O)Ph complex which is not stable in the presence of dithionite because of its weaker PGHS-FeII-N(O)R bond when compared to PGHS-FeII-nitrosoalkane complexes. The ability of various N-alkylhydroxylamines to form PGHS-FeII-N(O)R complexes greatly depends upon their hydrophobicity. Actually, CH3NHOH and C2H5NHOH are totally inactive whereas about 10 molar excess of N-hydroxyamphetamine and C6H5NHOH already lead to 50% complex formation. This is in favor of an hydrophobic environment of the heme in PGHS. Finally, PGHS engaged in such FeII-nitrosoalkane complexes completely loses its dioxygenase activity, suggesting that N-substituted hydroxylamines or compounds that can be metabolized in vivo to give such hydroxylamines could act as strong PGHS inhibitors.

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“…This convergent reaction sequence was utilized for the production of compounds 10e-k, which were designed for the exploration of structure-activity relationships. Initial biological screening data against COX-1, 33 COX-2, 33 5-LO, 34 and LPO 35 for these analogues and related derivatives ( 10a,b,d and 12d) are summarized in Table 1. All compounds at a test concentration of 300 µM effectively inhibited CCl 4 -induced LPO of polyunsaturated fatty acids from guinea pig liver microsomes as quantified by spectrophotometric analysis of MDA formation, with equivalent or better potency than that produced by R-tocopherol.…”

Section: Resultsmentioning

confidence: 99%

Design, Synthesis, and Biological Evaluation of Conformationally Constrainedaci-Reductone Mimics of Arachidonic Acid

1998

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An efficient and convergent synthesis has been developed for the production of 3,4-dihydroxy-5-[4-(2-((2Z)-hexenyl)phenyl)-3-(1Z)-but enyl]-2 (5H)-furanone (12d). This hydrophobic antioxidant is a stable conformationally constrained mimic of arachidonic acid (AA) (1) and its respective aci-reductone analogue (2). Pd(0)-catalyzed cross-coupling of 5-(3-butynyl)-3,4-dihydroxy-2(5H)-furanone (7) with 2-((2Z)-hexenyl)iodobenzene (8d) followed by Lindlar catalyzed hydrogenation produces 12d. Butynyl intermediate 7 is prepared from 2-(benzyloxy)-5-deoxyascorbic acid (15) by iodination (I2, PPh3, Imd), iodo substitution with lithium acetylide ethylenediamine complex (LiAEDA, HMPA, -5 degrees C), and benzyl group cleavage (Ac2O, Pyr, BCl3). The utility of this synthetic method was demonstrated by the synthesis of analogues 10e-k. Biological testing revealed that certain of these antioxidants inhibit both cyclooxygenase (COX) and 5-lipoxygenase (5-LO) with comparable efficacy as reported for aspirin and zileuton, respectively. The antioxidant activity of these aci-reductones, measured as a function of their inhibitory effect on CCl4-induced lipid peroxidation of hepatic microsomes, exceeds that produced by alpha-tocopherol. Synthetic routes and initial structure-activity relationships (SAR) for these novel mixed functioning antioxidants are presented.

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Purification and characterization of sheep platelet cyclo-oxygenase Acetylation by aspirin prevents haemin binding to the enzyme

Cited by 21 publications

References 50 publications

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: Formation of iron(III)-.sigma.-phenyl complexes

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: Formation of iron(III)-.sigma.-phenyl complexes

Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines

Design, Synthesis, and Biological Evaluation of Conformationally Constrainedaci-Reductone Mimics of Arachidonic Acid

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