Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid

Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.     

On the haemoprotein character of prostaglandin endoperoxide synthetase.

Prostaglandin endoperoxide synthetase is a membrane-bound glycoprotein isolated as a dimer of molecular weight of approximately 129 000 consisting of two identical polypeptide chains. Several research workers have reported that haemin (ferri-protoporphyrin-IX) is required to restore the full enzymic activity of the pure apoprotein. Difference spectroscopy shows association of haemin up to two molecules per polypeptide chain of molecular weight 70 000. Both the cyclooxygenase and the peroxidase activity displayed by the enzyme can be optimally stimulated by similar quantities of haemin. The restored haemin-enzyme complex has a millimolar absorption coefficient at 408 nm of 61 mM-1 . cm-1 per haem. Using this value, the presence of non-haem iron in the enzyme is virtually excluded. These findings and the spectra of the reassociated enzyme-haemin complex point to a haemoprotein character. The availability of haemin to the enzyme might play a regulating r?le in its action.     

Prostaglandin endoperoxide E2 isomerase is dissociated from prostaglandin endoperoxide synthetase in the renal cortex

The rate of arachidonic acid metabolism by prostaglandin (PG) endoperoxide synthetase by the rabbit renal cortex (approximately 200 pmol/mg of protein/min) is very slow compared to medulla (approximately 2000 pmol/mg/min). However, by using PGH2 as a substrate and limiting reaction times, we were able to directly measure the endoperoxide-dependent PGE2 isomerase and found that both the cortex and medulla possess high levels of this activity (approximately pmol/mg/min). The PG endoperoxide E2 isomerase is dependent on reduced glutathione, but not cysteine, and is inactivated by p-hydroxymercuribenzoate or boiling. Thus, the renal medulla appears to exhibit an efficient coupling of cyclooxygenase and PG endoperoxide E2 isomerase, whereas the cortex has a vast excess of endoperoxide-dependent enzyme.     

Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands.

The membrane-bound prostaglandin endoperoxide synthetase was purified until homogeneity, starting from sheep vesicular glands. The enzyme was obtained as a complex with Tween-20, containing 0.69 mg detergent per mg protein. No residual phospholipid could be detected. Prostaglandin endoperoxide synthetase appeared to be a glycoprotein, containing mannose and N-acetyl-glucosamine. No haemin or metal atoms were present. A molecular weight of 126 000 was found for the apoprotein by ultracentrifugation in 0.1% Tween solutions. The polypeptide chain without carbohydrate had a molecular weight of 69 000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pure enzyme displays both cyclooxygenase and peroxidase activity, thus converting arachidonic acid into prostaglandin H2. The isolated synthetase requires haemin, which possibly acts as an easily dissociable prosthetic group, and a suitable hydrogen donor to protect the enzyme from peroxide inactivation and which is consumed in stoichiometric amounts to reduce the intermediate hydroperoxy group.     

The nature of functional groups regulating the catalytic activity of prostaglandin endoperoxide synthetase

The influence of some reagents modifying NH2-, SH-groups or imidazole moiety, on the prostaglandin endoperoxide synthetase activity was studied. Acetaldehyde, pyridoxal phosphate, dithiobis (nitrobenzoic) acid and iodoacetamide were found not to affect the enzyme activity. The activity was abolished as a result of the interaction with p-chloromercuribenzoic acid and diethyl pyrocarbonate. The hemin completely protected the apo-enzyme against the inactivation with diethyl pyrocarbonate. The assumption about the presence of imidazole moiety in the active site of the enzyme was made.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Metabolism of prostaglandin endoperoxide by microsomes from cat lung

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.     

Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes.

The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion of the 15-hydro-peroxide of prostaglandin G1 to a 15-hydroxyl group, producing prostaglandin H1. The latter enzymatic reaction required heme and was stimulated by a variety of compounds, including tryptophan, epinephrine, and guaiacol, but not by glutathione. A peroxidatic dehydrogenation was demonstrated with epinephrine or guaiacol in the presence of various hydroperoxides, including hydrogen peroxide and prostaglandin G1. Higher activity and affinity were observed with the 15-hydroperoxide of eicosapolyenoic acid, especially those with the prostaglandin structure. Both the dehydrogenation of epinephrine or guaiacol and the 15-hydroperoxide reduction of prostaglandin G1 were demonstrated in nearly stoichiometric quantities. With tryptophan, however, such a stoichiometric transformation was not observed. The peroxidase activity as followed with guaiacol and hydrogen peroxide and the tryptophan-stimulated conversion of prostaglandin G1 to H1 were not dissociable as examined by isoelectric focusing, heat treatment, pH profile, and heme specificity. The results suggest that the peroxidase with a broad substrate specificity is an integral part of prostaglandin endoperoxide synthetase which is responsible for the conversion of prostaglandin G1 to H1.     

Unique peroxidase reaction mechanism in prostaglandin endoperoxide H synthase-2: compound I in prostaglandin endoperoxide H synthase-2 can be formed without assistance by distal glutamine residue

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln-->Arg), Asn (Gln-->Asn), and Val (Gln-->Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.     

Effect of the modification of carboxylic groups in protohemin IX on the properties of endoperoxide prostaglandin synthetase

The effect of protohemin IX and its modified analogs (monomethyl ester, dimethyl ester, as well as monoamides with Val-Phe-OCH3 or Leu-His-OCH3) has been examined on the activity of prostaglandin endoperoxide synthetase from sheep vesicular glands (PGH-synthetase, EC 1.14.99.1, isolated as apoenzyme). For holoenzymes having the above compounds as prosthetic groups, the dissociation constants, relative activities and the apparent inactivation constants in the course of the reaction have been determined. The effect of Tween 20 on the indicated parameters for holoenzymes with protohemin IX and its mono- and dimethyl esters has been studied. Modification of one of the two carboxylic groups of protohemin IX markedly increases the dissociation constant for the respective holoenzyme and virtually does not affect catalytic activity. Modification of both carboxylic groups of protohemin IX hinder the binding with the apoenzyme and strongly reduces the catalytic activity of the holoenzyme.     

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H₂ to thromboxane B₂ and 12L-hydroxy-5, 8, 10- heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 μM. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H₂ from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H₂ as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H₂ formation from arachidonic acid was also affected.     

Metabolism of prostaglandin endoperoxide in animal tissues

The endoperoxide PGH₂ serves as a common intermediate for the enzymatic production of prostaglandins (PGEs and PGFs), thromboxanes (Tx) and prostacyclin (PGI₂). These compounds have quite different physiological activities and apparently perform important regulatory functions in various tissues and organs. We have obtained information on the distribution of individual enzymes responsible for the bioconversion of PGH₂ into these compounds in various tissue preparations. [1-C¹⁴] PGH₂ was incubated with a membrane fraction from each tissue homogenate. The products were isolated and identified by radiometric TLC and gas chromatography-mass spectrometry. Short life intermediates were detected by their specific biological activities. With this approach, we have demonstrated the formation of thromboxanes in rhesus monkey platelets, spleen and bone marrow, guinea pig lung and spleen, rabbit lung, human platelets and thioglycollate stimulated peritoneal macrophage from rat. On the other hand, the membrane preparation of bovine and mare corpus luteum, uteri from rabbit, monkey and human, rat stomach and small intestine, and rabbit lung produced predominantly prostacyclin. In addition, a PGH₂ to PGD₂ isomerase was found in the homogenate of rat brain and polymorphonuclear leukocytes. In those tissues which possess more than one enzyme catalyzing the metabolism of prostaglandin endoperoxide, substrate availability appeared to be one factor controlling the metabolic fate of the endoperoxide. The wide occurrence of thromboxane and prostacyclin synthetases suggests that their biological roles are not limited to the cardiovascular system.     

Some biological effects of prostaglandin endoperoxide analogs.

The effects of two methano-epoxy analogs of the prostaglandin endoperoxides PGG₂ and PGH₂ were tested on human platelets and rabbit aorta strips. One of these analogs, 9α, 11α-methano-epoxy-15- hydroxy-prosta-5, 13-dienoic acid, was 3.7 times more potent than the endoperoxide, PGG₂, as aggregating agent and was 6.2 times more active than PGH₂ in eliciting contractions of the isolated rabbit aorta. The analog initiated the platelet release reaction, but was less active than the endoperoxide in this respect. Furthermore, the release of ¹⁴C-serotonin induced by this analog was inhibited by indomethacin, which indicated that generation of endoperoxide was required.The corresponding 9α, 11α, epoxy-methano-analog was less active than the 9α, 11α, methano-epoxy analog in the test systems employed.     

Activation of carbamoyl phosphate synthetase by cryoprotectants

Molecular and Cellular Biochemistry - Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryoprotectants. Their diverse chemical structure and the normal...     

Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin.

Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100- or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (+/-)-2-fluoro-alpha-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acetyl]NAI indicated incorporation of 1.7 +/- 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo- and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. The different biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase

We have investigated the time course of formation of thromboxane A₂, thromboxane B₂, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A₂, but rather thromboxane A₂ decomposes exclusively into thromboxane B₂. The kinetics of formation of thromboxane B₂ from the endoperoxide prostaglandin H₂ in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A₂ from prostaglandin H₂ and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H₂.