Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands.

The effect of methional on prostaglandin biosynthesis from 5,8,11,14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands

The effect of methional on prostaglandin biosynthesis from 5,8,11, 14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Stimulation of prostaglandin biosynthesis by lipoic acid.

Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.     

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase.

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants alpha-naphthol. guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited by specifically by alpha-naphthol. guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus alpha-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-alpha-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.     

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase,platelet aggregation and lipoxidase

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants α-naphthol, guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-α-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited specifically by α-naphthol, guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-α-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus α-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-α-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.     

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.     

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.     

Glucagon stimulates the A system for neutral amino acid transport in isolated hepatocytes of adult rat.

It has been found that both the peroxidase and synthetase activity of sheep vesicular gland microsomes catalyze the oxygenation of singlet oxygen trapping or quenching agents. Furthermore the synthetase was also readily inactivated by these agents, particularly bilirubin, and suggests that singlet oxygen formed by the peroxidase activity may initiate prostaglandin biosynthesis. The singlet oxygen agents also protected the synthetase from self-catalyzed destruction or inactivation by peroxides and suggest that singlet oxygen may also be responsible for the inactivation.     

Radical scavenging as the mechanism for stimulation of prostaglandin cyclooxygenase and depression of inflammation by lipoic acid and sodium iodide.

Certain radical-trapping reducing agents have been shown to stimulate prostaglandin biosynthesis in vitro (1--6) and to depress phorbol myristate acetate-induced mouse ear edema (16). The increased prostaglandin synthesis resulted from influences on the cyclooxygenase. To ascertain whether these alterations were due to direct interaction with the enzyme or to indirect scavenging of the oxidant released during PGG2 reduction, we report the effects of lipoic acid and sodium iodide. Both of these agents stimulated the enzymatic oxygenation of arachidonic acid, increased the reduction of PGG2 to PGH2, quenched the EPR signal induced by arachidonic acid and depressed mouse ear edema. In addition to discovering two unusual antiinflammatory agents, we have confirmed that materials with entirely different structures can have identical effects on the cyclooxygenase, suggesting indirect stimulation of this enzyme due to trapping of the oxidant.     

Prostaglandin biosynthesis and its regulation in the bovine endometrium: A comparison between nonpregnant and pregnant status

Prostaglandin F(2alpha) (PGF(2alpha)), an arachidonic acid metabolism product of the prostaglandin synthetase pathway, is synthesized and released from the endometrium during luteolysis in nonpregnant animals. When proper conception occurs, the synthesis and release pattern is changed to maintain the corpus luteum (CL) function. The biosynthesis of prostaglandins in the bovine endometrium was highest in the microsomes but of low order. In nonpregnancy, the formation of prostaglandins from labelled precursor acid was higher than in pregnancy. Besides the prostaglandin synthetase, an inhibiting activity on the conversion of arachidonic acid to prostaglandins was found in both the nonpregnant and pregnant endometrium. During luteolysis (Day 17), a low inhibiting capacity was seen in comparison with other days of the estrous cycle (Days 1, 4 and 14). The inhibitory capacity was very high on Days 16 to 20, 25, and 31 of pregnancy. In the nonpregnant endometrium at Day 17, a very low inhibitor potency, calculated as IC(50) values, was found both in the cytoplasma and in the microsomes, whereas during early pregnancy (Days 17, 18, and 20) both cytoplasma and microsomes possessed very high inhibitor potency. This finding indicates that the bovine endometrium contains both prostaglandin synthetase and an unknown potent inhibitor of prostaglandin biosynthesis that regulates prostaglandin biosynthesis both during the estrous cycle and early pregnancy.     

Lipoate metabolism in Pseudomonas putida LP: thiolsulfinates of lipoate and bisnorlipoate.

Lipoate thiolsulfinate and two bisnorlipoate thiolsulfinates, as well as the previously identified products of β-oxidation (bisnorlipoate, tetranorlipoate, and β-hydroxybisnorlipoate), were isolated and identified as catabolites of [¹⁴C]lipoate from cultures of Pseudomonas putida LP, an organism capable of growth on lipoic acid as a sole source of carbon and sulfur. The newly identified metabolites were characterized by ion-exchange and paper chromatography and infrared, ultraviolet, and mass spectroscopies. Comparison of the isolated catabolites with synthetic standards implies that the lipoic thiolsulfinate isolated is the S-1 monoxide of 1,2-dithiolane-3-valeric acid; one bisnorlipoic thiolsulfinate isolated is the S-1 monoxide, the other apparently the S-2 monoxide. Metabolic studies with P. putida show that lipoate thiolsulfinate is taken up by this microorganism in an energy-dependent process, but less readily than lipoate; lipoate thiolsulfinate supports oxygen consumption in short-term experiments but does not support growth. These results are interpreted as meaning that the thiolsulfinates are “dead-end” metabolites, not intermediates in the sulfur metabolism of this organism. Lipoate thiolsulfinate is not detectably β-oxidized to bisnorlipoate thiolsulfinate under the usual culture conditions.     

Radical scavenging as the mechanism for stimulation of prostaglandin cyclooxygenase and depression of inflammation by lipoic acid and sodium iodide

Certain radical-trapping reducing agents have been shown to stimulate prostaglandin biosynthesis (1–6) and to depress phorbol myristate acetate-induced mouse ear edema (16). The increased prostaglandin synthesis resulted from influences on the cyclooxygenase. To ascertain whether these alterations were due to direct interaction with the enzyme or to indirect scavenging of the oxidant released during PGG₂ reduction, we report the effects of lipoic acid and sodium iodide.Both of these agents stimulated the enzymatic oxygenation of arachidonic acid, increased the reduction of PGG₂ to PGH₂, quenched the EPR signal induced by arachidonic acid and depressed mouse ear edema. In addition to discovering two unusual antiinflammatory agents, we have confirmed that materials with entirely different structures can have identical effects on the cyclooxygenase, suggesting indirect stimulation of this enzyme due to trapping of the oxidant.     

Fatty acid and lipoic acid biosynthesis in higher plant mitochondria

Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP). We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl(C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors. Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria.     

Protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site."     

LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulence

The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the DeltalplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell.     

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplA·lipoyl-5′-AMP and LplA·octyl-5′-AMP·apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5′-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys¹³³ residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.     

Stabilization of prostaglandin synthetase by immobilization of goat seminal microsomes on silica gel-G

Prostaglandin synthetase was immobilized by adsorption of goat vesicular microsomes on silica gel containing CaSO4 (silica gel G). Repeated cycles of enzymatic conversion of arachidonic acid to prostaglandin by the immobilized microsomes increased the product yield by 1.5 fold, in comparison to the same by free microsomal particles. The presence of Ca2+ in silica gel is responsible for this improved yield of prostaglandin as the divalent metal ion stabilized prostaglandin synthetase activity in a remarkable way. Microsomal particles immobilized on solid supports like alumina G and controlled pore glass were not very effective.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.     

Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.