Mammalian lipoxygenases--are they only involved in the arachidonic acid cascade?
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R Wiesner
Publications and source records attributed to R Wiesner.
Formation of ketodienoic fatty acids by the pure pea lipoxygenase-1.
A pure lipoxygenase from dried green pea seeds (isoenzyme 1) oxygenates linoleic acid to 9(S/R)-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPODE) and 13(S/R)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE). Furthermore (10E,12Z)-9-keto-10,12-octadecadienoic acid (9-KODE) and (9Z,11E)-13-keto-9,11-octadecadienoic acid (13-KODE) in a ratio of 1:1 were formed. Uv-spectroscopic measurements and HPLC data indicated a hydroperoxy fatty acid: keto fatty acid ratio of about 2:1. The product mixture formed from arachidonic acid was even more complex. 15-, 11-, 9- and 5-H(P)ETE1 and their corresponding keto derivatives have been detected. The chemical structures of the compounds have been identified by HPLC analysis, by uv- and ir-spectroscopy and gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The data presented indicate that a pure lipoxygenase catalyzes the formation of both hydroperoxypolyenoic fatty acids and ketopolyenoic fatty acids from linoleic acid and arachidonic acid. The possible mechanism of the formation of the keto compounds is discussed.
Arachidonic Acids↗
On the reaction of wheat lipoxygenase with arachidonic acid and its oxygenated derivatives.
Lipoxygenase was purified from wheat kernels by means of ammonium sulfate precipitation, gel chromatography on Sephadex G-200 and anion exchange chromatography on DEAE-Sephadex A-50. Arachidonic acid was mainly converted by the wheat lipoxygenase to 5D-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5D8-HPETE) with other HPETE isomers including 8-HPETE being minor products. At higher concentrations of lipoxygenase, multiple oxygenation products such as 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and, to a lower extent, 8,15-diHETE and lipoxin isomers were detected after reduction of the hydroperoxy derivatives primarily formed. Similar results were obtained with 5D8- or 15L8-hydroxyeicosatetraenoic acid as substrate. Moreover, evidence was obtained for leukotriene A4 synthase activity of the wheat lipoxygenase.
Arachidonate 5-Lipoxygenase↗
Survival after liver transplantation in diabetics.
Explore the source record for details and available documents.
Diabetes Complications↗
Evaluation of Abbott TDx monoclonal assay of cyclosporine in whole blood.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Oxygenation of biological membranes by the pure reticulocyte lipoxygenase.
We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.
Animals↗
Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes.
Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid, 9S and 9R isomers of (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,11Z,13E)-(15S)-15-hydroxy-5,8,11,13-icosa tetraenoic acid, 9- and 13-oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co-chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15-lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific: a complex mixture of all positional and optical isomers was detected. The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.
Animals↗
Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase.
Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.
Animals↗
Metabolism of polyenoic fatty acids by rabbit reticulocytes. Intracellular action of the erythroid lipoxygenase on membrane lipids.
Rabbit reticulocytes metabolize exogenous polyenoic fatty acids via three different pathways. (i) incorporation into the cellular ester lipids, predominantly into phospholipids, (ii) beta-oxidation forming CO2 and (iii) lipoxygenase reaction. The lipoxygenase pathway contributes to about 30% to the metabolism of exogenously added linoleic acid. The endogenous substrates of the lipoxygenase pathway are not only the free polyenoic fatty acids bound to the cellular membranes but also the membrane phospholipids. The lipoxygenase products detected in the membrane lipids have been isolated and their complete chemical structure has been identified as 13-hydroxy-9Z,11E-octadecadienoic acid. 9-hydroxy-10E,12Z-octadecadienoic acid, their all E isomers and 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid. Subcellular fractionation of different cellular membranes indicated that these products occur in both the mitochondrial membranes and the plasma membrane. By quantitative HPLC analysis it has been shown that the mitochondrial membranes contain about 3 times more oxygenated fatty acids than the plasma membranes; one out of ten linoleic acid residues in the mitochondrial membranes is present as hydroxylated derivative. The pattern of lipoxygenase products detected in the mitochondrial membranes was much more unspecific than that of plasma membranes. These data are discussed in the light of the involvement of the lipoxygenase pathway in the degradation of mitochondria during the maturation of red blood cells.
Animals↗
Oxygenation of biological membranes by the reticulocyte lipoxygenase. Lack of stoichiometry between oxygen uptake and product formation.
The oxygenation of different types of biological membranes (rat liver mitochondria, rat liver endoplasmic membranes, inside-out erythrocyte ghosts, right side-out erythrocyte ghosts) was studied with respect to products formed during the reaction. In all cases a very similar product pattern was observed with 15S-hydroperoxy-5Z.8Z.11Z.13E-eicosatetraenoic acid (15-HETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid, (13-HODE) being the major products. Comparison of the amount of lipoxygenase products formed with the oxygen uptake measured during the reaction indicated an excessive oxygen uptake. With mitochondrial membranes the oxygen consumption was almost one order of magnitude higher than the amount of the products detected. The origin of the excessive oxygen uptake remains unclear. These data, however, indicate that the oxygen consumption with complex substrates is not a reliable measure for the lipoxygenase activity.
Animals↗
Oxygenation of mitochondrial membranes by the erythroid lipoxygenase. Consequences for membrane properties.
The reticulocyte lipoxygenase is able to oxygenate mitochondrial membranes. The main products formed during this reaction are 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HODE). The oxygenation of mitochondrial membranes is accompanied by an inactivation of enzymes localized in the outer and inner mitochondrial membranes, by a drastic change of the passive electric properties and by a destruction of the iron-sulfur clusters of the outer mitochondrial membrane. The possible mechanism of the processes leading to these functional changes is discussed.
Animals↗
Formation of oxygenase and hydroperoxidase products by the pure reticulocyte lipoxygenase.
The pure reticulocyte lipoxygenase oxygenates free arachidonic acid to 15S- and 12S-HPETE in a ratio of about 10:1 15S-H(P)ETE or its methyl ester can be further converted by the lipoxygenase via three different types of reactions: (i) oxygenase reaction, (ii) hydroperoxidase reaction and (iii) leukotriene synthase reaction. Here we summarize the products formed from arachidonic acid by the reticulocyte lipoxygenase via these three types of reactions.
Animals↗
Occurrence of free and esterified lipoxygenase products in leaves of Glechoma hederacea L. and other Labiatae.
Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid, (10E,12Z,15Z)-9-oxo-10,12,15-octadecatrienoic acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and (10E,12Z)-9-oxo-10,12-octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co-chromatography with authentic standards on various types of HPLC columns including chiral-phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy-polyenoic fatty acids. Air-drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids. The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.
Chemical Phenomena↗
Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B.
Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.
Animals↗
Lipoxygenase catalyzed oxygenation of hydroxy fatty acids to lipoxins.
The pure lipoxygenases from rabbit reticulocytes and soybeans convert a variety of substrates (arachidonic acid, 15-HPETE, 15-HETE, 5-HETE, various DiHETE isomers) to trihydroxy eicosanoids containing a conjugated tetraene system (lipoxins). In general, the methyl esters are better substrates for lipoxin formation than are the free acids. Lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid) strongly inhibit the lipoxin formation. The complete stereochemistry of the lipoxin B formed from 15S-HETE methyl ester has been established by co-chromatography with authentic standards on various types of HPLC columns, by GC/MS analysis, by gas liquid chromatography of the ozonolysis fragments of the menthoxy carbonyl derivatives and 1H-NMR studies. The molar absorption coefficient of the conjugated tetraenes was measured as epsilon 301 = 53,000. The lipoxins formed from 15-HETE and various DiHETE isomers are formed exclusively via the oxygenation pathway as shown by experiments under an 17O2 atmosphere and/or by anaerobic incubations. Our results indicate that lipoxins can be synthesized via lipoxygenase-catalyzed sequential oxygenation of polyenoic fatty acids and their hydro(pero)xy derivatives.
Animals↗
Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate.
The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.
Animals↗
Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography.
A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.
Animals↗
Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.
It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.
Adenosine Triphosphate↗