Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY

O₂ uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O₂ uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O₂, it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O₂ uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase.     

Biochemical and immunochemical evidences for the presence of lipoxygenase in plant mitochondria

In this paper, both biochemical and immunochemical evidence for the presence of lipoxygenase (LOX) in plant mitochondria is presented. Highly purified pea (Pisum sativum L., cv. Alaska) mitochondria show LOX activity, evaluated as conjugated diene formation, oxygen consumption, and hydroperoxide formation. Both 9- and 13-hydroperoxy-octadecadienoic acids are produced by the oxidation of linoleic acid. LOX activity is particularly evident in swollen mitochondria; it is inhibited by nordihydroguaiaretic acid, a pea anti-LOX B antibody, and has two pH optima (6.0 and 7.5). A mitochondrial protein of approximately 97 kDa cross-reacts with a pea seed anti-LOX B antibody. This reaction is detectable in both soluble (matrix fraction) and membrane-bound (submitochondrial particles) proteins. Considering that pea mitochondria were extracted from actively growing stems that were differentiating tube elements, it is suggested that the presence of LOX in these organelles may be related to their degradation linked to xylem differentiation.     

The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves

In order to understand the physiological significance of the in-vitro lipoxygenase (EC 1.13.11.12)-mediated ethylene-forming system (J.F. Bousquet and K.V. Thimann 1984, Proc. Natl. Acad. Sci. USA 81, 1724–1727), its characteristics were compared to those of an in-vivo ethylene-forming system. While oat (Avena sativa L.) leaves, as other plant tissues, preferentially converted only one of the 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) isomers to 1-butene, the lipoxygenase system converted all four AEC isomers to 1-butene with nearly equal efficiencies. While the in-vivo ethylene-forming system of oat leaves was saturable with ACC with a K_m of 16 M, the lipoxygenase system was not saturated with ACC even at 10 mM. In contrast to the in-vivo results, only 10% of the ACC consumed in the lipoxygenase system was converted to ethylene, indicating that the reaction is not specific for ethylene formation. Increased ACC-dependent ethylene production in oat leaves following pretreatment with linoleic acid has been inferred as evidence of the involvement of lipoxygenase in ethylene production. We found that pretreating oat leaves with linoleic acid resulted in increased ACC uptake and thereby increased ethylene production. A similar effect was observed with oleic acid, which is not a substrate of lipoxygenase. Since linoleic acid hydroperoxide can substitute for lipoxygenase and linoleic acid in this system, it is assumed that the alkoxy radicals generated during the decomposion of linoleic acid hydroperoxide are responsible for the degradation of ACC to ethylene. Our results collectively indicate that the reported lipoxygenase system is not the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - Epps N-(2-hydroxyethyl)-piperazine-N-3-propanesulfonic acid - LH linoleic acid - LOOH linoleic acid hydroperoxide - pyridoxal-P pyridoxal-phosphate This work was presented at the 12th International Conference on Plant Growth Substances, Heidelberg, FRG, August 1985 (Abstract No. PO 5-52)     

C6-aldehyde formation by fatty acid hydroperoxide lyase in the brown alga Laminaria angustata

Some marine algae can form volatile aldehydes such as n-hexanal, hexenals, and nonenals. In higher plants it is well established that these short-chain aldehydes are formed from C18 fatty acids via actions of lipoxygenase and fatty acid hydroperoxide lyase, however, the biosynthetic pathway in marine algae has not been fully established yet. A brown alga, Laminaria angustata, forms relatively higher amounts of C6- and C9-aldehydes. When linoleic acid was added to a homogenate prepared from the fronds of this algae, formation of n-hexanal was observed. When glutathione peroxidase was added to the reaction mixture concomitant with glutathione, the formation of n-hexanal from linoleic acid was inhibited, and oxygenated fatty acids accumulated. By chemical analyses one of the major oxygenated fatty acids was shown to be (S)-13-hydroxy-(Z, E)-9, 11-octadecadienoic acid. Therefore, it is assumed that n-hexanal is formed from linoleic acid via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase (HPL), by an almost similar pathway as the counterpart found in higher plants HPL partially purified from the fronds has a rather strict substrate specificity, and only 13-hydroperoxide of linoleic acid, and 15-hydroperoxide of arachidonic acid are the essentially suitable substrates for the enzyme. By surveying various species of marine algae including Phaeophyta, Rhodophyta and Chlorophyta it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected.     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

Oxidation of furan fatty acids by soybean lipoxygenase-1 in the presence of linoleic acid

The interaction of furan fatty acids (F-acids) with lipoxygenase was investigated by incubation experiments of a synthetic dialkyl-substituted F-acid with soybean lipoxygenase-1. Originally the oxidation of furan fatty acids was assumed to be directly effected by lipoxygenase. It is now demonstrated that this reaction is a two-step process that requires the presence of lipoxygenase substrates, e.g. linoleic acid. In the first step linoleic acid is converted by the enzyme to the corresponding hydroperoxide. This attacks, probably in a radical reaction, the furan fatty acid to produce a dioxoene compound that can be detected unequivocally by gas chromatography-mass spectrometry.     

Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.     

Rat pulmonary lipoxygenase: dioxygenase activity and role in xenobiotic metabolism.

1. Dioxygenase activity and the ability of pregnant rat lung lipoxygenase to oxidize xenobiotics were examined in vitro under a variety of experimental conditions. 2. More than 90% of the dioxygenase activity towards linoleic acid in the lung homogenate was found to be associated with the cytosolic fraction. The cytosolic enzyme exhibited pH optima at 6.5 and 9.5, the activity being two-fold greater at pH 9.5. To observe maximal dioxygenase activity (about 0.7 mumol of 13-hydroperoxylinoleic acid formed/min per mg protein) at pH 9.5, the presence of 6.0 mM linoleic acid was required. 3. Benzidine oxidation occurred at maximal rate of pH 6.5 when the reaction medium contained 1.0 mM benzidine and 13.5 mM linoleic acid. All eight xenobiotics tested were oxidized at significant rates by the lung cytosolic lipoxygenase. 4. Both dioxygenase activity and benzidine oxidation were inhibited by the inhibitors of lipoxygenase, viz. nordihydroguaiaretic acid, BHT, caffeic acid, esculetin, and gossypol, in a concentration-dependent manner. 5. The results suggest that oxidation of xenobiotics by lipoxygenase may be an important pathway of metabolism in the mammalian lung.     

Recent investigations into the lipoxygenase pathway of plants

The plant lipoxygenase (LOX) pathway is in many respects the equivalent of the 'arachidonic acid cascade' in animals. The LOX-catalyzed dioxygenation of the plant fatty acids, linoleic and linolenic acids, is followed by metabolism of the resulting fatty acid hydroperoxides by other enzymes. Although the physiological functions of the end-products do not appear to be fully defined at this time, hormonal and anti-fungal activities have been reported.     

Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods.

The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).     

Use of Tetraethylthiuram Disulfide to Discriminate between Alternative Respiration and Lipoxygenase

Mitochondria from axes of Glycine max (L.) Merr. cv. Chippewa 64 seedlings purified on discontinuous Percoll gradients exhibited classical cyanide-resistant respiration. These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 0.8 millimolar linoleic acid. This activity is inhibited by most known inhibitors of alternative respiration (i.e. hydroxamates and propyl gallate). Tetraethylthiuram disulfide (disulfiram) at 50 micromolar inhibited cyanide-resistant succinate oxidation by 90 per cent, whereas concentrations as high as 100 micromolar had no effect on lipoxygenase activity. Use of tetraethylthiuram disulfide allows discrimination between alternative respiration and lipoxygenase activity in mitochondria.     

Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.     

Oxodiene formation during the Vicia sativa lipoxygenase-catalyzed reaction: occurrence of dioxygenase and fatty acid lyase activities associated in a single protein

An enzyme with at least dual activities, lipoxygenase and fatty acid lyase, has been isolated from Vicia sativa seeds. The enzyme utilizes directly linoleic acid as substrate. The enzyme had a pH optimum at 5.8 for the two activities and converted linoleic acid into two products: 9-hydroperoxylinoleic acid and trans-2, cis-4 decadienal. The enzyme does not act on 13- or 9- fatty acid hydroperoxide isomers. An enzymatic reaction for the biogenesis of trans-2, cis-4- decadienal is proposed. This involves the synthesis of an intermediate peroxyl radical due to oxygen insertion in carbon 9 of linoleic acid. This intermediate peroxyl radical may be converted into 9-HPOD and 2,4-decadienal.     

Examination of the possible involvement of lipoxygenase and jasmonates in pollination-induced senescence of Phalaenopsis and Dendrobium orchid flowers

Application of linoleic and linolenic acids to Phalaenopsis and Dendrobium flowers enhanced their senescence and promoted ethylene production. This effect was specific to unsaturated fatty acids which serve as substrates for lipoxygenase action, and did not occur following similar treatments with saturated fatty acids. Several major lipoxygenase pathway metabolites including jasmonic acid methyl ester, traumatic acid, trans -2-hexenal and cis -3-hexenol also enhanced flower senescence. Jasmonic acid methyl ester promoted ethylene production by Phalaenopsis flowers. In contrast, treating flowers with the lipoxygenase inhibitors salicylhydroxamic acid and n -propyl gallate. which inhibite(d) lipoxygenase activity in vitro, had no effect on pollination-induced senescence of the flowers. Furthermore, during the 50-h period following pollination, there was no increase in lipoxygenase activity in Phalaenopsis flowers. During the 10-h period from pollination of Dendrobium flowers until the initiation of ethylene production, there was no effect of pollination on jasmonate levels in either the perianth or the columns. These results suggest that lipoxygenase activity and jasmonates are not directly involved in pollination-induced Phalaenopsis and Dendrobium flower senescence.     

Occurrence of a New Lipoxygenase Isoenzyme in Germinating Barley Embryos

Partially purified preparations of lipoxygenase from the germinating barley embryos converted linoleic acid to 9- and 13-hydroperoxy linoleic acids in the ratio of approximately 3:1, while the similar preparations from the ungerminated embryos converted linoleic acid mainly to 9-hydroperoxy linoleic acid.

Isoelectric focusing of the partially purified preparations of the germinating embryos revealed the presence of the two lipoxygenase active peaks, having isoelectric point at pH 4.9 and 6.6, respectively. The former peak (barley lipoxygenase-1) was identical to lipoxygenase of the ungerminated embryos, but the latter peak (barley lipoxygenase-2) was found only in the germinating embryos. The newly found isoenzyme, barley lipoxygenase-2, converted linoleic acid mainly to 13-hydroperoxy linoleic acid, and could oxidize esterified derivatives of linoleic acid (methyl linoleate and trilinolein) much strongly than barley lipoxygenase-1.     

Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway.

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z)-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S)-hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.     

On the mechanism of oxidation of cholesterol at C-7 in a lipoxygenase system.

Incubation of [7-2H2]cholesterol with soybean lipoxygenase and linoleic acid in the presence of oxygen gave a mixture of 5-cholestene-3 beta,7 alpha-diol, 5-cholestene-3 beta,7 beta-diol, 3 beta-hydroxy-5-cholesten-7-one,5 alpha,6 alpha-epoxycholestan-3 beta-ol, and 5 beta,6 beta-epoxycholestan-3 beta-ol. The conversion into the 7-oxygenated products was associated with a very high intermolecular isotope effect (KH/KD = 15-17), suggesting that the rate-limiting step in the overall conversion is likely to be the abstraction of hydrogen at C-7 in a radical reaction. Evidence that linoleic acid is to some extent directly involved was obtained with the use of [7-3H]cholesterol. Incubation of [7-3H]cholesterol resulted in a significant incorporation of 3H in the reisolated linoleic acid fraction. The isotope effect associated with conversion of [7 alpha-2H]cholesterol into 7-oxygenated products in the lipoxygenase system was 2-3, indicating that the extraction of hydrogen is nonstereospecific. Incubation of [7-2H2]cholesterol with 13-hydroperoxy-9,11-octadecadienoic acid gave the above 7-oxygenated products with relatively small isotope effects (KH/KD = 3-4). It is concluded that the most important mechanism for oxidation of cholesterol at C-7 in the lipoxygenase system involves participation of radicals and that a carbon-centered linoleic acid radical can extract hydrogen directly from cholesterol. Fatty acid hydroperoxides and their secondary products seem to be less important as initiators in connection with oxidation of cholesterol.