High Lipoxygenase Activity Research Articles

Molecular Identification of Alleles on Lpx-B1 Locus and Lipoxygenase Activity in Durum Wheat ( Triticum turgidum L.)

Lipoxygenase(LOX) has a close relationship to the processing quality of flour products in durum wheat(Triticum turgidum L.). Activity of LOX is greatly influenced by the locus of Lpx-B1. The Lpx-B1 alleles in 167 durum wheat varieties from different countries or regions were identified using gene-specific primers, and the Lpx-B1 genotypes were analyzed based on LOX activity data. The LOX activity varied greatly in the 167 varieties, ranging from 0.20 to 7.98 AU min–1 g–1. On Lpx-B1.1 locus, three alleles(Lpx-B1.1a, Lpx-B1.1b, and Lpx-B1.1c) were identified with frequencies of 55.1%, 7.8%, and 37.1%, respectively. Loci Lpx-B1.2 and Lpx-B1.3 were always reciprocally present in varieties, in which Lpx-B1.2 was presented in 146 varieties and Lpx-B1.3 in the remaining 21 varieties. This result indicates the allelic possibility of Lpx-B1.2 and Lpx-B1.3. In the three Lpx-B1.1 genotypes, the Lpx-B1.1b varieties had significantly higher LOX activity than the Lpx-B1.1a and Lpx-B1.1c varieties, and varieties carrying Lpx-B1.1c allele possessed the lowest LOX activity. The LOX activity in Lpx-B1.3 genotypes was significantly higher than that in Lpx-B1.2 genotypes. A total of six genotypic combinations were found on the three Lpx-B1 loci in 167 varieties. Among them, Lpx-B1.1b/Lpx-B1.3 combination had the highest LOX activity and combinations of Lpx-B1.1c/Lpx-B1.2 and Lpx-B1.1c/Lpx-B1.3 had the lowest LOX activity. These results are informative to quality breeding of durum wheat.

Enhanced lipoxygenase activity is involved in the stress response but not in the harmful lipid peroxidation and cell death of short-term cadmium-treated barley root tip

Root growth inhibition and radial root swelling were the characteristic symptoms of barley root tips after the short-term exposure of roots to 15 and 30μM Cd. Higher Cd concentrations caused extensive cell death and root growth arrest. Enhanced lipid peroxidation was observed as early as 1h after the short-term treatment in a Cd concentration-dependent manner. In contrast to lipid peroxidation, the induction of lipoxygenase activity was detected only 3h after the exposure of roots to 15 or 30μM Cd. In addition, it was not observed in 60μM Cd-treated root tips. The highest lipoxygenase activity was detected 6h after 15μM Cd treatment in the meristematic and elongation zone of root tip and was probably associated with the radial expansion of cells. Our results indicate that the upregulation of lipoxygenase is an important component of stress response in barley roots to toxic Cd. It is probably involved in the morphological stress response of root tips or/and in the alleviation of Cd-induced toxic alterations in plant cell membranes, but it is not responsible for the Cd-induced harmful lipid peroxidation and cell death.

Molecular characterization of a novel type of lipoxygenase (LOX) gene from common wheat (Triticum aestivum L.)

Lipoxygenases (LOXs) are key enzymes catalyzing the oxidation of polyunsaturated fatty acids, and play an important role in grain storage, seed vigor and bread-making. In this study, we report cloning and characterization of TaLOX3, the homolog gene of barley LoxB, from common wheat (Triticum aestivum L.). The full-length cDNA sequence of TaLOX3 had an open reading frame of 2,634 bp encoding a deduced polypeptide of 878 amino acid residues with a predicted molecular weight of 99,549.79 Da and an estimated isoelectric point of 6.26. TaLOX3 was assigned to chromosome 4A. The multiple alignment result showed that TaLOX3 protein had an identity of 91.3% to barley LOX3 (encoded by LoxB). Profile analyses revealed that the three TaLOX genes are expressed in unique patterns in various organs. Furthermore, the expression profiles of the TaLOX3 gene were determined by q-PCR using the high and low seed LOX activity of different wheat varieties. The result showed that TaLOX3 transcript levels increased significantly during the development of wheat grain. Further analysis indicated genotypes with high LOX activity were associated with higher expression levels than the low LOX activity ones. This indicates that TaLOX3 may contribute to the total LOX activity in common wheat seeds. Phylogenetic analysis conducted with LOXs from rice, maize, Brachypodium distachyon genome, barley and wheat seeds (TaLOX1, TaLOX2 and TaLOX3) revealed that LOXs could be well resolved into two clades (G1 and G2). Moreover, G1 can be divided to three subgroups, with each TaLOX in one subgroup. Collectively, these results can extent our knowledge of the molecular characterization of LOXs, and will provide a valuable resource for wheat quality improvement and breeding.

Enzyme activity in bioregulator-treated tomato (Solanum lycopersicon) genotypes.

The challenges of using exogenous plant bioregulators to manipulate plant responses by researchers and producers are increasing rapidly. In previous studies, we have demonstrated that 100 mg/l concentration of the bioregulators; indole-acetic acid (IAA), indole butyric acid (IBA) and naphthalene acetic acid (NAA) is suitable for tomato growth in the field. In this work, spectrophotometric analysis was conducted to determine the level of enzymes that could affect the keeping quality of the fruits harvested at the orange-red ripe stage following pre-sowing seed treatments with 100 mg/l solutions of the bioregulators. The NHLy 11, NHLy 12, NHLy 13, NHLy 15 and NHLy 16 genotypes were used and the activities of lipoxygenase (LOX), catalase, peroxidase, alcohol dehydrogenase and acid phosphatase determined. All treated genotypes showed high LOX activity with a range of 0.06 - 1.07 mmol.abs.units (A234). The NAA-treated NHLy13 genotype had complete loss of catalase activity after 210 s. Peroxidase activity was higher in NAA-treated genotypes with values between 0.07 and 0.62 abs. units/g (A430) while alcohol dehydrogenase activity suffered a decline in all IBA-treated genotypes. IAA gave consistently higher acid phosphatase activity than IBA and NAA in all the test genotypes. These findings have important implications on the biochemical changes associated with the fruits during storage and processing.

Phenolic Content Pattern, Polyphenol Oxidase and Lipoxygenase Activity in Relation to Albinism, Fruit Malformation and Nubbins Production in Strawberry (Fragaria x ananassa Duch)

Total phenol content, polyphenol oxidase (PPO) and lipoxygenase (LOX) activity were determined in strawberry fruits to establish a relationship between phenolic content, PPO and LOX activity with the production of abnormal fruits in 10 different strawberry cultivars. The study revealed that all strawberry cultivars produced abnormal fruits in varying proportion. Highest percentage of abnormal fruits was produced by Etna (21.8%), and the lowest by Senga Sengana (3.7%). Among cultivars, Etna produced maximum percentage of albino (25.8%), malformed (22.7%) or nubbin (button) berries (16.9%), while the lowest percentage of albino (5.2%), and malformed fruits (2.5%) were produced by Senga Sengana, and nubbin (button) (2.9%) by Camarosa. Total phenol content and PPO and LOX activity varied widely among different strawberry cultivars. The highest phenol content was recorded in Senga Sengana (14.4 mg g−1, fresh tissue weight) and the minimum in Etna (3.1 mg g−1 fresh tissue weight). Likewise, Etna exhibited the minimum PPO activity (0.503 ΔA400 g−1 min−1) and Senga Sengana, the maximum (0.732 ΔA400 g−1 min−1). Similarly, normal fruits exhibited higher PPO activity (0.753 ΔA400 g−1 min−1) than abnormal fruits. The highest LOX activity was exhibited by Senga Sengana (0.462 μ katal kg−1) and the lowest by Etna (0.338 μ katal kg−1) Similarly, LOX activity was very high in normal fruits (0.568 μ katal kg−1) in comparison to abnormal ones. The regression equations developed between total phenol content, PPO and LOX activity with abnormal fruits revealed that an inverse relationship existed between total phenol content, PPO and LOX activity and production of abnormal fruits in strawberry.

Oxidative Responses of St. Augustinegrasses to Feeding of Southern Chinch Bug, Blissus insularis Barber

Southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is a serious insect pest of St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, a turfgrass commonly grown in the southeastern United States. Resistance to southern chinch bug has been identified in the polyploid St. Augustinegrass varieties 'Floratam' and 'FX-10', and the diploid 'Captiva'. However, southern chinch bug in Florida and elsewhere has overcome Floratam's resistance. This research investigated the potential role of selected plant oxidative enzymes in resistance/susceptibility to southern chinch bug in two polyploid varieties (FX-10 and Floratam) and two diploid varieties (Captiva and Palmetto). Oxidative enzyme activity was estimated spectrophotometrically from plant samples collected 1, 3, 5, and 8 days after southern chinch bug infestation and from uninfested control plants. Resistant FX-10 and Captiva had significantly higher peroxidase activity, while Captiva had significantly higher polyphenol oxidase activity 5 and 8 days after infestation compared to uninfested controls. FX-10 had higher lipoxygenase activity 3, 5, and 8 days after infestation compared to uninfested controls. Catalase activities did not differ between infested and control plants in any of the varieties tested. Native gels stained for peroxidase indicated that certain isozymes in FX-10 and Captiva were induced 5 and 8 days after infestation. Isozyme profiles of polyphenol oxidase and lipoxygenase did not differ between control and infested FX-10, Floratam, Captiva, and Palmetto. Potential mechanisms to explain the correlation of resistance to southern chinch bug in FX-10 and Captiva with higher activities of oxidative enzymes are discussed.

Correlation between the Flavor and Quality of Radix Astragali: The Extraction and Characterization of Lipoxygenase in Radix Astragali

Abstract Lipoxygenase (LOX) is a key biotic factor producing the bean flavor in Radix Astragali. LOX was studied through spectro-photometry and Linolic acid. Extraction samples and detection methods were developed to investigate a range of biotic and abiotic factors, including the temperature, metal ion, chelate and age, and to evaluate the LOX activity of Radix Astragali. The results showed that LOX exists in the Radix Astragali grown in the Hunyuan County of Shanxi Province, China. Radix Astragali was ground into fine powder using liquid nitrogen, followed by being soaked for 1 h in the borate buffer. LOX crude extracts were obtained through centrifugation at 4000 r/min for 10 min at 4°C. LOX crude extracts and the substrate solution were mixed at a ratio ranging from 1:10 to 1:3 when the LOX activity was detected at a wavelength of 236 nm. The results also suggested that the enzyme could be stable for 15 min at the temperature from 0°C to 50°C, but the enzymes rapidly became inactivated after 2 min at 70°C. The enzymes were activated by Fe 3+ , Fe 2+ and Ca 2+ , but inhibited by Zn 2+ , Mg 2+ and tartaric acid. The Radix Astragali plants of 5 years old had the highest LOX activity, suggesting that LOX activity was associated with the growth age. The findings were useful for further study of the mechanism of producing the bean flavor in Radix Astragali and for searching for authentic growing sites.

Response of Endogenous Salicylic Acid and Jasmonates to Mechanical Wounding in Pea Leaves

The roles of on endogenous jasmonates (JAs) and salicylic acid (SA) in wounding response were investigated. Pea ( Pisum sativum L.) seedlings were treated with three different methods including mechanical wounding, JAs application, and SA application. The contents of endogenous JAs and SA, as well as the activities of the related enzymes were detected by enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), and spectrophotometer, respectively. The results showed that endogenous JA rapidly accumulated within 30 min after wounding. The increase in the activities of both lipoxygenase (LOX) and allene oxide synthase (AOS) lagged behind JAs burst. A second slight increase in JAs level was observed at 24 h after wounding treatment, and at the same time point, higher activities of LOX and AOS were also detected. Endogenous free SA content decreased accompanied with JAs burst. Effects of exogenous JA application were similar to those of wounding treatment on endogenous SA level and phenylalanine ammonia lyase (PAL) activity, whereas exogenous SA application led to the significant inhibition of LOX and AOS activities and the decrease of endogenous JAs level at the early stage of treatment. It is thus suggested that JAs burst and SA decrease in early response to wounding may constitute an important mechanism by which plant starts the related defense reaction and adapts to wounding stress.

Soybean variety and storage effects on soymilk flavour and quality

SummarySoymilk prepared from five soybean cultivars, grown in Ontario, were analysed for protein, oil, mineral composition, viscosity, colour, lipoxygenase (LOX) activities and flavour profile. Among the five soybean cultivars, the Vinton 81 variety contained the highest protein and the lowest fat. The yield of soymilk from all five cultivars was similar. Major differences were observed in viscosity and in the composition of both the soymilk and the okara. Higher protein and fat extractability was found in soymilk made from S08‐80 and Vinton 81 varieties. Their okara protein contents were also among the highest. Minimum extractability was observed with S03W4 cultivar. Soymilk made from S 03W4 and Vinton 81 cultivars had the whitest colour (lowest ΔE values). Viscosity values were the highest for S08‐80, FG1 and S20‐20 varieties. Headspace solid‐phase microextraction gas chromatography was used to analyse volatile compounds in soymilk. A total of fourteen volatiles were identified, among which aldehydes and their corresponding alcohols were the major compounds. Similar volatile compounds were identified in all the samples analysed but at different concentrations. The highest LOX activity was observed in the Vinton 81 and S20‐20 soybean cultivars, which had the highest total volatile and hexanal contents. A positive correlation (correlation coefficient = 0.82) between enzymatic activity and the total volatiles was observed. Vinton 81 cultivar was subjected to storage (at 18 °C and 50% relative humidity) for a period of 10 months. Soymilk was prepared at different times during storage. The results showed that the soymilk colour, LOX and total volatiles were significantly (P < 0.05) affected by the storage of the soybeans over time.

Effect of lipoxygenase activity in defatted soybean flour on the gelling properties of soybean protein isolate

Soybean lipoxygenase was inactivated to different degrees by dry heating of defatted soybean flour for 0, 5, 10, 15, 20 and 25 min and soy protein isolates were prepared thereof by isoelectric precipitation of the water extract of the defatted soybean flour. The fluorescence emission intensity at 420 nm of the chloroform–methanol extract of soy protein isolates, which was indicator of the existence of peroxidized lipid, varied in parallel with the lipoxygenase residual activity in defatted soybean flours. The dispersion of soy protein isolate showed an increasing turbidity with the increase of lipoxygenase residual activity in the starting defatted soybean flour, suggesting an elevated tendency to form insoluble aggregates during the preparation of soy protein isolate. Small deformation rheological test revealed that the gelling times were shorter for those soy protein isolates derived from low lipoxygenase activity defatted soybean flours than that of high lipoxygenase activity. Frequency sweep showed that G′ of soy protein isolate derived from low lipoxygenase defatted soybean flour was independent of oscillation frequency in contrast to that of derived from non dry-heated defatted soybean flour, the latter showed a marked frequency dependence. Large deformation test revealed that the gel hardness increased about 10 times after dry heating of defatted soybean flour for 20 min. As the increase of the lipoxygenase residual activity, the gel permeability increased markedly, suggesting that soy protein isolate from high lipoxygenase defatted soybean flour produced coarser textured gel, which corresponded well with the results of scanning electron microscopy.

Wheat Lipoxygenase Activity Induces Greater Loss of Carotenoids than Vitamin E during Breadmaking

The current study was undertaken to provide solutions to optimize the unsaponifiable antioxidants content of bread. We report a complete description of changes in wheat carotenoids and vitamin E content from grain to bread and highlight the most important processing steps affecting their level in wheat bread. Major carotenoids losses occurred during kneading. A close correlation (r(2) = 0.97; P = 0.05) was found between carotenoid pigment losses and lipoxygenase (LOX) activity, both parameters depending on wheat genotype. The use of wheat species exhibiting high carotenoid contents and low LOX activity was shown to preserve significant carotenoid level in the bread. No relation was found between vitamin E losses during doughmaking and LOX activity. In addition, moderate kneading resulted in higher vitamin E retention in comparison with carotenoids (12% and 66% losses, respectively). It is concluded that carotenoids are more susceptible to oxidation by endogenous lipoxygenase than vitamin E during breadmaking. This study showed that bread nutritional quality, in terms of antioxidant content, could be improved by selecting suitable cereal genotypes, if this potential is preserved by milling and baking processes.

Analysis of 15-Lipoxygenase Activity in Irradiated Thymocytes

We studied the effect of gamma-radiation on 15-lipoxygenase activity in rat thymocytes. The enzyme activity was determined as the rate of linoleic acid oxidation by the protein fraction isolated from the control and irradiated thymocytes under standard conditions. We demonstrate lipoxygenase activation immediately after irradiation of thymocytes. High lipoxygenase activity is observed in the cells for no more than an hour after irradiation. No high lipoxygenase activity later indicates that its synthesis is not directly induced by irradiation. Irradiation-induced generation of lipid peroxides can be the factor of lipoxygenase activation.

Release of deuterated (E)-2-nonenal during beer aging from labeled precursors synthesized before boiling.

Although lipid autoxidation in the boiling kettle is a key determinant of the cardboard flavor of aged beers, recent results show that mashing is another significant source of wort nonenal potential, the well-known indicator of how a beer will release (E)-2-nonenal during storage. Although unstable, deuterated (E)-2-nonenal nitrogen adducts created during mashing can in some cases partially persist in the pitching wort, to release deuterated (E)-2-nonenal during beer aging. In the experiment described here, the relative contributions of mashing and boiling were estimated at 30 and 70%, respectively. The presence of oxygen during mashing and, to a lesser extent, high lipoxygenase activity can intensify the stale cardboard flavor.

Characterization of the lipoxygenases in some olive cultivars and determination of their role in volatile compounds formation.

Enzymatic extracts from olive pulp (Olea europea L.) were used to characterize lipoxygenase (LOX) activity in order to determine its role in the biogenesis of the volatile compounds that influence the aroma of extra virgin olive oil. The LOX activity was tested spectrophotometrically at an optimal pH of 6.0 in three olive cultivars, Ascolana Tenera, Kalamata, and FS17. The trend of the LOX activity was determined as a function of pH and temperature; the kinetic constants of the enzyme were also determined. The highest LOX activity was observed in the FS17 fruit, which had the highest concentrations of C(5) and C(6) compounds (aldehydes, alcohols, and ketones), followed by Kalamata and Ascolana T., respectively. Given the direct relationship between enzymatic activity and the quantity of aromas measured in the fruit, it is hypothesized that olive LOX is involved in the formation of C(5) and C(6) volatile compounds. To study the mechanism of the movement of the aromas from the fruit to the oil, which was obtained by simple mechanical extraction, the headspace of the oil for each cultivar was analyzed as well as the aromatic composition in order to compare it with the aromas of the fruit.

Effect of overexpression of fatty acid 9-hydroperoxide lyase in tomatoes (Lycopersicon esculentum Mill.).

To modify the flavor properties of tomato fruits, cucumber fatty acid hydroperoxide lyase (HPL), which can act on 9-hydroperoxides of fatty acids to form volatile C9-aldehydes, was introduced to tomato plants. Through enzyme assay, high activity of the introduced HPL could be found in either the leaves or fruits of transgenic tomatoes; however, the composition of volatile short-chain aldehydes and alcohols in the transgenic tomato fruits was little modified. This was unexpected because tomato fruits have high lipoxygenase activity to form 9-hydroperoxides. When linoleic acid was added to a crude homogenate prepared from the transgenic tomato fruits, a high amount of C9-aldehyde was formed, but the amount of C6-aldehyde was almost equivalent to that in nontransgenic tomatoes. Through quantification of fatty acid hydroperoxides, it has been revealed that 13-hydroperoxides of fatty acids are preferably formed from endogenous substrate, whereas 9-hydroperoxides are formed from fatty acids added exogenously. From these observations, possible mechanisms to regulate metabolic flow of the lyase pathway are discussed.

Reaction of soybean lipoxygenase-3 in emulsions as affected by emulsifiers, salts and phospholipids

Soybean lipoxygenase-3 reaction in emulsions stabilized by proteins and low molecular weight surfactants was investigated. High lipoxygenase activity was observed in the emulsions stabilized by proteins, whereas the reaction rates were very low in the surfactant-stabilized emulsions. The specificity of hydroperoxide isomers produced by lipoxygenase reaction was independent of the type of emulsifier. The effects of neutral salts and phospholipids addition on lipoxygenase reaction in protein-stabilized emulsions were also studied. Lipoxygenase activity decreased with increasing concentration of neutral salts. The efficiency of inhibition of lipoxygenase activity by salts was related to the activity of salts to decrease ζ-potential at the surfaces of substrate oil droplets. With respect to the effect of addition of phospholipids, it was shown that the lowering of interfacial tension and the fall in ζ-potential at the oil droplet surfaces were responsible for the inhibition of lipoxygenase activity.

Membrane lipid peroxidation, free radical scavangers and ethylene evolution in Amaranthus as affected by lead and cadmium

Membrane lipid peroxidation, activity of free radical scavangers and ethylene evolution of Amaranthus lividus seedlings were used to determine the lead and cadmium (1, 10, 100 and 1000 µM) induced phytotoxicity. Malondialdehyde (MDA) accumulation and higher lipoxygenase activity (LOX) was found in the 7-d-old treated seedlings. The activities of free radical scavangers like peroxidase, catalase and superoxide dismutase declined considerably with the concomitant rise in hydrogen peroxide level. Heavy metal treatment also caused decline in ethylene evolution in germinating seedlings.

Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate.

A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.C. 1.13.11.12) in wheat (Triticum aestivum) leaves. Immunoblot analysis using anti-LOX antibodies revealed the induction of 92- and 103-kD LOX species after Pgt elicitor treatment. In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants. The effects of chitin oligomers and chitosan resembled those caused by MJ. In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action. LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions. It is most active at pH 5.5 to 6.0, and product formation from linoleic and [alpha]-linolenic acid is clearly in favor of the 9-LOOHs. It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves.

Mass spectrometric identification of N-phenyllinoleamide metabolites in mouse peritoneal macrophages.

N-Phenyllinoleamide (NPLA), the anilide of linoleic acid, has been regarded as a marker of the case oils associated with toxic oil syndrome, but the mechanisms of toxic injury remain enigmatic. Experimental data have related an increased systemic toxic effect of heated linoleic anilides to chemical structural modifications that might also be possible by in vivo metabolism; however, little is known about their metabolism. Taking into account that NPLA is a derivative of linoleic acid, a fatty acid that can be metabolized by lipoxygenase activity to a vast array of derivatives possessing biological activity, the objective has been to elucidate the oxidative metabolism of NPLA by mouse peritoneal macrophages, a cellular model with high lipoxygenase activity. Cells were incubated with 0.1 mM NPLA spiked with N-phenyl[1-14C]linoleamide. The metabolites were separated by high-performance liquid chromatography and individually collected prior to GC/MS analysis. Identification of trihydroxy-, monohydroxy- and epoxy-derivatives of NPLA, suggests that this xenobiotic can be metabolized via the same oxidative processes as for linoleic acid. Furthermore, identification of the non-amidated monohydroxylated and trihydroxylated derivatives of linoleic acid arising from NPLA suggests an amidase-like activity with release of aniline and the free fatty acid. These results provide information about possible biological structures arising from NPLA, and open the way to evaluate the biological significance of these metabolites in the inflammatory reactions associated with toxic oil syndrome.

Lipoxygenase is an abundant protein in cucumber exudates

The presence of lipoxygenase (LOX) has been reported in many plant organs. High LOX activity (1–2 μkatal/mg protein) was detected in exudates from cut cucumber (Cucumis sativus L.) stems and petioles. Exudate LOX had a pH optimum of 5.0, an estimated molecular weight of 95 kDa and cross-reacted on sodium-dodecyl-sulfate gels with anti-LOX antibodies raised against soybean leaf LOX isoenzymes. Lipoxygenase activity was detected on native gels stained with o-dianisidine using linoleic acid as a substrate. Enzyme activity was similar with linoleic and linolenic acid and 2 times greater with arachidonic acid as substrate. At pH 6.8, LOX metabolized linoleic acid into 13- and 9-hydroperoxides at a ratio of 1∶2. Linolenic acid was preferentially oxidized at carbon 13. Lipoxygenase activity was inhibited by n-propyl gallate (IC50 300 nM) and nordihydroguaiaretic acid (IC50 25 nM), but not by nonsteroidal anti-inflammatory drugs. LOX activity was enhanced 4.5-fold by 300 mM Ca2+. Spermine at 1 mM, and putrescine and spermidine at 2 mM completely inhibited LOX activity, but at low concentrations spermine (100 mM) and spermidine (100–500 mM) significantly stimulated LOX activity: 8- and 4.5-fold, respectively. Tissue printing of stem, petiole and hypocotyl sections with subsequent incubation with the antiserum raised against soybean leaf LOX revealed the presence of LOX in the internal and external phloem and in the sieve tubes.