Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.     

On the reaction specificity of the lipoxygenase from tomato fruits

A lipoxygenase was purified 300-fold from a homogenate supernatant of ripe tomato fruits by fractionated ammonium sulfate precipitation and anion exchange fast protein liquid chromatography. The specific linoleate oxygenase activity of the final enzyme preparation was 1300 nkat per mg protein at pH 6.8 and 25°C in the absence of any detergent. The enzyme oxygenated linoleic acid and α-linolenic acid at comparable rates, whereas γ-linolenic acid, arachidonic acid, 11,14-eicosadienoic acid and 11,14,17-eicosatrienoic acid were poor substrates. Linoleic acid was converted to 9(S)-hydroperoxy-10E,12Z-octadecadienoic acid, whereas 5(S)-HpETE, 11(S)-HpETE and 8(S)-HpETE were identified as major oxygenation products from arachidonic acid. The tomato lipoxygenase did not react with either dilinoleyl phosphatidylcholine or the lipid extract from beef heart mitochondria. The possible biological importance of the reaction of tomato lipoxygenase with arachidonic acid is discussed.     

Identification and characterization of lipoxygenase isoforms in senescing carnation petals

A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents.     

Properties of a Lipoxygenase in Green Algae (Oscillatoria sp.)

A lipoxygenase preparation was obtained from green algae Oscillatoria sp. and was shown to differ from previous described lipoxygenases in the positional specificity and pH characteristics of the dioxygenation reaction. The enzyme had a pH optimum at 8.8 and was inactive at pH 6. Oscillatoria lipoxygenase converted linoleic acid into two products: 13-hydroperoxylinoleic acid (52%) and 9-hydroperoxylinoleic acid (48%). The molecular weight of the enzyme was estimated at 124,000. Esculetin was found to be the best inhibitor of the enzyme activity.     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Nitrogen and methyl jasmonate induction of soybean vegetative storage protein genes

Vegetative storage protein (VSP) and VSP mRNA levels in soybean (Glycine max) leaves correlated with the amount of NH₄NO₃ provided to nonnodulated plants. The mRNA level declined as leaves matured, but high levels of N delayed the decline. This is consistent with the proposed role for VSP in the temporary storage of N. Wounding, petiole girdling, and treatment with methyljasmonate (MeJA) increased VSP mRNA in leaves 24 hours after treatment. The magnitude of the response depended on leaf age and N availability. N deficiency essentially eliminated the response to wounding and petiole girdling. MeJA was almost as effective in N-deficient plants as in those receiving abundant N. Inhibitors of lipoxygenase, the first enzyme in the jasmonic acid biosynthetic pathway, blocked induction by wounding and petiole girdling but not by MeJA. This supports a role for endogenous leaf jasmonic acid (or MeJA) in the regulation of VSP gene expression.     

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. ¹⁸O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.     

Prominent ornaments and rapid color change: use of horns as a social and reproductive signal in unicornfish (Acanthuridae: <Emphasis Type="Italic">Naso</Emphasis>)

The behavior of two unicornfishes, Naso unicornis with a horn-shaped protuberance on the forehead and Naso vlamingii with a round protuberance, was observed in social and reproductive contexts in an aquarium. Males of both species performed displays that were associated with quick changes in the colors of the protuberance and other body parts, highlighting the protuberance by color contrast. The displays with color changes of the protuberance took place when a male courted a female in the evening or in the night. The same displays were occasionally performed by males toward females throughout the daylight hours. In N. vlamingii, dominant males displayed the protuberance toward subordinate smaller males. Although the sizes and shapes of the protuberance were sexually monomorphic, females of both species rarely made displays of the protuberance. A hypothesis about the function of the protuberance in unicornfishes is proposed: that males use the protuberance as a conspicuous signal in courtship and contests among males by emphasizing it with quick changes of its color contrasts. We argue that the combination of morphologically distinct protuberances and quick changes of their color is a sexually selected trait among unicornfishes, because these characters play important roles in intersexual and intrasexual interactions.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Hormone-dependent lipoxygenase activity in Achlya ambisexualis

Homogenates of male Achyla ambisexualis oxidize exogenously added [¹⁴C]arachidonic acid to an unidentified lipoxygenase product. Synthesis occurs at a rate of 10.6 ± 1.3 μg mg⁻¹ protein 30 min⁻¹. Activity in homogenates of female mycelium is only 2.1 ± 1.2. Conversion is eliminated by the lipoxygenase inhibitor nordihydroguaiaretic acid (10⁻⁴ M). Homogenates prepared from the male grown in chemical but not physical contact with female mycelium had decreased lipoxygenase activity (3.1 ± 1.5), suggesting that antheridiol produced by the female decreases lipoxygenase activity in the male. To confirm this, actively growing male cultures were exposed to 10⁻⁹ M 7-deoxy-7-dihydroantheridiol, a stable analog of antheridiol, for 24 h. Homogenates from these cultures also had diminished lipoxygenase activity (2.7 ± 1.0). 7-Deoxy-7-dihydroantheridiol added to the incubation mixture at 10⁻⁹ M had no effect on lipoxygenase activity (9.0 ± 1.8), excluding a direct action of 7-deoxy-7-dihydroantheridiol on the enzyme. These data support the hypothesis that lipoxygenase products are associated with vegetative growth and suggest the antheridiol initiation of reproductive growth suppresses lipoxygenase activity.     

Partial purification and characterization of the lipoxygenase from grains of Hordeum distichum

Lipoxygenase activities in ungerminated and germinating barley grains were found to be associated exclusively with the embryos. A lipoxygenase was extracted from ungerminated embryos and partially purified by fractional precipitation with ammonium sulfate and gel-filtration. Both the crude extracts and the purified preparation appeared to contain only a fatty acid type lipoxygenase which mainly converted linolele acid to 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid. The purified enzyme was inhibited by its own products, hydroperoxides, but not by 1 mM cyanide, EDTA, Hg²⁺ or Cu²⁺.     

An Enzymatic Conversion of Lipoxygenase Products by a Hydroperoxide Lyase in Blue-Green Algae (Oscillatoria sp.)

An enzyme has been isolated from blue-green algae Oscillatoria sp. which utilizes the product, 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPOD), of lipoxygenase for its substrate. This enzyme, termed hydroperoxide lyase, converts the conjugated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9, 11-dienoic acid. The structure of the latter has been determined by ultraviolet spectroscopy and mass spectrometry. 9-HPOD is not a substrate for this enzyme. The hydroperoxide lyase from Oscillatoria sp. has a maximum of activity at pH 6.4 and 30°C. The molecular weight of the enzyme was estimated at 56,000. The enzyme was not inhibited by BW 755C, but was inhibited by molecules containing more than one hydroxyl group. Quercetin was found to be the best inhibitor of the enzyme activity. The purified hydroperoxide lyase from Oscillatoria sp. showed an apparent K_m of 7.4 micromolar and a V_max of 35 nanomoles per minute per milligram of protein for 13-HPOD. An enzymatic pathway for the biogenesis of oxodienoic acid from linoleic acid is proposed. This involves the sequential activity of lipoxygenase and hydroperoxide lyase enzymes.     

Substituted furans as potent lipoxygenase inhibitors: Synthesis,in vitro and molecular docking studies

A number of 2-methyl-4-(2-oxo-2-phenyl-ethyl)-5-phenyl-furan-3-carboxylic acid alkyl ester derivatives (3a–j) were synthesized and evaluated for their in vitro inhibitory activity on soybean lipoxygenase enzyme. Among the screened compounds, 5-(4-bromo-phenyl)-4-[2-(4-bromo-phenyl)-2-oxo-ethyl]-2-methyl-furan-3-carboxylic acid methyl ester (3g) has been found to exhibit potent inhibitory activity with IC₅₀12.8 μM using nordihydroguaiaretic acid (NDGA) as standard. Molecular modeling was employed for better understanding of the binding between compounds and soybean lipoxygenase enzyme. The predicted binding energy values correlated well with the observed in vitro data.     

Identifying a Carotenoid Cleavage Dioxygenase (ccd4) Gene Controlling Yellow/White Fruit Flesh Color of Peach

Peach flesh color is a monogenic trait with the white phenotype being dominant over the yellow; its expression has been reported to be determined by a carotenoid degradative enzyme. In the present study, a carotenoid cleavage dioxygenase (ccd4) gene was analyzed to test whether it can be responsible for the flesh color determinism. The analysis was conducted on chimeric mutants with white and yellow sectors of the fruit mesocarp; it was then extended to a pool of cultivars and a segregating F₁ population. A ccd4 functional allele is consistently associated with the ancestral white flesh color; on the other hand, the yellow phenotype originated from at least three independent mutations disrupting ccd4 function, thus preventing carotenoid degradation. In addition, retro-mutations recovering ccd4 function and re-establishing the ancestral white flesh color were detected. Our results show that ccd4 is the gene controlling flesh color in peach; its expression results in the degradation of carotenoids in white-fleshed genotypes, while the yellow color arises as a consequence of its inactivation.     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.     

A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads

Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR.     

Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene

The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons.     

In vitro,fluorescence-quenching and computational studies on the interaction between lipoxygenase and 5-hydroxy-3′,4′,7-trimethoxyflavone from Lippia nodiflora L.

Abstract

Lippia nodiflora L. is extensively used in traditional medicine for several medicinal purposes, including their use in inflammatory disorders. In this study, the folk use of L. nodiflora was validated using the isolated natural compound, 5-hydroxy-3′,4′,7-trimethoxyflavone (HTMF) by in vitro, fluorescence spectroscopic and molecular modeling studies with lipoxygenase (LOX), because LOX plays an essential role in inflammatory responses. In this perspective, the methanol extract and HTMF are shown to demonstrate prominent inhibitory activity against soybean lipoxygenase, with an IC₅₀ value of 21.12 and 23.97?µg/ml, respectively. The data obtained from the spectroscopic method revealed that the quenching of intrinsic fluorescence of LOX is produced as a result of the complex formation of LOX–HTMF. The binding mode analysis of HTMF within the LOX enzyme suggested that hydrogen bond formation, hydrophobic interaction and π–π stacking could account for the binding of HTMF. Molecular dynamics results indicated the interaction of HTMF with LOX and the stability of ligand–enzyme complex was maintained throughout the simulation. The computational results are reliable with experimental facts and provided a good representation for understanding the binding mode of HTMF inside the active site of lipoxygenase enzyme.     

Genosensor for rapid,sensitive, specific point-of-care detection of H1N1 influenza (swine flu)

A 5′ amine group-linked haemagglutinin (HA) gene-specific probe was attached over the surface of a working electrode to develop a rapid, specific, and sensitive point of care detection assay for H1N1 (swine flu) in human respiratory nasal swabs. The probe was attached with a cysteine covered screen-printed gold electrode via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). The electrochemical assay was performed using differential pulse voltammetry with the use of the redox indicator methylene blue for the detection of different concentrations of the single-stranded viral genome. The developed genosensor showed high sensitivity for H1N1 influenza virus with a detection limit of 0.002 ng/6 μL of viral nucleic acid in the sample. Samples were analysed by quantitative real-time Polymerase Chain Reaction as well as by conventional PCR. The genosensor showed high specificity, as no cross-reaction was observed with the heterologous nucleic acid of different pathogens (Salmonella typhi, Neisseria meningitides, and Streptococcus pyogenes) and human DNA, and it was specific for H1N1 with a sensitivity of ∼49 μA cm⁻² ng^-1. Genosensor is based on a very simple methodology that can be followed based on its easy-to-access approach. It is quick and could be used as a point-of-care test for the detection of influenza virus within 30 min.