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Plant Physiol. (1998) 116: 1359-1366
Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results & Discussion
References
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In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O2 uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.
In the past animal researchers have spent considerable effort on
4-HNE research because of its cytotoxicity and mutagenicity (Esterbauer
et al., 1991 Despite the interest in 4-HNE, a biosynthetic pathway in animals has
not been found. In plants the biosynthetic route originates with
oxidation of linoleic acid by 9-specific LOX leading to NON by
hydroperoxide lyase cleavage. Subsequently, NON is oxidized by a
"3Z-alkenal oxygenase" and the resultant 4-HPNE is
reduced by a hydroperoxide-dependent peroxygenase (Gardner et al.,
1991 LOX-1 from soybean (Glycine max cv Williams) seeds was
prepared by the method of Axelrod et al. (1981) Enzyme Assays
INTRODUCTION
Top
Abstract
Introduction
Methods
Results & Discussion
References
). More recently, 4-HNE has been implicated as a lipid
signal because it activates phosphoinositide-specific phospholipase-C
(Rossi et al., 1994) and phospholipase-D (Natarajan et al., 1993), and
triggers Ca2+ influx in hepatocytes (Carini et
al., 1996).
; Gardner and Hamberg, 1993; Takamura and Gardner, 1996). In a
second pathway, the higher oxidation state of 4-HPNE is utilized by
peroxygenase to oxidize NON to 3,4-epoxynonanal, which subsequently
rearranges into 4-HNE (Gardner and Hamberg, 1993). To our knowledge,
the specific enzymes involved in 4-HNE formation have not previously been isolated and characterized. In an attempt to isolate the proposed
first enzyme, a 3Z-alkenal oxygenase, we found strong evidence that 3Z-alkenal oxidizing activity was identical
with soybean (Glycine max) LOX-1 activity. Additional data
pointed to the possible existence of other NON oxidizing activity,
including the other LOX isozymes of soybean. This research was
communicated in part at Plant Biology '97 in Vancouver, British
Columbia (Gardner and Grove, 1997).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results & Discussion
References
. The LOX-1 isolates were stored as a suspension in 2.3 m
(NH4)2SO4
at 3°C; protein concentration of the isolates used ranged from 12 to
30 mg/mL. NON was prepared from 3Z-nonen-1-ol by the
procedure of Ratcliffe and Rodehorst (1970), and the product was
purified by silica chromatography (Takamura and Gardner, 1996) followed
by open-column chromatography on Sephadex LH-20 (4 × 23 cm) with
hexane elution to remove contaminants, principally
3Z-nonenyl 3Z-nonenoate. 4-HNE was prepared as
described (Gardner et al., 1992), and 4-ONE was synthesized from 4-HNE
by oxidation with pyridinium chlorochromate (Corey and Suggs, 1975), followed by TLC isolation with hexane: diethyl ether (1:1, v/v) development (RF = 0.68); UV
max = 216 nm.
GC-MS and Flame-Ionization Detection GC
GC-MS was accomplished with a gas chromatograph (model 5890, Hewlett-Packard) interfaced with a mass selective detector (model 5971, Hewlett-Packard) operating at 70 eV. The capillary column used was an HP-5MS (Hewlett-Packard) cross-linked 5% phenyl methyl silicone, 0.25 mm × 30 m, film thickness, 0.25 µm. The aldehydes were separated by temperature programming from 65 to 260°C at a rate of 10°C/min. (-)-Menthoxycarbonyl derivatives were separated by programming from 160 to 260°C at 5°C/min (He flow rate = 0.67 mL/min).Chiral Analysis
For chiral analysis 4-HPNE was produced by incubating 1 mm NON and 0.09 mm 13S-HPODE with 0.48 mg of LOX-1 in 8 mL of 50 mm potassium borate, pH 8.3, for 15 min on ice with an O2 stream of bubbles. Incubations with the other LOX isozymes were similar, except the reaction volumes were doubled, and additionally, 0.1 m potassium Pipes, pH 6.5, was used. The amount of LOX-2 and LOX-3 used was 25 and 52 mg, respectively; the relatively larger amount of protein required reflects in part the nonhomogeneity of the LOX-2 and -3 fractions. The diethyl ether-extracted product was reduced with 10 mg of KI dissolved in 200 µL of methanol for 1 h in the dark, and then extracted into CHCl3 by addition of water/CHCl3. 4-HNE was isolated from the extracted material by TLC (Silica Gel 60 F254 precoated plates, Merck, 20 cm × 20 cm × 0.25 mm) using hexane:diethyl ether (1:1, v/v) development. The 4-HNE band was located by UV absorbance and a separately spotted standard (RF = 0.26-0.31). The scraped material was extracted with diethyl ether, the solvent was evaporated, and residual 4-HNE was derivatized with (-)-menthoxycarbonyl chloride for chiral analysis using a slightly modified procedure of Hamberg (1971).
The (-)-methoxycarbonyl derivative of 4-HNE was isolated from
Silica Gel 60 F254 TLC (hexane:ethyl acetate
[95:5, v/v] development) immediately above the by-product menthol
band at a RF of about 0.2. This isolate separated
by GC-MS into two peaks (retention times = 14.45 and 14.55 min)
giving virtually identical mass spectra: m/z (relative
intensity) 200 (4); 199 (1); 156 (10); 139 (40); 138 (60); 123 (20);
109 (8); 95 (52); 83 (100); 81 (65); 69 (38); and 55 (47).
). The resultant methyl
esterified (-)-menthoxycarbonyl derivatives of methyl 2R-
and 2S-hydroxyheptanoate were analyzed by GC-MS giving two
peaks (retention time = 12.42 and 12.49 min) with virtually
identical mass spectra: m/z (relative intensity) 205 (5);
173 (4); 139 (50); 138 (100); 123 (33); 111 (13); 95 (65); 83 (98); 81 (65); 69 (33); and 55 (48). The relative percentage of each of the
(-)-menthoxycarbonyl derivatives of methyl 2R- and
2S-hydroxyheptanoate isomer was determined by
flame-ionization detection GC as described above for the
flame-ionization equipment. Retention times for the S- and
R-isomers were 11.30 and 11.43 min, respectively. The
identites of these derivatives were determined to be authentic by
subjecting known 13S-HPODE, as its reduced methyl ester, to
the same chiral analysis affording mainly the (-)-menthoxycarbonyl
derivative of methyl 2S-hydroxyheptanoate.
Preparation of Crude Soybean Enzyme
A crude soybean enzyme was prepared by homogenizing 2 g of hexane-defatted soybean flour (seeds from cv Williams) for 30 s with a Polytron homogenizer after soaking in 20 mL of 0.1 m potassium borate buffer (either pH 8.3 or 9.0) for 10 min. The homogenate was filtered through cheesecloth and centrifuged at 9300g for 15 min. The resultant supernatant was diluted 10-fold with the same buffer giving a protein concentration of 3.4 to 3.5 mg/mL. The diluted supernatant (20 mL) was used to oxidize NON (1 mm) for 5 min on ice while bubbling pure O2 through the solution. In certain experiments the diluted supernatant was preincubated for 5 min at 25°C with 20 mm H2O2 before incubation with NON to inactivate hydroperoxide-dependent peroxygenase (Takamura and Gardner, 1996). Products were extracted into
CHCl3 after adding a 3-fold volume of
CHCl3:CH3OH (2:1, v/v).
Either the product mixture or isolated 4-HPNE was reduced with KI,
4-HNE isolated, and subjected to chiral analysis as described above.
Protein Determination and Other Methods
Protein was determined by the bicinchoninic acid assay (Smith et al., 1985). All other chemical methods, including the preparation of
13S-HPODE, and preparation of derivatives are reviewed
(Gardner, 1997).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Methods
Results & Discussion
References
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LOX-1 Gives 3Z-Alkenal Oxygenase Activity
During preliminary attempts to isolate NON-oxidizing activity by ionic-exchange and gel-filtration methods, it was noted that activity consistently coincided with LOX-1 activity using linoleic acid as a substrate. Utilizing an activity assay at pH 9.0 that was determined to be optimum for NON oxidation (Takamura and Gardner, 1996), coelution of
the two activities occurred employing columns of Sephracyl S-200 and
DEAE-Sephacel using procedures similar to those reported previously
(Salch et al., 1995) (data not shown). Therefore, we resorted to a
method used to prepare highly purified soybean LOX-1 (Axelrod et al.,
1981). A check by SDS-electrophoresis showed that the isolate was a 90- to 100-kD protein with a minor contaminant of a slightly lower
molecular mass, which was probably due to degradation of LOX-1. As seen
in Figure 1, the final chromatographic separation employed by this method gave superimposed oxidation activities of NON and linoleic acid (LOX). It can also be seen in
Figure 1 that there was an immense difference in the amount of relative
activity; that is NON was 400- to 1000-fold less effective as a
substrate, compared with linoleic acid. Dependency of pH on NON
oxidation activity was also reminiscent of literature data (Christopher
et al., 1970) for linoleic acid oxidation by LOX-1 (Fig.
2).
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Products of NON Oxidation
The oxidation products of NON catalyzed by LOX-1 were separated by HPLC (Fig. 3), collected, and identified by GC-MS before and after preparation of appropriate derivatives. The GC-MS of HPLC-isolated 4-ONE was identical to the 4-ONE standard (retention time, 8.05 min) as follows: m/z (percent abundance, fragment identity) 154 (1, M+); 139 (2, M+
CH3); 125 (100, M+ CHO); 98 (92, M
[CH2]3CH3 + H+); 83 (68, M+
[CH2]4CH3);
70 (40); and 43 (45). Isolated 4-ONE was also derivatized with
methoxylamine hydrochloride (Aldrich) giving three separable bis methoxime peaks (due to syn and
anti isomerism, four isomers are possible). The spectra of
the three isomeric peaks were similar, but they differed mainly in
fragment ion intensities: m/z (fragment identity) 212 (M+); 181 (M+
CH3O); 166 (M+
CH3O CH3); 156; 154;
141 (M+
[CH2]4CH3);
125; and 110 (M+
[CH2]4CH3
CH3O). GC-MS of 4-HPNE isolated from HPLC gave
a peak with a mass spectrum identical to 4-ONE on the front side of the
peak, and on the back side of the peak the mass spectrum was identical
to an unknown rearrangement product of 4-HNE. Selected ion monitoring
confirmed this assessment. Since heat decomposition of 4-HPNE in the
injector port was anticipated, the isolate was reduced with neutral
methanolic KI. For GC-MS, the reduced compound was treated either with
OTMS reagent or benzylhydroxylamine followed by OTMS reagent. The GC-MS
of the first of the two derivatives proved to be the OTMS of 4-HNE
(retention time, 10.1 min) as follows: m/z (relative
intensity and ion structure) 228 (1, M+); 213 (5, M+ CH3); 199 (17, M+ CHO); 184 (5, M+
CHO CH3); 157 (100, M+
[CH2]4CH3);
143 (5); 129 (23); and 73 (73, TMS+). The second
derivative, the syn and anti benzyloxime-OTMS of 4-HNE (retention times, 12.2 and 13.1 min), gave essentially the same
mass spectrum as reported previously (Gardner and Hamberg, 1993). The
last-eluting HPLC peak was identified as 4-HNE by making its
benzyloxime-OTMS derivative for GC-MS of the syn and
anti isomers, as was completed above for KI-reduced 4-HPNE.
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Threshold Requirement and Product Shift
There were two intriguing aspects of NON activity. First, there was a requirement for a threshold concentration of LOX-1 to trigger the reaction (Fig. 4). This threshold phenomenon was ascertained by determining initial rates of O2 uptake with an O2 electrode (Fig. 5). Second, when sufficient LOX-1 was present, the product was principally 4-ONE, not the expected 4-HPNE (Fig. 3). The observation of a threshold enzyme requirement was believed to be due to a "lag phase" observed when LOX is incubated with substrate in the absence of hydroperoxide. That is, native LOX, an Fe2+ species, needs to be oxidized to Fe3+ to start the catalytic cycle. The length of the lag is inversely dependent on the amount of both LOX and hydroperoxides present (Smith and Lands, 1972). This need for a
threshold amount of LOX is undoubtedly the reason soybean LOX-1 was
previously found to be inactive with NON (Gardner and Hamberg, 1993).
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Chiral Analysis of 4-HPNE
Possible Origin of the 4R-Isomer of 4-HNE in Crude
Preparations
). O2-starved
reactions also can be triggered by excessive amounts of LOX (H.W.
Gardner, personal observation). Therefore, we spiked the reaction with
a small amount of a LOX-1 product, 13S-HPODE, to oxidize the
enzyme to the active Fe3+ state. This spiked
activity was compared with oxidation of NON in the absence of
13S-HPODE. Under these conditions NON-oxidizing activity was
enhanced and the threshold phenomenon disappeared (Fig. 4). In the
presence of 13S-HPODE at relatively low LOX-1 concentration,
the main product was 4-HPNE instead of 4-ONE (Fig. 6). Apparently, NON, being a
comparatively poor substrate, is inefficient in electron cycling the Fe
active site by substrate and O2 and,
consequently, 4-HPNE is consumed in the process of keeping the active
site oxidized. This tendency to further oxidize to the ketone is
reminiscent of LOX-1 oxidation of another 
,
-unsaturated carbonyl,
12-oxo-9Z-octadecenoic acid, to afford
9,12-dioxo-10E-octadecenoic acid (Kühn et al., 1991).
View larger version (19K):
[in a new window]
Figure 6.
HPLC separation of NON oxidation products after
addition of a below-threshold amount of LOX-1 (left), and an identical
treatment spiked with 55 µg of 13S-HPODE (right).
Conditions, methods, and abbreviations are the same as in Figure 3.
). The
structural similarities of the two substrates, and how they might fit
in the active site of LOX-1, is illustrated in Figure
8 using a model suggested for the
oxidation of linoleic acid (de Groot et al., 1975).
View larger version (12K):
[in a new window]
Figure 7.
Chiral analysis by GC separation of
R- and S-isomers of (-)-menthoxycarbonyl
derivatives of methyl 2-hydroxyheptanoates obtained from 4-HNE by
chemical modification. Top, Analysis of synthetic 4-HNE; bottom,
analysis of 4-HNE derived from LOX-1 oxidation of NON.
View this table:
Table I.
Analyses of stereoconfiguration of 4-HNE and 4-HPNE
View larger version (26K):
[in a new window]
Figure 8.
Mechanism of LOX oxidation of linoleic acid (top)
compared with oxidation of NON (bottom) showing the redox cycling of
the Fe active site and the hydrophobic pocket (hatched marks) that accommodates the 
6 tail-end of linoleic acid or NON.
), it was necessary to
ascertain the stereochemistry of the KI-reduced product of the crude
preparation to determine if there was evidence for a significant
contribution of LOX-1 in the biogenesis of 4-HNE. As seen in Table I,
the 4R-isomer was unexpectedly predominant at pH 8.3 (56.0%
4R-isomer) and at pH 9.0 (57.1% 4R-isomer).
). Using the procedure
of Axelrod et al. (1981), two peaks of activity eluted early from the
first of two DEAE-Sephadex columns used. These peaks showed LOX
activity at pH 6.5, but negligible activity at pH 9.0. The material
from the two peaks were tested for activity with 0.09 mm
13S-HPODE at both pH 8.6 and 6.5, giving oxidation ranging
from 27 to 54% of total NON. 4-HNE was isolated from the KI-reduced
product to complete stereochemical analyses. The first eluting isozyme
peak (presumably LOX-3 [Axelrod et al., 1981]) gave 55.5 and 71.4%
4S-enantiomeric excess at pH 8.6 and 6.5, respectively
(Table I). The second isozyme peak (presumably LOX-2 [Axelrod et al.,
1981]) furnished 60.8 and 86.5% 4S-enantiomeric excess at
pH 8.6 and 6.5, respectively (Table I). As might be expected,
stereochemical purity was improved at lower pHs where these isozymes
have their optima. However, even at pH 8.6 these isozymes afforded a
slight excess of the 4S-isomer. Thus, the 56 to 57%
enantiomeric excess of 4R-isomer obtained from crude preparations was still difficult to attribute solely to the combined action of LOX isozymes.
). 3,4-Epoxynonanal originates from oxidation
of NON by peroxygenase utilizing the oxidation potential derived from
4-HPNE in its reduction to 4-HNE. According to Blée and Schuber
(1990), linoleic acid is oxidized by soybean peroxygenase to
predominantly the 9R,10S- and
12R,13S-epoxides. Based on these data, one might
predict that 3R,4S-epoxynonanal could be the
preferred isomer formed. Soybean epoxide hydrolase hydrolyzed
9R,10S-epoxystearic acid to
9R,10R-dihydroxystearic acid (Blée and
Schuber, 1992), thus inverting the stereoconfiguration of
carbon-10. Applied to 4-HNE formation by the peroxygenase/hydrolase
route, one might predict the preferential formation of the
4R-hydroxyl with loss of the 3R-hydroxyl by
rearrangement/dehydration. It is known that peroxygenase is inactivated
by a 5-min preincubation with
H2O2 (Hamberg and
Fahlstadius, 1992). Therefore, a crude preparation was treated with 20 mm H2O2 for 5 min, then incubated with 1 mm NON as above. The 4-HNE
obtained after KI reduction of the products was 52.4 and 52.2% of the
4S-isomer at pH 8.3 and 9.0, respectively (Table I).
) for
chiral analysis to determine if there was any segregation of
configuration. Without H2O2
preincubation, the crude preparation afforded 4-HPNE that was 56.3% of
the 4S-isomer, whereas 4-HNE was 61.6% of the
4R-isomer (Table I). With
H2O2 preincubation, 4-HPNE
was 52.5% 4S-isomer, and the small amount of 4-HNE isolated (2% yield compared with the absence of
H2O2 preincubation) was analyzed at 50.4% of the 4S-isomer (Table I). In the
absence of H2O2, the
prevalence of the 4R-isomer in 4-HNE, compared with the
4S-isomer excess in 4-HPNE, further suggested the
participation of a 4R preference of the
peroxygenase/hydrolase pathway. Since H2O2 preincubation
inactivates peroxygenase, the stereoconfigurations of 4-HPNE and 4-HNE
isolated from the H2O2
treatment should reflect the stereoconfiguration of 4-HPNE from the
absence of H2O2 treatment, which was largely the result obtained. However, it is noted that the 52 to 56% 4S-isomeric excess in 4-HPNE obtained with the crude preparations does not seem to compare with the 55 to 83%
4S-isomeric excess obtained with LOX isozymes at pH 8.3 to
8.6. Since LOX-1 is the most active LOX isozyme at pH 8.0 to 9.0, one
would expect a result approaching 83% 4S-isomer. Therefore,
the origin of a significant percentage of the 4R-isomer
seems to be unresolved. In this regard, we have not yet ruled out the
existence of a membrane-bound 3Z-alkenal oxygenase as
originally proposed (Gardner and Hamberg, 1993; Takamura and Gardner,
1996). In these investigations significant 3Z-alkenal-oxidizing activity occurred with washed
microsomal fractions. In the present work we found that enzyme
extraction of defatted soybean flour with 0.5% Triton X-100 enhanced
NON oxidation activity 3-fold, but had little influence on LOX
activity with linoleic acid. This suggests that
4R-oxidizing activity may reside in membranes.
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CONCLUSIONS |
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There are two reasons that one might question the physiological
significance of LOX-1 oxidation of NON. One is the 400- to 1000-fold
less activity of NON oxidation, compared with the preferred substrate,
linoleic acid. Another is the threshold requirement and formation of
mainly 4-ONE with the highly purified LOX-1. However, this contrasts
with the facile production of 4-HPNE and 4-HNE from NON in crude
preparations of soybean (Takamura and Gardner, 1996). In addition,
9S-hydroperoxy-10E,9Z-octadecadienoic acid is readily converted in vitro to 4-HNE via hydroperoxide lyase
cleavage to give 4-HNE through the intermediate NON (Gardner et al.,
1991). This implies that activation of the LOX pathway would certainly
lead to 4-HPNE and 4-HNE in soybean. Since the 9-hydroperoxide of
linoleic acid is a precursor, catalytic amounts of hydroperoxide
needed to overcome the threshold of reaction would always be present in
crude preparations, but hydroperoxides would be initially very low in
pure LOX-1 systems in the absence of trace amounts of polyunsaturated
fatty acids. In the case of a wounded or stressed plant, it seems
certain that facile oxidation of polyunsaturated fatty acid would
"prime the pump" to form the active Fe3+-LOX
to make it active in NON oxidation. Thus, fatty acid hydroperoxide stimulated oxidation of NON to 4-HPNE and subsequent conversion by
peroxygenase would yield 4-HNE. Soybean seed is one of the richest
sources of LOX known, and LOX-1 is the most active of the soybean LOXs.
From the data of Axelrod et al. (1981), one can calculate that LOX-1
amounts to about 2.5% of the protein extractable from defatted soybean
flour. This suggests that fatty acid hydroperoxide-aided oxidation of
NON by this relatively large quantity of LOX is certainly possible, if
not probable. For example, even in the absence of catalytic
13S-HPODE, the threshold of NON oxidation was 0.125 mg
LOX-1/mL (Fig. 4), whereas the crude preparation contained 3.4 to 3.5 mg protein/mL (3.5 mg/mL × 2.5% = about 0.09 mg/mL LOX-1). In
view of the oxidation of NON by all three soybean LOX isozymes,
3Z-alkenal oxidation may be a general reaction for all LOXs,
including those of animal origin. 3Z-Alkenals might be
oxidized by LOX particularly under conditions of oxidative stress
necessary to overcome the threshold requirement.
). Although 3Z-hexenal and
3Z,6Z-nonadienal were not tested with the LOX
isozymes, 3Z-hexenal was found to be oxidized to
4-hydroxy-2E-hexenal by soybean preparations (Takamura and
Gardner, 1996).
). The hydroperoxide lyase branch of the LOX pathway,
leading to aldehydes and alcohols from aldehyde reduction, is often
thought to only give generalized protection by inhibiting the growth of
pathogenic microbes and fungi (Gardner, 1995). However, by extending
the pathway to include 4-oxo-, 4-hydroperoxy-, and 4-hydroxy-2-nonenals
and hexenals, consideration should be given to their possible effects
on both the physiology of plants and pathogenic organisms. One such
study showed that 4-HNE inhibited the growth of soybean pathogens
(Vaughn and Gardner, 1993).
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FOOTNOTES |
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Received October 9, 1997;
accepted December 3, 1997.
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ABBREVIATIONS |
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Abbreviations: 4-HNE, 4-hydroxy-2E-nonenal. 4-HPNE, 4-hydroperoxy-2E-nonenal. 4-ONE, 4-oxo-2E-nonenal. 13S-HPODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid. LOX, lipoxygenase. LOX-1, -2, and -3, soybean seed lipoxygenase-1, -2, and -3 isozymes. NON, 3Z-nonenal. OTMS, trimethylsilyloxy derivative of hydroxyls.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results & Discussion
References
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Axelrod B, Cheesbrough TM, Laakso S (1981) Lipoxygenase from soybeans. Methods Enzymol 71: 441-451
Blée E, Schuber F (1990) Stereochemistry of the epoxidation of fatty acids catalyzed by soybean peroxygenase. Biochem Biophys Res Commun 173: 1354-1360 [Medline]
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Schuber F
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Regio- and enantioselectivity of soybean fatty acid epoxide hydrolase.
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Christopher JP, Pistorius EK, Axelrod B (1970) Isolation of an isozyme of soybean lipoxygenase. Biochim Biophys Acta 198: 12-19 [Medline]
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Gardner HW,
Hamberg M
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Oxygenation of (3Z)-nonenal to (2E)-4-hydroxy-2-nonenal in the broad bean (Vicia faba L.).
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Hamberg M (1971) Steric analysis of hydroperoxides formed by lipoxygenase oxygenation of linoleic acid. Anal Biochem 43: 515-526 [CrossRef][Medline]
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Fahlstadius P
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, Linoleic acid assay, µmol
min
1 mL
, NON, µmol
min
), compared
with the same conditions plus 55 µg (0.09 mm) of
13S-HPODE (
). NON (1 mm) in 0.1 m potassium borate buffer (pH 8.6) was incubated for
5 min on ice in the presence of pure O2; reaction volume
was 2 mL.
