Full text of DOI:10.1007/s11745-001-0766-9

Effect of 4-Hydroxy-2(E )-nonenal on Soybean Lipoxygenase-1
a,
b
Harold W. Gardner * and Nigel Deighton
a
b
Mycotoxin Research, NCAUR, ARS, USDA, Peoria, Illinois 61604, and Scottish Crop
Research Institute, Invergowrie, Dundee DD2 5DA, Scotland
ABSTRACT: The oxidation of linoleic acid by soybean lipoxy- cess the active site of LOX-1 and other lipoxygenase isozymes
genase-1 (LOX-1) was inhibited in a time-dependent manner by (12). Three essential histidines ligate the iron active site of
4
-hydroxy-2(E)-nonenal (HNE). Kinetic analysis indicated the
LOX-1 (14). It has been shown that HNE forms a Michael
adduct with histidine residues (15,16), thereby implying that
HNE can react with and cause suicide inhibition of LOX-1 by
compromising the active site. In addition, it has been shown
that HNE can react with other amino acid residues, such as cys-
teine (17) and lysine (18), which also can contribute to the in-
activation of LOX-1. The literature abounds with other exam-
ples of enzyme inactivation or activation by HNE. Those en-
effect was due to slow-binding inhibition conforming to an
affinity labeling mechanism-based inhibition. After 25 min of
preincubation of LOX-1 with and without HNE, Lineweaver-
Burk reciprocal plots indicated mixed noncompetitive/competi-
tive inhibition. Low concentrations of HNE influenced the elec-
tron paramagnetic resonance (EPR) signal of 13(S)-hydroper-
oxy-9(Z),11(E)-octadecadienoic acid (13-HPODE)-generated
3+
Fe -LOX-1 slightly, but higher concentrations completely elim-
inated the EPR signal indicating an active site hindered from ac- zymes inactivated include glucose-6-phosphate dehydrogenase
+
+
cess by 13-HPODE. HNE may compete for the active site of (19), microsomal cytochrome P450 (20), Na -K -ATPase (21),
LOX-1 because its precursor, 4-hydroperoxy-(2E)-nonenal, is a aldose reductase (22), cytochrome C oxidase (23), α-ketoglu-
product of LOX-1 oxidation of (3Z)-nonenal. Also, it was an at-
tractive hypothesis to suggest that HNE may disrupt the active
site by forming a Michael adduct with one or more of the three
histidines that ligate the iron active site of LOX-1.
tarate dehydrogenase (24), pyruvate dehydrogenase (24),
2+
2+
plasma membrane (Ca + Mg )-ATPase (25), glutathione
peroxidase (26), and glyceraldehyde-3-phosphate dehydroge-
nase (27). Interestingly, multicatalytic proteinase, which clears
cells of oxidized protein, is inhibited by HNE-cross-linked pro-
tein (28), implying a mechanism for accumulation of age-re-
lated lipofuscin. Enzymes activated by HNE are those involved
Paper no. L8721 in Lipids 36, 623–628 (June 2001).
In the animal kingdom it is generally acknowledged that in lipid signaling, phospholipase D (29), and phosphoinositide-
-hydroxy-2(E)-nonenal (HNE) originates from lipid peroxida- specific phospholipase C (30).
tion of n-6 polyunsaturated fatty acids (1). Recently, specific au-
In this work we demonstrate that HNE inactivates LOX-1
4
toxidative pathways from both 9(S)-hydroperoxy-10(E),12(Z)- by a mixed noncompetitive/competitive mechanism and that
octadecadienoic acid (9-HPODE) and 13(S)-hydroperoxy- at least part of the competitive mechanism may be due to the
9
(Z),11(E)-octadecadienoic acid (13-HPODE) have been decomposition of HNE in buffer.
defined (2). Once formed, HNE has been documented to act as
a lipid signal (3). Involvement of HNE in mammalian patholo-
gies is diverse, including Alzheimer’s disease (4), Parkinson’s
MATERIALS AND METHODS
disease (5), cancer (6), atherosclerosis (7), sporadic amy- Materials. HNE was synthesized by a modification of a previ-
otrophic lateral sclerosis (8), and alcoholic liver disease (9).
ously described method (31). Instead of oxidizing 3,4-epoxy-
In plants, a metabolic pathway has been found in faba beans nonan-1-ol to the aldehyde by periodinane, the oxidation of
and soybeans that involves a sequence of 9-lipoxygenase oxi- Ratcliffe and Rodehorst was used (32), making certain to wash
dation of linoleic acid to 9-HPODE, hydroperoxide lyase the ethyl ether solution of product HNE with NaOH, HCl, and
cleavage of 9-HPODE to give 3(Z)-nonenal, and lipoxygenase NaHCO solutions as prescribed by the method. The product
3
and hydroperoxide peroxygenase action on 3(Z)-nonenal to HNE was somewhat less pure than that obtained with periodi-
produce HNE (10–12). However, there is evidence that the last nane. Thus, HNE was purified by silicic acid (100 mesh;
step of oxidation of 3(Z)-nonenal readily occurs by autoxida- Mallinckrodt, Phillipsburg, NJ) column chromatography (40 g,
tion, even with plant enzymes (13). Since soybean lipoxyge- column i.d. 2.5 cm) by sequentially eluting with 300 mL each
nase-1 (LOX-1) oxidized 3(Z)-nonenal stereospecifically to of 7.5, and 10% acetone in hexane. HNE eluted between 200
4(S)-hydroperoxy-2(E)-nonenal, it is obvious that HNE can ac- and 300 mL in 95.5% purity. Linoleic acid was obtained from
Nu-Chek-Prep (Elysian, MN). LOX-1 was isolated as previ-
*
1
To whom correspondence should be addressed at NCAUR, ARS, USDA,
815 N. University St., Peoria, IL 61604.
ously described (33) and was stored at 3°C as a suspension in
2.3 M (NH ) SO ; protein concentration was 34.5 mg/mL. 13-
E-mail: gardnehw@mail.ncaur.usda.gov
4
2
4
Abbreviations: cmc, critical micelle concentration; EPR, electron paramag-
netic resonance; HNE, 4-hydroxy-2(E)-nonenal; 9-HPODE, 9(S)-hydroper-
oxy-10(E),12(Z)-octadecadienoic acid; 13-HPODE, 13(S)-hydroperoxy-
HPODE was prepared by the method of Gardner (34).
Time-dependent inactivation of LOX-1 by HNE. The oxy-
gen electrode assay (Gilson 5/6H oxygraph, Middleton, WI)
9(Z),11(E)-octadecadienoic acid; LOX-1, soybean lipoxygenase-1.
Copyright © 2001 by AOCS Press
623
Lipids, Vol. 36, no. 6 (2001)

624
H.W. GARDNER AND N. DEIGHTON
was completed in a 2.4-mL cell held at 25˚C. The cell was center field 130 mT, sweep width 100 mT, microwave power
filled with 0.1 M potassium borate buffer (pH 9.86) contain- 63 mW, modulation amplitude 1.2 mT, and sweep time 80 s.
ing 0.36 µg LOX-1/mL. For zero-time measurement, 10 µL of
HNE dissolved in methanol and 10 µL linoleic acid substrate trapped as the benzoxime and analyzed by flame-ionization
240 mM) in methanol were injected into the cell in rapid se- detection gas chromatography as the trimethylsilyloxy ether
Analyses of HNE decrease in buffered solutions. HNE was
(
quence. The final concentration of linoleic acid was 1 mM. as described (10). Methanolic HNE (15 µL of 60 mM HNE)
Various concentrations of HNE were tested, 10 µL of 4.8, 15, was added to 3 mL 0.1 M potassium borate buffer (pH 9.86)
24, 30, 60, 120, and 240 mM in methanol per 2.4 mL cell, giv- or 3 mL potassium borate buffer containing 1.04 mg LOX-1.
ing final concentrations of 0.02, 0.0625, 0.1, 0.125, 0.25, 0.5, These solutions were incubated at 25°C, and 0.5 mL aliquots
and1 mM, respectively. For the measurements requiring pre- were taken at 0, 10, 40, 90, and 120 min to make the O-ben-
cise timed incubation of HNE with the LOX-1 borate solution, zoxime derivative of HNE. Aliquots were added to 0.1 mL
a batch solution was prepared [24 mL 0.1 M potassium borate reagent containing 50 mM O-benzylhydroxylamine-HCl in
buffer (pH 9.86) containing 0.36 µg LOX-1/mL was stirred 100 mM Na piperazine-N,N′-bis-(2-ethanesulfonate) (pH 6.5)
with 100 µL of methanolic HNE solution at the various con- and 0.4 mL methanol. After permitting the reagent to react
centrations described above]. The batch solution was incu- for 10 min, 27.5 µg methyl nonadecanoate was added as an
bated at 25°C for the prescribed time of 5.5, 11, 17, 30, 55, and internal standard, and the derivative was extracted with 1 mL
9
0 min, after which 2.4 mL was transferred to the oxygen elec- chloroform. The chloroform extract was evaporated with a
trode cell and 10 µL of 240 mM methanolic linoleic acid was stream of nitrogen and reacted with trimethylchlorosilane/
injected into the cell to start the oxygen uptake measurement. hexamethyldisilazane/dry pyridine (3:2:2, by vol). After 10
The control measurements were the same as described min, the reagent was evaporated with a stream of nitrogen and
above, except the same volume of methanol replaced the taken up in 100 µL hexane for gas chromatographic analysis
methanolic HNE solution.
as reported previously (11). Values were relative based on
Lineweaver-Burk kinetic analyses of HNE inhibition. 100% HNE content at zero time.
LOX-1 activity assays were measured by oxygen electrode
without or with HNE inhibitor after preincubation for exactly
RESULTS AND DISCUSSION
2
5 min at 25°C by adding either 0.417% methanol (10 µL
methanol/2.4 mL LOX-1 solution) or 0.417% methanolic An examination of the persistence of HNE in 0.1 M borate
HNE solution (methanolic HNE tested were 30, 60, and 120 buffer (pH 9.86) revealed the loss of HNE (Table 1) with the
mM, giving 0.125, 0.25, and 0.5 mM final concentration, re- appearance of seven unidentified compounds having a greater
spectively). The LOX-1 solution was 0.97 µg LOX-1/mL in retention time by gas chromatography. The four main com-
0
.1 M potassium borate (pH 9.86). After the 25-min preincu- pounds had related mass spectra indicating that they were iso-
bation, 2.4 mL was transferred to the oxygen electrode cell meric. The disappearance of HNE was not markedly affected
and 10 µL methanolic linoleic acid was injected to start the by the presence of LOX-1, which was not too surprising as
reaction. Methanolic linoleic acid was injected at concentra- the concentration of LOX-1 was many orders of magnitude
tions of 24, 12, 8, 6, 4.8, or 4 mM giving final concentrations less than HNE. That is, HNE was present at 0.3 mM, and
of 0.1, 0.05, 0.0334, 0.025, 0.02, or 0.01667 mM, respec- LOX-1 was only at 0.011 µM. Since HNE was present at only
tively, all of which are below the critical micelle concentra- 57% of its original concentration after 40 min, the potential
tion (cmc) of linoleic acid at pH 9.86 (35). As many as 10 effect of HNE on LOX-1 was diminished at longer times.
replicates were determined for the “without HNE” activities, However, during short incubation times, when the rate of in-
and “with HNE” activities were replicated four to eight times. hibition was greatest (Fig. 1), the amount of HNE was only
Lineweaver-Burk reciprocal data were plotted and interpreted slightly depleted. At 5.5 and 11 min, the amount of HNE was
according to Dixon and Webb (36).
Electron paramagnetic resonance (EPR) analysis of Fe - the rate of inhibition was the greatest.
LOX-1 in presence of HNE. A LOX-1 stock solution (109
projected to be about 95 and 91%, respectively, at which time
3+
µM) in potassium borate buffer (0.1 M, pH 9.9) was prepared.
To 120 µL aliquots of this enzyme stock, dilute solutions of
TABLE 1
Loss of HNE with Time When Incubated in 0.1 M Potassium Borate
HNE in ethanol (20 µL) were added such that the final con-
centrations of HNE were 0, 0.21, and 1.57 mM. These solu-
tions were incubated for 1 h (25°C), at which point the en-
zyme was oxidized through addition of a 37 µL aliquot of 13-
HPODE (2 mM) in methanol. Samples were immediately
frozen in liquid nitrogen and EPR spectra acquired at 77 K in
a quartz finger Dewar flask. The samples (177 µL) analyzed
as follows: LOX-1 (74 µM), 13-HPODE (418 µM), and HNE
a
Buffer (pH 9.86) at 25°C With or Without LOX-1
Percentage, relative to zero time
Without LOX-1 With LOX-1
100 100
86.4 92.8
Time (min)
0
10
40
90
57.3
27.3
26.3
55.7
25.1
18.8
120
=
0, 0.17, and 1.24 mM. All spectra were acquired on a
aThe concentrations of 4-hydroxy-2(E)-nonenal (HNE) and soybean lipoxy-
Bruker ESP-300E X-band spectrometer. Key parameters were genase-1 (LOX-1) were 0.3 mM and 0.35 mg/mL, respectively.
Lipids, Vol. 36, no. 6 (2001)

4-HYDROXY-2-NONENAL AND LIPOXYGENASE
625
k3[I]
k4
k5
E
E[I]
E-I
SCHEME 1
For this type of inhibitor the rate equation is:
k5[I]
kobs
=
[1]
K +[I]
i
where kobs, apparent (observed) rate constant; K , apparent
i
concentration of inhibitor required to reach the half-maximal
rate of inactivation, and [I], inhibitor concentration.
Since this rate equation is reminiscent of the Michaelis-
Menton equation, a reciprocal plot of kobs vs. [I] ideally should
give a linear plot. After correcting for the control, exponential
extrapolation of the rate of inactivation obtained during the first
11 min (Fig. 1) kobs was derived from 0.693/t1/2 min. The rate
for 1 mM HNE was omitted as being too rapid to obtain rea-
sonable data. The linear reciprocal plot shown in Figure 2 with
a nonzero intercept conforms to the inhibitor type shown in
Scheme 1. The intercept with the y-axis gave the 1/maximal
FIG. 1. Time-dependent inactivation of soybean lipoxygenase-1 (LOX-
1
) activity by various concentrations of 4-hydroxy-2(E)-nonenal (HNE).
I, Control containing methanol only in place of methanolic HNE; G,
.02 mM HNE; ✕, 0.0625 mM HNE; ꢀ, 0.125 mM HNE; +, 0.25 mM
HNE; G, 0.5 mM HNE; ꢁ, 1 mM HNE. All points represent mean val-
0
rate of k , or the maximal rate of inactivation, kinact., that is,
5
−
1
ues of replicates. The range in coefficient of variation in percentages 1/kinact. = 5 or kinact. = 0.2 min . Intersection of the plot with
was as follows: control, 13 to 18 (n = 19); 0.02 mM HNE, 3 to 31 (n =
the x-axis gave –1/K = −1/3.4 or K = 0.294 mM. This type of
i
i
4
0
); 0.0625 mM HNE, 5 to 18 (n = 3); 0.125 mM HNE, 9 to 36 (n = 3);
.25 mM HNE, 10 to 26 (n = 3); 0.5 mM HNE, 0 to 28 (n = 3); and 1
inhibition is reasonable in view of the fact that HNE is known
to form Michael adducts with cysteine (17), lysine (18), and
histidine (15,16). It is noted that LOX-1 possesses three essen-
tial histidines that ligate the iron active site. It is known from
the literature that 4(S)-hydroperoxy-2(E)-nonenal is a LOX-1
product of 3(Z)-nonenal oxidation (12); thus, it seems reason-
able that the smaller and structurally related HNE should be ca-
pable of accessing the active site.
mM HNE, 22 to 81 (n = 3).
It was determined that HNE in concentrations ranging be-
tween 20 µM and 1 mM was an effective inhibitor of LOX-1
activity as assayed with 1 mM linoleic acid (Fig. 1). Previous
studies with other enzymes showed that the effective concen-
trations of HNE required for inhibition ranged from a low for
+
+
cytochrome P450 (0.24 µM to 0.8 mM) (20) and Na -K -
ATPase (1 µM to 1 mM) (21) to a high for glucose-6-phos-
phate dehydrogenase (1 to 8 mM) (19). Other enzymes were
reported to be effective at HNE concentrations between the
foregoing values; thus, the inhibition of LOX-1 by 20 µM to
1
mM was comparable to the effect on many other enzymes.
Interestingly, we did not see inhibition when HNE and
linoleic acid were added simultaneously; that is, no signifi-
cant slowing of the rate was seen over the course of the assay
of 4 to 5 min. This result indicated that linoleic acid was a
more effective competitor for the active site than HNE. How-
ever, when HNE was incubated with LOX-1 for a period of
time in the absence of linoleic acid, subsequent activity with
linoleic acid substrate was markedly reduced. After the data
shown in Figure 1 were compensated for the control, the loss
of activity was found to be complete after about 17 min of in-
cubation with HNE.
The data presented in Figure 1 are indicative of a slow-
binding inhibitor. As outlined by Copeland (37), slow-bind-
ing inhibitors are categorized into three types, which can be
distinguished by kinetic analysis. The data derived from Fig-
ure 1 best fit the “affinity labeling and mechanism-based in-
hibition” according to Scheme 1, where E, enzyme; k, rate
constant; and [I], inhibitor.
FIG. 2. Reciprocal plot of apparent (observed) rate constant, k
obs
−1
(min ), as a function of inhibitor [I] concentration (mM). Intercept with
the x-axis is equivalent to – 1/K , and the intercept with the y-axis is
i
1
/kinact (reciprocal of the maximal rate of inactivation). Derived from
the data shown in Figure 1.
Lipids, Vol. 36, no. 6 (2001)

626
H.W. GARDNER AND N. DEIGHTON
For this reason, Lineweaver-Burk kinetic analyses of the product, 13-HPODE, followed by immediate liquid N freez-
2
inhibitor effect of HNE were completed to confirm the fore- ing (Fig. 4). In the presence of low HNE concentrations (0.21
going assessment. In order to determine these data, LOX-1 mM incubation concentration, 0.17 mM after 13-HPODE ad-
was preincubated with HNE for 25 min, a time determined to dition) the EPR signal from oxidized LOX-1 was slightly af-
be safely beyond the effective time required for inhibition; fected. A 22% decrease in peak-to-peak width of the g (g =
⊥
however, after 25 min the concentration of HNE was reduced 6) signal was observed: 11.3mT in the absence of HNE and
to about 75% of its original value by decomposition in buffer. 8.8 mT in the presence of HNE. In view of the half-maximal
Also, all activity assays had to be completed below the cmc inhibitor concentration (K ) for HNE of 0.294 mM (Fig. 2),
i
concentration of linoleic acid, because the true substrate of this result was not unreasonable. When the concentration of
LOX-1 has been determined to be monomeric linoleic acid, HNE was increased (1.57 mM incubation concentration, 1.24
not substrate trapped in micelles (38). This severely restricts mM after 13-HPODE addition), the EPR signal completely
the usable range of substrate concentrations, but reasonable disappeared. Since 1.24/1.57 mM HNE is substantially
data can be collected by the use of replicates. Thus, the K of greater than K , complete inhibition was anticipated; however,
M
i
LOX-1 was determined to be 23 µM (Fig. 3), which compares the relatively low HNE/LOX-1 incubation ratio of 21 may
with 15 ± 3 µM reported by Schilstra et al. (39). Reciprocal have depleted the actual HNE concentration by noninhibitory
plot analyses in the presence of various concentrations of reactions with LOX-1 to some extent. In the present EPR
HNE revealed mixed competitive/noncompetitive inhibition study, the HNE/LOX-1 incubation ratio of 21 compared to lit-
at the two lowest concentrations of HNE tested. Since 3(Z)- erature HNE/protein ratios of 15–143 that were utilized to
nonenal served as a substrate of LOX-1 and 4(S)-hydroper- modify histidine residues (15). From these observations it
oxy-2(E)-nonenal was a product (12), it was expected that was concluded that HNE caused the active site to be suffi-
2
+
3+
HNE would occupy the active site of LOX-1. The competi- ciently hindered to prevent oxidation of Fe -LOX-1 to Fe -
tive aspect of HNE can be readily explained. The noncompet- LOX-1 by 13-HPODE.
itive nature of the inhibition was expected due to the slow-
A potential physiological function of HNE is suggested by
binding kinetic analysis outlined above. Surprisingly, at the the results. It is well known that pathogens activate the
highest inhibitor concentration tested (0.5 mM), where one oxylipin pathway (e.g., Ref. 40). More specifically, Deighton
would expect the greatest degree of noncompetitive inhibi- et al. (41) found that HNE accumulates at levels as high as
tion, the plot showed an almost classical competitive inhibi- 19,000 pmol/g tissue at the site of fungal infection of sweet
tion. In fact, the trend toward competitive inhibition occurred peppers. In certain experiments HNE was found at even
as the inhibitor concentration progressively increased.
higher levels of 30,000 pmol/g (Deighton, N., unpublished
This observation of classical competitive inhibition at 0.5 data). Because of HNE reaction with protein and/or glu-
mM HNE does not have a straightforward explanation. Some tathione, it is plausible that the amount produced was actu-
competitive inhibition could be caused by decomposition ally higher. The concentrations found by Deighton translate
products of HNE, which would afford higher concentrations
after 25 min preincubation with 0.5 mM HNE. To test this
possibility, HNE was preincubated in buffer for 2 to 4 h be-
fore testing for its effect on LOX-1 activity. Pretreated HNE
was then incubated with LOX-1 for 10 min prior to adding 1
mM linoleic acid. Preincubated HNE, 0.5 and 1 mM, inhib-
ited LOX-1 at 17 and 58%, respectively, compared to con-
trols. Little significant difference in activity could be seen
with the time of preincubation between 2 and 4 h. By contrast,
a 10 min exposure of LOX-1 to HNE (not preincubated in
buffer) at 0.5 and 1 mM inhibited activity 73 and 85%, re-
spectively. As seen in Table 1, the amount of HNE remaining
after 2 h preincubation in buffer was less than 25%. It is con-
ceivable that the inhibition observed with preincubation is
partly due to residual HNE, but the lack of significant effect
over time is suggestive of the contribution from degraded
HNE. It is conceivable that the competitive aspect of the
Lineweaver-Burk plots observed at higher HNE concentra-
FIG. 3. Lineweaver-Burk reciprocal plots of LOX-1 activity with and
without HNE inhibitor. Preincubation with or without HNE was 25 min
at 25°C before measurement of activity with linoleic acid. Reciprocals
tions was due to degraded HNE from 25 min preincubation are activity, V, in µmol/min/µg LOX-1, and linoleic acid substrate, S, in
mM. I, Activity without HNE; G, activity with 0.125 mM HNE ꢀ, ac-
tivity with 0.25 mM HNE; and ꢁ, activity with 0.5 mM HNE. All points
are mean values of replicates. The ranges in coefficient of variation in
percentages were as follows: “without HNE,” 14 to 35 (n = 10); 0.125
of HNE. Nevertheless, some degree of mixed competitive/
noncompetitive inhibition should be expected.
Disruption of the active site was supported by EPR results.
An EPR signal of LOX-1 was generated by oxidizing native
mM HNE, 9 to 22 (n = 4); 0.25 mM HNE, 11 to 24 (n = 6); 0.5 mM, 7
2
+
3+
Fe -LOX-1 to Fe -LOX-1 by exposing the enzyme to its to 22 (n = 4). For abbreviations see Figure 1.
Lipids, Vol. 36, no. 6 (2001)

4-HYDROXY-2-NONENAL AND LIPOXYGENASE
627
W.R. (1997) Elevated 4-Hydroxynonenal in Ventricular Fluid
in Alzheimer’s Disease, Neurobiol. Aging 18, 457–461.
. Selley, M.L. (1998) (E)-4-Hydroxy-2-nonenal May Be Involved
in the Pathogenesis of Parkinson’s Disease, Free Radicals Biol.
Med. 25, 169–174.
. Zarkovic, N., Ilic, Z., Jurin, M., Schaur, R.J., Puhl, H., and Es-
terbauer, H. (1993) Stimulation of HeLa Cell Growth by Physi-
ological Concentrations of 4-Hydroxynonenal, Cell Biochem.
Func. 11, 279–286.
5
6
7
8
9
. Uchida, K., Itakura, K., Kawakishi, S., Hiai, H., Toyokuni, S.,
and Stadtman, E.R. (1995) Characterization of Epitopes Recog-
nized by 4-Hydroxy-2-nonenal Specific Antibodies, Arch.
Biochem. Biophys. 324, 241–248.
. Smith, R.G., Henry, Y.K., Mattson, M.P., and Appel, S.H.
(
1998) Presence of 4-Hydroxynonenal in Cerebrospinal Fluid of
Patients with Sporadic Amyotrophic Lateral Sclerosis, Ann.
Neurol. 44, 696–699.
. Ohhira, M., Ohtake, T., Matsumoto, A., Saito, H., Ikuta, K., Fu-
jimoto, Y., Ono, M., Toyokuni, S., and Kohgo, Y. (1998) Im-
munohistochemical Detection of 4-Hydroxy-2-nonenal-Modi-
fied-Protein Adducts in Human Alcoholic Liver Diseases, Alco-
hol. Clin. Exp. Res. 22, 145S–149S.
1
1
1
1
0. Gardner, H.W., and Hamberg, M. (1993) Oxygenation of (3Z)-
Nonenal to (2E)-4-Hydroxy-2-nonenal in the Broad Bean (Vicia
faba L.), J. Biol. Chem. 268, 6971–6977.
1. Takamura, H., and Gardner, H.W. (1996) Oxygenation of (3Z)-
Alkenal to (2E)-4-Hydroxy-2-alkenal in Soybean Seed (Glycine
max L.), Biochim. Biophys. Acta 1303, 83–91.
FIG. 4. Electron paramagnetic resonance spectra of oxidized LOX-1.
The enzyme was oxidized by 13(S)-hydroperoxy-9(Z),11(E)-octadeca-
dienoic acid in the absence (A) and presence of HNE at 0.17 (B) and
2. Gardner, H.W., and Grove, M.J. (1998) Soybean Lipoxy-
genase-1 Oxidizes 3Z-Nonenal. A Route to 4S-Hydroperoxy-
2
E-nonenal, Plant Physiol. 116, 1359–1366.
1.24 mM (C). For abbreviations see Figure 1.
3. Noordermeer, M., Feussner, I., Kolbe, A., Veldink, G.A., and
Vliegenthart, J.F.G. (2000) Oxygenation of (3Z)-Alkenals to 4-
Hydroxy-(2E)-alkenals in Plant Extracts: A Nonenzymatic
Process, Biochem. Biophys. Res. Commun. 277, 112–116.
4. Prigge, S.T., Boyington, J.C., Gaffney, B.J., and Amzel, L.M.
(1996) Lipoxygenases: Structure and Function, in Lipoxygenase
and Lipoxygenase Pathway Enzymes (Piazza, G.J., ed.) pp.
to 0.019 to 0.03 mM, which brackets the lowest concentra-
tion of HNE tested in this study (Fig. 1). Thus, it seems pos-
sible that the downstream oxylipin HNE, and possibly related
oxylipins, may serve to switch off the lipid signal pathway
through inactivation of one of the initiating enzymes. Because
linoleic acid effectively competes for the active site, this
1
1
1–32, AOCS Press, Champaign, IL.
5. Uchida, K., and Stadtman, E.R. (1992) Modification of Histi-
dine Residues in Proteins by Reaction with 4-Hydroxynonenal,
Proc. Natl. Acad. Sci. USA 89, 4544–4548.
“
feedback suicide” of LOX is suggested to occur only after
substrate depletion.
Further research is planned to locate the specific site of
LOX-1 inactivation by HNE.
16. Tsai, L., and Sokoloski, E.A. (1995) The Reaction of 4-Hy-
droxy-2-nonenal with N -Acetyl-
Biol. Med. 19, 39–44.
L
-Histidine, Free Radicals
a
1
1
1
2
7. Uchida, K., and Stadtman, E.R. (1992) Selective Cleavage of
Thioether Linkage in Proteins Modified with 4-Hydroxynone-
nal, Proc. Natl. Acad. Sci. USA 89, 5611–5615.
8. Nadkarni, D.V., and Sayre, L.M. (1995) Structural Definition of
Early Lysine and Histidine Adduction Chemistry of 4-Hydroxy-
nonenal, Chem. Res. Toxicol. 8, 284–291.
9. Szweda, L.I., Uchida, K., Tsai, L., and Stadtman, E.R. (1993)
Inactivation of Glucose-6-phosphate Dehydrogenase by 4-Hy-
droxy-2-nonenal, J. Biol. Chem. 268, 3342–3347.
0. Bestervelt, L.L., Vaz, A.D.N., and Coon, M.J. (1995) Inactiva-
tion of Ethanol-Inducible Cytochrome P450 and Other Microso-
mal P450 Isozymes by trans-4-Hydroxy-2-nonenal, a Major
Product of Membrane Lipid Peroxidation, Proc. Natl. Acad. Sci.
USA 92, 3764–3768.
ACKNOWLEDGMENT
The Scottish Crop Research Institute is grant-aided by the Scottish
Executive Rural Affairs Department.
REFERENCES
1
. Pryor, W.A., and Porter, N.A. (1990) Suggested Mechanisms
for the Production of 4-Hydroxy-2-nonenal from the Autoxida-
tion of Polyunsaturated Fatty Acids, Free Radicals Biol. Med.
8
, 541–543.
2
. Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R., 21. Siems, W.G., Hapner, S.J., and van Kuijk, F.J.G.M. (1996) 4-
+
+
Two Distinct Pathways of Formation of 4-Hydroxy-Nonenal:
Mechanisms of Nonenzymatic Transformation of the 9- and 13-
Hydroxynonenal Inhibits Na -K -ATPase, Free Radicals Biol.
Med. 20, 215–223.
Hydroperoxides of Linoleic Acid to 4-Hydroxyalkenals, J. Biol. 22. Del Corso, A., Dal Monte, M., Vilardo, P.G., Cecconi, I., Mos-
Chem., in press.
chini, R., Banditelli, S., Cappiello, M., Tsai, L., and Mura, U.
(1998) Site-Specific Inactivation of Aldose Reductase by 4-Hy-
droxynonenal, Arch. Biochem. Biophys. 350, 245–248.
3
4
. Dianzani, M.U. (1998) 4-Hydroxynonenal and Cell Signaling,
Free Radical Res. 28, 553–560.
. Lovell, M.A., Ehmann, W.D., Mattson, M.P., and Markesbery, 23. Chen, J., Schenker, S., Frosto, T.A., and Henderson, G.I. (1998)
Lipids, Vol. 36, no. 6 (2001)

628
H.W. GARDNER AND N. DEIGHTON
Inhibition of Cytochrome c Oxidase Activity by 4-Hydrox-
ynonenal (HNE). Role of HNE Adduct Formation with the En-
Oxidations with the Chromium Trioxide-Pyridine Complex, J.
Org. Chem. 35, 4000–4002.
zyme Subunits, Biochim. Biophys. Acta 1380, 336–344.
4. Humphries, K.M., and Szweda, L.I. (1998) Selective Inactiva-
33. Axelrod, B., Cheesbrough, T.M., and Laakso, S. (1981) Lipoxy-
genase from Soybeans, Methods Enzymol. 71, 441–451.
2
2
tion of α-Ketoglutarate Dehydrogenase and Pyruvate Dehydro- 34. Gardner, H.W. (1997) Analysis of Plant Lipoxygenase Metabo-
genase: Reaction of Lipoic Acid with 4-Hydroxy-2-nonenal,
Biochemistry 37, 15835–15841.
lites, in Advances in Lipid Methodology—Four (Christie, W.W.,
ed.) pp. 1–43, The Oily Press, Dundee, Scotland.
5. McConnell, E.J., Bittelmeyer, A.M., and Raess, B.U. (1999) Ir- 35. Verhagen, J., Vliegenthart, J.F.G., and Boldingh, J. (1978) Mi-
2
+
2+
reversible Inhibition of Plasma Membrane (Ca + Mg )-
celle and Acid-Soap Formation of Linoleic Acid and 13-L-Hy-
droperoxylinoleic Acid Being Substrates of Lipoxygenase-1,
Chem. Phys. Lipids 22, 255–259.
2
+
ATPase and Ca Transport by 4-OH-2,3-trans-Nonenal, Arch.
Biochem. Biophys. 361, 252–256.
2
6. Bosch-Morell, F., Flohé, L., Marín, N., and Romero, F.J. (1999) 36. Dixon, M., and Webb, E.C. (1979) Enzymes, 3rd edn., Acade-
4
-Hydroxynonenal Inhibits Glutathione Peroxidase: Protection
mic Press, New York.
by Glutathione, Free Radicals Biol. Med. 26:1383–1387.
7. Uchida, K., and Stadtman, E.R. (1993) Covalent Attachment of
37. Copeland, R.A. (2000) Enzymes, A Practical Introduction to
Structure, Mechanism, and Data Analysis, 2nd edn., pp.
318–349, Wiley-VCH, New York.
2
4
-Hydroxynonenal to Glyceraldehyde-3-phosphate Dehydroge-
nase. A Possible Involvement of Intra- and Intermolecular 38. Schilstra, M.J., Veldink, G.A., and Vliegenthart, J.F.G. (1994)
Cross-linking Reaction, J. Biol. Chem. 268, 6388–6393.
8. Friguet, B., and Szweda, L.I. (1997) Inhibition of the Multicat-
alytic Proteinase (proteasome) by 4-Hydroxy-2-nonenal Cross- 39. Schilstra, M.J., Veldink, G.A., Verhagen, J., and Vliegenthart,
linked Protein, FEBS Lett. 405, 21–25.
9. Natarajan, V., Scribner, W.M., and Taher, M.M. (1993) 4-Hy-
Effect of Nonionic Detergents on Lipoxygenase Catalysis,
Lipids 29, 225–231.
2
2
J.F.G. (1992) Effect of Lipid Hydroperoxide on Lipoxygenase
Kinetics, Biochemistry 31, 7692–7699.
droxynonenal, a Metabolite of Lipid Peroxidation, Activates 40. Croft, K.P.C., Jüttner, F., and Slusarenko, A.J. (1993) Volatile
Phospholipase D in Vascular Endothelial Cells, Free Radicals
Biol. Med. 15, 365–375.
0. Rossi, M.A., Di Mauro, C., Esterbauer, H., Fidale, F., and Dian-
Products of the Lipoxygenase Pathway Evolved from Phaseolus
vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv
phaseolicola, Plant Physiol. 101, 13–24.
3
zani, M.U. (1994) Activation of Phosphoinositide-specific Phos- 41. Deighton, N., Muckenschnabel, I., Goodman, B.A., and
pholipase C of Rat Neutrophils by the Chemotactic Aldehydes
-Hydroxy-2,3-trans-nonenal and 4-Hydroxy-2,3-trans-octenal,
Cell Biochem. Func. 12, 275–280.
Williamson, B. (1999) Lipid Peroxidation and the Oxidative
Burst Associated with Infection of Capsicum annuum by Botry-
tis cinera, Plant J. 20, 485–492.
4
3
3
1. Gardner, H.W., Bartelt, R.J., and Weisleder, D. (1992) A Facile
Synthesis of 4-Hydroxy-2(E)-nonenal, Lipids 27, 686–689.
2. Ratcliffe, R., and Rodehorst, R. (1970) Improved Procedure for cepted May 14, 2001]
[Received December 22, 2000, and in final revised form and ac-
Lipids, Vol. 36, no. 6 (2001)