Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal

Article abstract of DOI:10.1248/bpb.26.787

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na⁺-K⁺-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na⁺-K⁺-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca²⁺- and Mg²⁺-ATPase activities. The results of the kinetic analysis of the Na⁺-K ⁺-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na ⁺-K⁺-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na⁺-K⁺-ATPase activity associated with alterations in the membrane lipid organization.

Full text of DOI:10.1248/bpb.26.787

June 2003
Biol. Pharm. Bull. 26(6) 787—793 (2003)
787
Contribution of Lipid Dynamics on the Inhibition of Bovine Brain
Synaptosomal Na؉-K؉-ATPase Activity Induced by 4-Hydroxy-2-nonenal
Akinori KADOYA, Hiromi MIYAKE, and Takao OHYASHIKI*
Department of Clinical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University; Kanagawa-machi,
Kanazawa, Ishikawa 920–1181, Japan. Received January 29, 2003; accepted February 9, 2003
The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondi-
aldehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were
examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na؉-K؉-ATPase ac-
tivity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the
synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The de-
creases in ATPase activity and SH content by treatment with HNE were also observed, as a Na؉-K؉-ATPase
preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptoso-
mal Ca2؉- and Mg2؉-ATPase activities. The results of the kinetic analysis of the Na؉-K؉-ATPase activity indi-
cated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP
completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a de-
crease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was
found that there is a good relationship between the lipid fluidity and the Na؉-K؉-ATPase activity under the pres-
ence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced in-
hibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity.
HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, re-
spectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal
Na؉-K؉-ATPase activity associated with alterations in the membrane lipid organization.
Key words 4-hydroxy-2-nonenal; malondialdehyde; lipid peroxidation product; Naϩ-Kϩ-ATPase; lipid fluidity; brain synapto-
some
Lipid peroxidation in cell membrane phospholipids in- reported that HNE disrupts neuronal ion homeostasis by
duced by reactive oxygen species and/or free radicals leads to impairing the function of membrane-bound iron-motive
alterations in the membrane structure and functions of cells, ATPases such as Naϩ-Kϩ-ATPase and Ca2ϩ-ATPase.14—16)
and has been proposed as a major mechanism for the onset of These findings strongly suggest that the aldehydes generated
several pathological events in vivo, including postischemic- by lipid peroxidation of the membrane are serious toxic prod-
reperfusion injury, cancer, senile dementia and aging.1)
A
ucts for the onset of oxidative injury. However, the exact
number of studies have shown that peroxidation of mem- mechanism of the onset of cellular injury induced by HNE is
brane lipids in cells gives rise to highly reactive a,b-unsatu- still unclear at present.
rated aldehydes from the lipid hydroperoxides as secondary
In the present study, we examined the effects of HNE on
oxidation products such as 4-hydroxy-2-nonenal (HNE) and membrane-bound ATPases of bovine brain synaptosomes in
malondialdehyde (MDA).2—4) Pryor and Porter have reported the context of their membrane lipid dynamics in order to ob-
that HNE can be formed from omega-6-polyunsaturated fatty tain further information concerning mechanisms of the onset
acids.5) Among the aldehydes generated as the result of of HNE toxicity. This paper suggests the possibility that
membrane lipid peroxidation, HNE can be produced in rela- changes in the lipid dynamics are related to HNE-induced in-
tively large concentrations up to 5 mM in cells, and is the activation of the synaptosomal Naϩ-Kϩ-ATPase.
most damaging to cells.3,6) Koster et al. also reported that the
concentration of HNE in the lipid bilayer of peroxidized mi- MATERIALS AND METHODS
crosomes is about 4.5 mM.7)
HNE directly interacts with several amino acid residues of
Materials Pepstatin A (microbial source), aprotinin
protein, i.e., the sulfhydryl (SH) group of cysteine, the imida- (bovine lung, 5.2 TIU/mg solid), leupeptin hydrochloride
zole moiety of histidine and the e-amino group of lysine (microbial source), soybean trypsin inhibitor (type 1-S), 1,6-
through a Michael-type nucleophilic addition.2) Several in- diphenyl-1,3,5-hexatriene (DPH), phenylmethylsulfonyl fluo-
vestigators have reported that this reaction leads to the struc- ride (PMSF), 1-dimethylaminonaphthalene-5-sulfonyl chlo-
tural modification and functional impairment of proteins.3.8.9) ride (DNS-Cl), p-hydroxy-mercuribenzoic acid (PHMB) and
Previous studies have shown that free and/or protein- Naϩ-Kϩ-ATPase (EC 3.6.1.3; porcine cerebral cortex,
bound HNE accumulate in a variety of non-neural cells ex- 0.42 U/mg protein) were purchased from Sigma (St. Louis,
posed to oxidative insult,3) in pyramidal neuron cytoplasm MO, U.S.A.). 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB),
and on neurofibrillary tangles in the brain of Alzheimer’s dis- sodium dodecyl sulfate (SDS), N-ethylmaleimide (NEM) and
ease patients.10—12) Mark et al. have also recently reported dithiothreitol were obtained from Wako Pure Chemical Co.
that amyloid b-peptide-mediated neuronal cell death is re- (Osaka, Japan). n-Heptyl-b-D-thioglucoside and 1-(4-
lated to a large increase in the levels of free and protein- trimethylammonium
phenyl)-6-phenyl-1,3,5-hexatriene
bound HNE.13) In addition, several investigators have also (TMA-DPH) were from Dojin Chemical Co. (Kumamoto,
* To whom correspondence should be addressed. e-mail: t-ohyashiki@hokuriku-u.ac.jp
© 2003 Pharmaceutical Society of Japan

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Vol. 26, No. 6
Japan). ATP (disodium salt) was purchased from Oriental value (subtracted) was determined in the presence of 0.2 mM
Yeast Co. (Tokyo, Japan). (Ϫ)-Ouabain octahydrate was from EGTA. (3) Mg2ϩ-ATPase activity was measured in the same
Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). HNE and assay medium employed for Ca2ϩ-ATPase activity, except
malonaldehyde bis(dimethylacetal) were obtained from Cay- 0.2 mM MgCl2 was used in place of CaCl2. The control value
man Chemical Co. (Ann arbor, MI, U.S.A.) and Tokyo Kasei (subtracted) was determined in the presence of 0.2 mM
Co. (Tokyo, Japan), respectively. A MDA stock solution EGTA. In the latter two ATPases, other experimental condi-
(100 mM) was prepared by dissolving malonaldehyde bis(di- tions of the activity assays were the same as those described
methylacetal) in 1 N HCl and adjusted by 1 N NaOH to pH for the Naϩ-Kϩ-ATPase activity assay.
7.4. All other chemicals were of the highest grade purity.
Measurements of SH Content The SH content of the
Synaptosome Preparation Isolation of synaptosomes synaptosomal membrane proteins was determined according
from bovine brain was performed according to the procedure to the procedure described by Ellman.19) The synaptosomes
described by Hensley et al.17) with a slight modification. (0.3 mg protein/ml) were treated with 5 mM DTNB for
Bovine brain cortices (5 g) were homogenized in 10 volumes 10 min at 37 °C in 20 mM Tris–HCl buffer (pH 8.0) in
of 20 mM Hepes–NaOH buffer (pH 7.4) containing 0.32 M su- the presence of 3% SDS. The SH content was calculated
crose, 4 mg/ml leupeptin, 4 mg/ml pepstatin, 5 mg/ml apro- using the molar extinction coefficient of 13600 MϪ1 cmϪ1 at
tinin, 0.2 mg/ml PMSF, 20 mg/ml trypsin inhibitor, 2 mM 412 nm.
EDTA and 1 mM dithiothreitol (Buffer A) in a Potter-Elve-
Fluorescence Measurements of DPH- and TMA-DPH-
hjem glass homogenizer (six strokes with 1000 rev/min). The Labeled Synaptosomes The aldehyde-modified synapto-
homogenate was centrifuged at 1500ϫg for 10 min at 4 °C, somes (0.1 mg protein/ml) were incubated with 20 mM DPH
and the supernatant obtained was then centrifuged at or TMA-DPH for 10 min at 37 °C in buffer C. The fluores-
20000ϫg for 10 min at 4 °C. The pellets were washed twice cence measurements were carried out at 25 °C using a Hi-
and suspended in Buffer A (9 ml). Samples were layered onto tachi F-4500 spectrofluorometer. The excitation and emission
a discontinuous gradient containing 10 ml of 1.18 M sucrose, wavelengths used in the fluorescence measurements of DPH-
10 ml of 1 M sucrose and 10 ml of 0.85 M sucrose, then cen- and TMA-labeled synaptosomes were 330 and 430 nm, re-
trifuged at 82500ϫg for 2 h at 4 °C. Synaptosome fraction spectively. The steady-state fluorescence anisotropy (g) is de-
was obtained at the 1 M sucrose/1.18 M sucrose interface, and fined as the value of (IVϪIH)/(IVϩ2IH), where IV and IH rep-
washed twice in 25 volumes of 20 mM phosphate buffer (pH resent the fluorescence intensities of the vertically and hori-
7.4) containing 4.6 mM KCl, 0.6 mM MgCl2 and 117 mM zontally polarized emitted lights with vertically polarized ex-
NaCl (Buffer B). After centrifugation at 20000ϫg for citation, respectively.20) The contribution of the products
10 min, the final synaptosome fraction was re-suspended in formed by modification of the synaptosomes with HNE or
the same buffer.
MDA to the fluorescence measurements was negligible.
Modification of Synaptosomes with Aldehydes The
Separation of Aldehyde-Modified Protein and Lipid
synaptosomes (1 mg protein/ml) were treated with 500 mM Fractions HNE- or MDA-modified synaptosomes (3 mg
aldehyde in Buffer B in the presence of 0.5% n-heptyl-b-D- protein/ml) were extracted with 3 ml of a chloroform/
thioglucoside for 30 min at 37 °C, unless otherwise specified. methanol (1 : 2 v/v) mixture and centrifuged at 1600ϫg for
The reaction was terminated by dilution with a large volume 10 min at room temperature to separate the protein and lipid
of the same buffer and centrifugation at 20000ϫg for 10 min fractions. The protein fraction obtained at the organic sol-
at 4 °C. The pellets obtained were washed twice in Buffer C vent/aqueous phase interface was collected and washed at
containing 20 mM Hepes–NaOH buffer (pH 7.4), 4.6 mM once with the same chloroform/methanol mixture (3 ml), and
KCl, 0.6 mM MgCl2, 1.1 mM KH2PO4 and 137 mM NaCl. then dissolved in 1 ml of 3% SDS. On the other hand, the
After centrifugation at 20000ϫg for 10 min, the pellets were combined chloroform layer (lipid fraction) was dried under
suspended in the same buffer, and used as aldehyde-modified N2 gas flow and then dissolved in 1 ml of chloroform. The
synaptosomes.
emission spectra of aldehyde-modified protein and lipid frac-
ATPase Activity Assay All assays were carried out in a tions were monitored at 25 °C, with an excitation wavelength
final volume of 1 ml with the use of 30 mg synaptosomal pro- of 355 nm.
tein. (1) Naϩ-Kϩ-ATPase activity was measured in the assay
Protein Determination The protein determination was
medium containing 50 mM Tris–HCl buffer (pH 7.4), 110 mM performed according to the procedure of Lowry et al.21)
NaCl, 10 mM KCl, 0.01 mM EDTA, 5 mM MgCl2 and synapto- using bovine serum albumin as the standard.
somes. The reaction was started by the addition of 3 mM ATP
Statistical Analysis Data were represented as the
(as the final concentration) to the assay medium, and incuba- meanϮS.D. of three independent determinations. To deter-
tion was carried out at 37 °C for 30 min, unless otherwise mine statistical significance between groups, the data were
specified. Liberated inorganic phosphate was measured ac- analyzed by an ANOVA Bonferroni’s multiple t-test.
cording to the procedure described by Fiske and Subbarow.18)
The control value (subtracted) was determined in the pres- RESULTS
ence of 1 mM ouabain. In the kinetic studies of Naϩ-Kϩ-
ATPase activity, the ATP concentration was varied from
Changes in Synaptosomal ATPase Activities and SH
0.3 mM to 3 mM, and the assay was performed within 2 min, Content by Aldehyde-Modification As shown in Table 1
because the ATP hydrolysis proceeds linearly within 5 min. and Fig. 1, treatment of the synaptosomes with 500 mM HNE
(2) Ca2ϩ-ATPase activity was measured in 50 mM Tris–HCl resulted in a marked decrease in Naϩ-Kϩ-ATPase activity in
buffer (pH 7.4) containing 0.2 mM EGTA, 2 mM CaCl2, 2 mM a time-dependent manner. On the other hand, the activities of
sodium azide, 1 mM ouabain and synaptosomes. The control Ca2ϩ- and Mg2ϩ-ATPases were almost not inhibited (less

June 2003
789
Table 2. Effects of SH Reagents on Naϩ-Kϩ-ATPase Activity and SH
Content
Table 1. Effects of HNE Treatment on Synaptosomal ATPase Activities
Specific activites (mmol Pi/mg protein/min)
Naϩ-Kϩ-ATPase activity
(mmol Pi/mg protein/min)
SH content
(nmol/mg protein)
HNE
SH reagent
Naϩ-Kϩ-ATPase
Ca2ϩ-ATPase
Mg2ϩ-ATPase
Ϫ
ϩ
0.506Ϯ0.006
0.137Ϯ0.002*
0.213Ϯ0.005
0.180Ϯ0.004*
0.224Ϯ0.003
0.175Ϯ0.014*
Control
NEM
PHMB
0.476Ϯ0.003
0.020Ϯ0.002*
0.011Ϯ0.001*
49.3Ϯ1.3
12.7Ϯ2.0*
8.3Ϯ1.5*
The concentration of HNE was 500 mM. Other experimental conditions are described
under Materials and Methods. * pϽ0.05 vs. control (no HNE treatment) in each system.
The concentrations of SH reagents were 500 mM. The conditions and procedure of
modification of the synaptosomes with NEM or PHMB are the same as those with alde-
hydes described under Materials and Methods. * pϽ0.05 vs. control (no HNE treat-
ment) in each system.
Fig. 1. Time Course of HNE-Induced Inhibition of Naϩ-Kϩ-ATPase Ac-
tivity
The synaptosomes were treated with 500 mM HNE. * pϽ0.05 vs. the activity at 0
time.
Fig. 3. Effects of Increasing Concentrations of HNE on the Activity and
SH Content of Naϩ-Kϩ-ATPase Preparation
Naϩ-Kϩ-ATPase (1 mg/ml) was treated with various concentrations of HNE (50—
750 mM) for 30 min at 37 °C. The reaction was terminated by the addition of 2 mM NAC,
followed by dialysis against 30 mM Tris–HCl buffer (pH 7.4). The concentrations of the
enzyme employed in the ATPase activity and SH content measurements were 10 mg and
1 mg/ml, respectively. The SH content was represented as mol per mol of the enzyme,
assuming that the molecular weight of the enzyme is 294000.36) The control ATPase ac-
tivity is 0.75Ϯ0.01 mmol Pi/mg protein/min. Symbols: ᭺, Naϩ-Kϩ-ATPase activity; ᭹,
SH content. *,** pϽ0.05 vs. the control (no HNE treatment) in each system.
tion required to induce half-maximal inhibition of the AT-
Pase activity is approximately 250 mM (Fig. 2). In contrast,
MDA did not affect the Naϩ-Kϩ-ATPase activity, even at
high concentrations (500 mM) of the aldehyde under the same
conditions (0.495Ϯ0.033 and 0.479Ϯ0.021 mmol Pi/mg pro-
tein/min for control and MDA-treated synaptosomes, respec-
tively). The SH content also did not change by treatment of
the synaptosomes with MDA (51.5Ϯ1.6 and 52.7Ϯ1.1
nmol/mg protein the systems of control and MDA-treated
ones, respectively).
Fig. 2. Concentration Dependence of HNE on Naϩ-Kϩ-ATPase Activity
and SH Content in Synaptosomal Membranes
Effects of SH Reagents on Na؉-K؉-ATPase Activity
The effects of several SH reagents on the synaptosomal Naϩ-
Kϩ-ATPase activity were examined.
The HNE concentration was varied from 100 to 750 mM. The experimental conditions
and procedures are described under Materials and Methods. Symbols: ᭺, Naϩ-Kϩ-AT-
Pase activity; ᭹, SH content. *,** pϽ0.05 vs. the control (no HNE treatment) in each
system.
As shown in Table 2, the ATPase activity was almost com-
pletely inhibited by treatment of the synaptosomes with
NEM or PHMB. And, the SH content of the synaptosomal
membrane proteins also markedly decreased by the treat-
ment. From these results, it is suggested that modification of
the SH groups in the membrane proteins is closely related to
HNE-induced inactivation of synaptosomal Naϩ-Kϩ-ATPase.
Effects of HNE-Modification on Enzyme Activity and
SH Content in Na؉-K؉-ATPase Preparation To further
confirm the involvement of SH-modification in HNE-in-
duced inhibition of Naϩ-Kϩ-ATPase activity, we used a Naϩ-
than 20% inhibition) by the treatment (Table 1).
Figure 2 shows the concentration dependence profiles of
HNE on the Naϩ-Kϩ-ATPase activity and SH contents in the
membrane proteins. In these experiments, the synaptosomes
were treated with various concentrations of HNE (100—
750 mM). As can be seen in the figure, binding of HNE to the
synaptosomes led to a dose-dependent inactivation of the
ATPase activity with the loss of SH content in the membrane
proteins. In addition, it was found that the HNE concentra-

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Fig. 4. The Double Reciprocal Plots of Naϩ-Kϩ-ATPase Activity and ATP
Concentration
The concentrations of HNE and MDA were 500 mM. Other experimental conditions
are the same as those described in the legend to Fig. 2. Symbols: ᭺, control (no HNE
treatment) synaptosomes; ᭹, HNE-treated synaptosomes; ᭝, MDA-treated synapto-
somes.
Table 3. Effects of Aldehyde-Modification on the Kinetic Parameters of
Naϩ-Kϩ-ATPase Activity
Km
(mM)
Vmax
Addition
(mmol Pi/mg protein/min)
No
HNE
HNEϩATP
0.60Ϯ0.03
2.37Ϯ0.51*
0.60Ϯ0.03
0.68Ϯ0.01
0.24Ϯ0.04*
0.65Ϯ0.02
MDA
0.52Ϯ0.02*
0.66Ϯ0.05
* pϽ0.05 vs. control (no HNE treatment) in each system.
Fig. 5. Effects of HNE-Modification on Fluorescence Anisotropy of DPH-
or TMA-DPH-Labeled Synaptosomes
Kϩ-ATPase preparation in place of the synaptosomes.
As shown in Fig. 3, the ATPase activity and SH content
were decreased by treatment of the enzyme preparation with
HNE in concentration-dependent manners, indicating that the
loss of SH groups in the enzyme molecule plays an impor-
tant role in HNE-induced inhibition of Naϩ-Kϩ-ATPase ac-
tivity.
(A) HNE Concentration Dependence. The concentration of HNE was varied from
100 to 750 mM. The procedure and conditions of the fluorescence anisotropy measure-
ment were described under Materials and Methods. * pϽ0.05 vs. control synaptosomes
(no HNE treatment). (B) Relationship between Naϩ-Kϩ-ATPase Activity and Fluores-
cence Anisotropy of TMA-DPH-Labeled Synaptosomes. The data of Naϩ-Kϩ-ATPase
activity and fluorescence anisotropy were obtained from Figs. 2 and 5A, respectively.
Symbols: ᭺, DPH-labeled synaptosomes; ᭹, TMA-DPH-labeled synaptosomes.
Kinetic Parameters of Na؉-K؉-ATPase Activity In
order to identify the mechanism of HNE-induced inhibition
of synaptosomal Naϩ-Kϩ-ATPase activity, the effects of alde-
hyde-modification on the kinetic parameters of the ATPase
activity were examined (Fig. 4), and the results are summa-
rized in Table 3. It is clear that treatment of the synapto-
somes with HNE resulted in an increase of the Km value for
ATP and a decrease of the Vmax value. In contrast, treatment
of the synaptosomes with MDA did not affect these enzy-
matic parameters of Naϩ-Kϩ-ATPase. In addition, treatment
of the synaptosomes with 500 mM HNE in the co-presence of
10 mM ATP completely protected the Naϩ-Kϩ-ATPase activ-
ity from the HNE toxicity (0.158Ϯ0.035 and 0.502Ϯ
0.019 mmol Pi/mg protein/min for the systems without and
with ATP). In this case, the ATPase activity of control synap-
tosomes was 0.492Ϯ0.035. Similar phenomena were also ob-
served in the kinetic parameters of the enzyme activity
(Table 3).
ined in terms of fluorescence anisotropy measurement.
As shown in Fig. 5A, the fluorescence anisotropy (g) of
DPH-labeled synaptosomes did not change by HNE-modifi-
cation over the concentration range tested (100—750 mM). In
contrast, the g value of TMA-DPH-labeled synaptosomes in-
creased depending on the concentration of HNE. On the
other hand, the extent of the fluorescence anisotropy of
DPH-labeled (gϭ0.246Ϯ0.003) and TMA-DPH-labeled
(gϭ0.268Ϯ0.002) control synaptosomes did not change
by treatment with 500 mM MDA (gϭ0.242Ϯ0.002 and
0.264Ϯ0.001 for DPH- and TMA-DPH-labeled ones, respec-
tively). These results suggest that HNE-modification induce
decrease in the lipid fluidity at the membrane surface rather
than membrane lipid interior. In addition, it was found that
there is a good relationship between the Naϩ-Kϩ-ATPase ac-
tivity and the g value of TMA-DPH embedded in the synap-
tosomal membranes (Fig. 5B).
Effects of Aldehydes on Lipid Fluidity of Synaptosomal
Membrane The effect of HNE- or MDA-modification on
the membrane lipid fluidity of the synaptosomes was exam-
Localization of Bound Aldehydes As shown in Fig. 6A,
HNE- and MDA-modified synaptosomes showed characteris-
tic emission spectra exhibiting the maximum at 425 nm and

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791
Fig. 7. Effects of Increasing Concentrations of HNE on Fluorescent Prod-
uct(s) Formation in Lipid Fraction
The concentration of HNE was varied from 100 to 750 mM. The excitation and emis-
sion wavelengths were 355 and 430 nm, respectively. Other experimental conditions
were the same as those described in the legend for Fig. 6. ᭝ Fluorescence intensity is
represented as the difference in the intensities of the systems with and without HNE-
modification.
in the lipid fractions was dependent on the concentration of
HNE (Fig. 7).
DISCUSSION
Treatment of the synaptosomes with HNE resulted in a
time- and concentration-dependent inhibition of Naϩ-Kϩ-
ATPase activity (Figs. 1 and 2). The concentration of HNE
required to induce half-maximal inhibition of the ATPase ac-
tivity was determined to be approximately 250 mM (Fig. 2),
which is in the range of the values previously reported for
other enzymes.3) On the other hand, HNE did not induce an
appreciable inhibition of Ca2ϩ- or Mg2ϩ-ATPase activity
(Table 1), suggesting that HNE-modification mainly affects
Naϩ-Kϩ-ATPase, rather than Ca2ϩ- and Mg2ϩ-ATPases, in
the synaptosomes.
It was also found that HNE-modification causes the loss of
SH groups in the membrane proteins (Fig. 2). In addition,
modification of the synaptosomes with SH-directed reagents
such as NEM and PHMB markedly inhibited the ATPase ac-
tivity (Table 2). These results suggest that the inhibition of
synaptosomal Naϩ-Kϩ-ATPase activity by HNE treatment is
mainly due to modification of the SH groups in the ATPase
molecule. The results with the Naϩ-Kϩ-ATPase preparation
also supported this hypothesis (Fig. 3). As is well known,
HNE can also react with lysine at both the double bond and
the carbonyl moiety of the aldehyde to form secondary
amine or Schiff’s bases.22) Xu has reported that Naϩ-Kϩ-AT-
Pase possesses a number of lysine residues in the active site,
and that the ATPase is deactivated by modification of lysine
residues.23) However, a preliminary experiment using DNS-
Cl showed that treatment of the synaptosomes with DNS-Cl
Fig. 6. Emissions Spectra of Fluorescent Products Formed by HNE- or
MDA-Modification of Synaptosomes
The concentrations of HNE and MDA were 500 mM. The emissions spectra were
monitored with the excitation wavelength at 355 nm. Other experimental conditions and
the procedure of separating protein and lipid fractions from aldehyde-modified synapto-
somes are described under Materials and Methods. (A) aldehyde-modified synapto-
somes; (B) protein fractions; (C) lipid fractions. a, control (no HNE treatment); b,
HNE-treated synaptosomes; c, MDA-treated synaptosomes.
457 nm, respectively. In addition, the analysis using the pro- (500 mM) for 30 min at 37 °C did not induce an appreciable
tein and lipid fractions separated from aldehyde-modified inhibition of the Naϩ-Kϩ-ATPase activity (0.476Ϯ0.025 and
synaptosomes showed that HNE and MDA were mainly in- 0.395Ϯ0.036 mmol Pi/mg protein/min for control and DNS-
corporated into the lipid and protein fractions, respectively treated ones, respectively). From this finding, we have ruled
(Figs. 6, B and C). In addition, the concentration dependence out the possibility that modification of lysine residues located
profile of HNE showed that fluorescent product(s) formation in the active site of Naϩ-Kϩ-ATPase is directly involved in

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Vol. 26, No. 6
age of cellular membranes.3,31) And, the emission maximum
of MDA-modified synaptosomes was 457 nm, which is sig-
nificantly different from the emission maximum (425 nm) of
HNE-modified ones (Fig. 6A), suggesting that the fluores-
cence is due to chromophores of another structure.32) From
these findings, it seems that the discrepancy observed be-
tween HNE- and MDA-modified synaptosomes may reflect
the difference in action mechanisms of these aldehydic com-
pounds on the synaptosomal Naϩ-Kϩ-ATPase described
above, although the exact mechanisms are unclear at present.
Recent studies have proposed that lipid peroxidation prod-
ucts of membranes, especially HNE, are involved in the onset
of a variety of neuronal disorders.33,34) In addition, several in-
vestigators have reported that HNE impairs the function of
proteins relating to the regulation of ion homeostasis in cul-
tured hippocampal neurons13,16) and in non-neuronal cells
such as liver cell microsomes.35) Recently, Keller et al. also
reported that HNE inhibits ion-motive ATPase and glutamate
transport activities of hippocampal neurons.14) In the present
report, we have proposed the possibility that alterations in
membrane lipid dynamics play an important role in the onset
of HNE-induced inhibition of synaptosomal Naϩ-Kϩ-ATPase
activity. Although further detailed experiments using recon-
stituted membranes including Naϩ-Kϩ-ATPase are necessary
to elucidate the precise mechanisms, it seems that the present
findings give us insight into the analysis of the mechanisms
of HNE toxicity.
HNE-induced inhibition of the ATPase. The kinetic data in-
dicated a decreased binding affinity of the substrate for the
enzyme (Fig. 4, Table 3). In addition, the inhibitory effect of
HNE against Naϩ-Kϩ-ATPase activity was completely pre-
vented by pre-treatment with the substrate, suggesting that
inhibition of the enzyme activity associated with HNE-modi-
fication may reflect alterations in or near the active site in the
enzyme molecule.
The results of the fluorescence anisotropy measurements
showed that the g value of TMA-DPH molecules embedded
in the synaptosomal membrane lipids increased by treatment
with HNE in a concentration-dependent manner (Fig. 5A). In
contrast, the g value of DPH-labeled synaptosomes did not
change in the concentration range of HNE tested. Because
TME-DPH is a positively charged probe, these results sug-
gest that HNE-modification of the synaptosomal membranes
cause a change in their lipid dynamics at the lipid/water in-
terface on the membrane surface, not in the lipid hydrocar-
bon interior. An increased fluorescence anisotropy reflects a
restricted motion of the fluorescence probes located in the
membrane lipid layers due to increased lipid-lipid interac-
tions.24) From these findings, therefore, it is suggested that
the lipid fluidity at the membrane surface decreased by modi-
fication of the synaptosomes with HNE. This finding is not
consistent with the reports by Subramanian et al.25) and Buko
et al.,26) in which they reported that modification of synapto-
somal membranes25) and liver plasma membranes26) with
HNE caused an increased lipid fluidity of the membranes.
As shown in Fig. 5B, there is good correlation between the
Naϩ-Kϩ-ATPase activities and the g values of TMA-DPH-la-
beled synaptosomes in the presence of various concentra-
tions of HNE. It has been well known that the dynamic envi-
ronment around membrane proteins plays an important role
in regulation of the activities of membrane-bound en-
zymes via alterations of lipid-lipid and lipid-protein interac-
tions.27—29) Based on these findings, it is suggested that lipid
dynamics are an important factor in the inactivation mecha-
nism(s) of synaptosomal Naϩ-Kϩ-ATPase by HNE treatment,
and we speculate that HNE-induced inhibition of the synap-
tosomal Naϩ-Kϩ-ATPase activity may be related to changes
in the reactivity of the SH groups located in or near the ac-
tive site in the enzyme via changes in their membrane lipid
dynamics.
Acknowledgement This work was partly supported by
the Specific Research Fund of Hokuriku University.
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