49
:Vlem. Fac. Agr. Kinki Univ. 30:
49~56
(997)
Effects of Light on Induction of Ascorbate Peroxidase
and Enzymes Involved in the Ascorbate-Glutathione
Cycle in Euglena gracilis Z
Takahiro ISHIKAWA ...., Toru TAKEDA·, and Shigeru SHIGEOKA·
Synopsis
We investigated the effects of light on antioxidant enzymes of the ascorbate·glutathione cycle
including superoxide dismutase in Englena cells adapted to the dark. Illumination of the cells for
24 hat 55 pE m~2 sec' caused 3.5-fold increase in ascorbate peroxidase activity. The effect of
illumination was saturated with this light intensity. This increase in activity was accompanied
by increases in the activities of superoxide dismutase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. The induction of ascorbate peroxidase and
the other enzymes was inhibited by cycloheximide, but not chloramphenicol or streptomycin. The
concentration of ascorbate peroxidase protein increased in parallel with the increase in the
enzyme activity, according to results of immunoblotting. These results showed that the increase
in concentrations of the enzymes of the ascorbate-glutathione cycle, including superoxide dismutase, arose because of de nul'O synthesis of their proteins.
Introduction
In the Mehler reaction of illuminated chloroplasts, the univalent reduction of oxygen at the
reducing side of photosystem I leads to production of the superoxide anion radical, which
disproportionates rapidly into H 20 2 by the action of superoxide dismutase (SOO) .1.,. In addition,
respiratory chain of mitochondria produces superoxide anion radical followed by thr formation
of H 2 0, by dismutation at the sites of ;>.;ADH dehydrogenase, NADH-ubiquinone reductase, and
the ubiquinone-cytochrome b segment. 31 Environmental stresses such as high light intensity:> low
temperature,"J and drought·· n are related with increased production of active oxygen specie::; in
photosynthetic organisms. The toxicity of active oxygen arises becauses it reacts with cell
components, causing inactivation of enzymes, pigment bleaching, lipid peroxidation, and protein
degradation. Photosynthetic organisms have developed defense systems of two kinds against such
toxicity: one is of low-molecular-weight antioxidants such as ascorbate (AsA), glutathione
(GSH). tocopherol, and carotenoid pigments and the other is of antioxidant enzymes such as SOD,
ascorbate peroxidase (AsAP), and catalase. 2l Figure 1 is a diagram of the AsA-GSH cycle,
consisting of A AP, monodehydroascorbate reductase, dehydroascorbate reductase., and GSH
reductase. 8 ) AsAP, which is widely distributed in higher plants and algae, catalyzes the reduction
6
Department of Food and Nutrition. Faculty of J\RTicuhure. Kinkt
•
nivcrsit)". Nak:ullilchi. 'ara 631, Japlln (.{jJlI.f:M.
~1l1
1ft•
{t1tillJ\:~)
··Present addr
: l'lepartmcnt of Biochemistry. W3kayama Medical ··ollCRe. \Vaka)'ama 640. Japan
Abbrt.'\'ialions: AsA. ascorbale: A P. ascorbale peroxidase: J)AsA, dehydroascorbalc; ~IDA monodehydroascorbale; GSH. Rlutalhi oe: SOU. super.
oxide dismutt\sc
(3)
Monodehydro(3)
DehydroAscorbate ...O(E-----'-- ascorbate
----~~~ ascorbate
~
NAD(P) +
NAD(P)H (5)
GSSG
GSH
~
NADPH
NADP+
Fig. 1 Ascorbate-glutathione cycle in photosynthetic organisms.
(l) Superoxide dismutase,
ascorbate peroxidsae. (3) nonenzymatic reaction, (4) monodehydroascorbate
reducta:-e, (5) dehydroascorbate reductase, (6) glutathione reductase.
(2)
of H 20 2 to water by the reducing power of AsA. Monodehydroascorbate (MDAsA) is the
primary product of the AsAP reaction and can be directly reduced to AsA by a :-.lAD(P)H
dependent MDAsA reductase. MDAsA can also spontaneously disproportionate to AsA and
dehydroascorbate (DAsA), the latter being converted back into AsA by DAsA reductase with
GSH as the electron donor. DAsA reductase is coupled with GSH reductase, thus completing the
AsA-GSH cycle. The cycle serves to maintain AsA in its reduced form and minimize oxidative
damage caused by active oxygen species to chloroplasts and other cell compartments.
Eugltma cells contain SOD and the AsA-GSH cycle. a.9 ) The enzymes in the cycle are found only
in the cytosol.'O-121 A site in photosystem II of Euglena chloroplasts is where active oxygen
forms 131 H 20 2 generated in chloroplasts and mitochondria diffuses from the organelles into the
cytosol and is decomposed by AsAP.'·) Euglena cytosolic AsAP has enzymological and im·
munological properties that resemble those of AsAP isozymes from higher plants, and other
properties that are not shared (e.g., the enzyme reduces lipid peroxidt:~. has a high molecular
weight, and has an amino acid sequence at the N·terminus that is nut similar to these other
enzymes) .'5"61 Reduced forms of AsA and GSH are present in large amounts in Euglena cells.
When dark-grown Euglena cells are illuminated, the cellular contents of AsA and GSH increase
by 7 and 4.5·fold, respectively; the increases were due mainly to increases in the reduced
forms.'7.18l On the base of findings reported so far, the question can be raised as to whether in
illuminated Euglena cells the increase in buth compounds is accompanied by the changes in the
enzymes involved in the AsA-GSH cycle. including SOD.
Here we report the effect of light on the enzyme activities of the AsA·GSl-I cycle and discuss
the physiolugical function of antioxidant enzymes in Euglena cells.
ISHIKAWA, TAKEDA, SHIGEOKA: Effects of Light on Induction of Ascorbate Peroxidase
II
Materials and Methods
Chemicals. AsA, HzO z, AsA oxidase, GSH reductase, and glucose oxidase were purchased from
Sigma Chemical Co. DAsA was prepared by the bubbling of Br z vapour through a freshly
prepared solution of 500 J,lmol of AsA and then bubbling with N z gas to remove excess Brz as
described elsewhere. II)
Organism and culture conditions. Euglena gracilis Z was cultured in the dark in the Koren·
Hutner medium at 26"C for 6 days, by which time the stationary phase was reached.'6) The cells,
kept at 26 C, were illuminated with white light (20-150 J,lE m- z sec-I) supplied by fluorescent
lamps for timed periods. Before illumination began, the dark·adapted cells were manipulated
under a green safe light. The number of cells was counted by a hemocytometer.
Preparation of crude extract. EI/J?lena cells were harvested by centrifugation, washed twice,
suspended in 50 mM potassium phosphate buffer (pH 6.3) containing 10% (w/v) sucrose, 1 mM
EDTA, and 1 mM AsA and sonicated at 10 kHz for 2 min. The lysate was centrifuged at 10000 X
g for 10 min and the supernatant was used as the crude extract. 161
Enzyme assays. The activity of AsAP mc 1.11.1.11) was assayde at 32'C in 2 ml of a reaction
mixture containing 50 m!VI: potassium phosphate buffer (pH 6.3), 0.4 mM AsA, 0.1 mM HzO z, and
the enzyme. The reaction was started by the addition of the enzyme. The oxidation of AsA was
monitored by measurement of the decrease in the absorbance at 285 nm (5.8 m:vl- ' cm-') .'1)
SOD (EC 1.15.1.1) was assayed spectrophotometrically in terms of the inhibition of xanthine
oxida~e·dependentreduction of 10 J,l M ferricytochrome c (Sigma) monitored at 550 nm in 50 mM
phosphate buffer (pH 7.8) containing 1 mM EDTA, and 50 J,lM xanthine. One unit of SOD was
defined as the amount of enzyme needed to inhibit the rate of ferricytochrome c reduction of 0.025
Asso min-' by 50%.9)
MDAsA for the :vJDAsA reductase (EC 1.6.5.4) assay was generated by AsA and an AsA
oxidase system as described previously") The reaction mixture (2 ml) comprised 50 mM
phosphate buffer (pH 7.0), 1 m:\<1 AsA, 1 unit AsA oxidase, 0.2 mM !'\ AD (P) H, and the enzyme.
The reaction was started by the addition of AsA oxidase. The progress of the reaction was
monitored by measurement of the decrease in absorbance of NAD(P) H at 340 nm (6.22 mM-'
cm-').
The activity of DAsA reductase (EC 1.8.5.4) was monitored spectrophotometrically by coupling
with the reduction of DAsA by GSH oxidation with GSH reductase" l . The reaction mixture (2
ml) contained 50 mM phosphate buffer (pH 7.0), 2.5 mM DAsA, 2.5 mM GSH, 1 unit GSH
reductase, 0.2 mM ;--JADPH, and the enzyme.
GSH reductase (EC 1.6.4.2) was assayed in a reaction mixture (2 ml) comprising 50 mM
phosphate buffer (pH 8.2), 1 mM EDTA, 0.2 mM N ADPH, 0.2 mM GSSG (oxidized form), and the
enzyme by measurement of the decrease in absorbance of j ADPH at 340 nm" l .
Immunoblot analysis. Western blotting with monoclonal antibodies raised against purified
Euglena AsAP was carried out as described earlier'91. The crude extracts were treated by
SDS·PAGE on a 10% slab gel. The gel was then equilibrated for 20 min in transfer buffer [25 mM
Tris, 192 mM glycine and 20% (v/v) methanol]. Proteins were blotted onto an Immobilon·P
transfer membrane (PVDF, pore size 0.45 J,lm, No. IPVH 304FO, Millipore, Bedford, MA) with a
semidry electroblotting system (Bio·Rad). Immunodetection was done at room temperature with
0.1% (\\'Iv) bovine serum albumin in phosphate· buffered saline as a blocking reagent. Goat
anti·mouse Igs antibodies conjugated with peroxidase was used as the secondary antibody.
Protein assay. Protein was determined by the method of Bradford ZOJ with bovine serum albumin
as the standard.
51
52
III
Results and Discussion
Effects of light on activities of AsAP and enzymes related to the AsA-GSH cycle Euglena
cells grown in the dark were switched to growth under various light·conditions. Figure 2 shows
the effects of light on AsAP activity in Euglena cells. When cells were cultured in the dark, the
AsAP activity was 25 ± 1 nmol min-I 10- 6 cells. Illumination at 55 Ji E m- 2 sec- I for 24 h caused
3.5-fold increase in the enzyme activity with a lag phase of 4 h. Simi liar results were obtained
by illumination at 150 Ji E m- 2 sec-I. Illumination at 20 Ji E m- 2 sec- I caused a slower increase in
the activity with a lag phase of 8 h, but by 72 h, the same level was reached as with 55 JiE m- 2
sec-'. These results suggested that the effects of light are saturating with an intensity of about
55JiE m- 2 sec-I.
The SOD activity increased 5.5-fold by 8 h of illumination ;:;t 55 Ji E m- 2 sec-I and later
decreased to the base· line level (Fig. 3). The activities of GSH reductase, MDAsA reductase, and
DAsA reductase increased 6-, 3-, and 4-folds, respectively. until they reached their maximum
levels at about 2·1 h.
Effects of antibiotics on AsAP and enzymes related to the AsA-GSH cycle. Effects of three
antibiotics on the activity of AsAP in Euglena cells kept 24 h in the light are shown in Table 1.
Cycloheximide, an inhibitor of protein synthesis on the 875 cytoplasmic ribosomes,2'.22) prevented
any increase in AsAP activity, whereas chloramphenicol and streptomycin, specific inhibitors of
protein synthesis on the 685 plastid ribosomes,2I.,,! had little effect. When cycloheximide was
added to the medium at different time during illumination, the increase in the AsAP activity was
completely suppressed (data not shown). This drug prevented any increase in the activities of
SOD, GSH reductase, MDAsA reductase, and DAsA reductase, as well (Fig. 3). These results
showed that the increase in activities of enzymes of the As:\·G5H cycle resulted from their de
nol'o protein synthesis.
Effect of light on AsAP protein translation. In an examination of the relationship between the
light dependent induction of AsAP activity and the translation of A"AP protein, immunoblotting
!!!.
Qi 80
u
'I'
....
0
";'
c:: 60
'E
'0
E 40
c::
~
~
:~
20
ti
III
CL
c(
I/)
c(
0
0
24
48
72
Illumination time (h)
Fig.2 Effects of light on activity of AsAP in Euglena cells grown first in the dark. O. dark;
•. 20,u E • sec-I; •. 55,u E-' sec'; .... 150,u E-' sec' Each value is the mean of three
a 'says (coefficient of variation <5%).
ISHIKAWA, TAKEDA, SHIGEOKA: Effects of Light on Induction of Ascorbate Peroxidase
~
SOD
0.6
Qj
(J
~
'I'
...
0
Qj
(J
'I'
-
0.4
~
0.2
...
0
'7
c: 6
'e
'c
2-
"0
E
oS 3
:~
~
u
:~
u
~
0
~
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B
F
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'0::
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Illumination time (h)
Fig,3
DAsA reductase
"0
E
"0
E
~
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c:
c:
~
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6
'I'
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9
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Illumination time (h)
Effects of light on activities of SOD and other enzymes of the AsA -GSH cycle, . ,
illumination at 55 Ii E- 2 sec' ; 0, illumination cycloheximide. The drug was added in
the dark to the culture medium at a final concentration of :) X 10- 0 M, which did not
affect cell viability. Each value is the mean of three assay (coefficient of variation
<6%).
Table 1. Effects of antibiotics on increases
caused by illumination in AsAP activo
ity of Euglena cells. A drug was added
in the dark to the culture medium at a
final concentration shown, which did
not affect cell viability.'o, The effects
are expressed as the percentage by
which the increase in A AI' activity in
the control without antibiotic was
inhibited.
Antibiotic
Cycloheximide
Chloramphenicol
Streptomycin
Concentration
Inhibition
(M)
(%)
5 10-'
3 10- 3
10-'
100
7.5
1.5
53
54
Fig.4
~58kDa
o
3 6 12 24
Time (h)
Eff cts of light on the concentration of
AsAP protein found by immunoblot analy·
sis, Eug/ena cells were illuminated at 55
Jl E-2 sec-' for O. 3, 6, 12, or ~4 h.
Total
soluble proteins were extract d from the
cells, resolved by 50S-PAGE, and electrob·
lotted on a PVOF membrane, Each lane
cuntained 100 Jlg of soluble protein,
that used monoclonal antibodies raised against purified Euglena AsAP'S) was performed. Euglpllfi
cells were harvested at different times after exposure to light (55 JL E m- 2 sec ') b('~an, The
antibodies reacted with a 58-kDa protein band corresponding to purified Euglmw AsAP (Fig, 4).
The amount of AsAP protein increased in parallel to the increase in the AsAP activity when c 'lIs
were illuminated.
IV
Conclusion
Shigeoka et a1. 17 ,181 have found that when Euglena cells grown in the dark are illuminated at
55 JL E m- 2 seC', the concentrations of AsA and GSH increase 7- and 4,5-fold, respectively, and
reach a peak after 7 h. The light·dependent increases of the enzymes involved in the AsA-GSH
cycle were closely connected with those in the concentrations of AsA and GSH, The increases
in activities of the enzymes of the AsA-GSH cycle arose because of de nUl'U synthesis of the
proteins. These results su~gested that the cycle including SOD operates efficiently as a system
conjugated to light intensity. Chloroplasts isolated from pea leaves grown at a high light
intensity, 400 JL E m- 2 sec-I, contain increased activities of AsAP, GSH reductase, and DAsA
reductase and higher concentrations of AsA than chloroplasts at a low light intensity of 100 JL E
m- 2 sec') ." Thomsen et al. 23 ) have reported that the appearance of AsAP is regulated by light
via phytochrome. Furthermore, in higher plants, high light intensity in combination with chilling,
magnesium deficiency treatment, or high temperature, increases the concentration of AsA and
activities of antioxidant enzymes including AsAP,.. -261 These results suggest that light is
important in the regulation of antioxidant ~ystems in photosynthetic organisms. The transition
of Euglena cells from the dark to the light apparently involves increased metabolic activity in
each organelle, causing to the generation of active oxygen species, Accordingly, increased levels
of antioxidative components seem to be an early physiological response of illuminated Euglena
cells, helping to prevent oxidative damage caused by active oxygen species.
V
Acknowledgements
This work was supported in part by a grant from Kinki University.
V I References
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ISHIKAW;\, TAKEDA, SHIGEOKA: Effects of Light on Induction of Ascorbate Peroxidase
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D.). GILLHAM
and A.D. Dorx;E:
55
5
6
近畿大学農学部紀墳
第3
0号
(
1
9
9
7
)
ユー グ レナにお ける光照射 のアス コル ビン酸- グル タチオ ン サイ クルの
アスコル ビン酸ペルオキシダーゼ と関連酵素 に及 ぼす影響
石川孝博,武 田 徹,婁岡
要
約
成
群 の活性 も上昇 した｡ タンパ ク質合成 阻啓剤 やイム
暗適応 したユーグ レナに光照射 した ときのアスコ
ノプロッティングの結果, これ らの活性上昇 はおの
ル ビン酸 - グル タチオ ン サ イ クルに関与 す る抗 酸
おのの タ ンパ ク質 の d
enou
o合成 に起 因 して い る
化帯架への影響 を検討 した｡ アスコル ビン酸ペルオ
ことが明かになった｡以上 よ り,光照射 を受 けたユ
キシダーゼ (
As
AP)は光照射 によ り2
4
時間で約3.
5
ー プレナ細胞 は代 謝活性 の上昇 に伴 ってお こる酸化
倍 に増加 した｡ この増加 と共 に, スーパーオキシ ド
ディスムターゼ
(
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的 ス トレスに応答す るために,防御系 としての抗酸
化酵紫群 を合成 す ることが,
'
7唆
: されたO