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Subcellular distribution and activities of superoxide dismutase catalase glutathione peroxidase and glutathione reductase in the southern armyworm Spodoptera eridania.

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Archives of Insect Biochemistry and Physiology 2173-186 (1988)
Subcellular Distribution and Activities of
Superoxide Dismutase, Catalase, Glutathione
Peroxidase, and Glutathione Reductase in the
Southern Armyworm, Spodoptera eridania
S a m i Ahmad, C h r i s A. Pritsos, Susan M. Bowen, Charles R. Heisler,
Gary J. Blomquist, a n d Ronald S. Pardini
Department of Biochemistry, University of Nevada, Reno
I n mid-fifth-instar larvae of the southern armyworm, Spodoptera eridania, the
subcellular distribution of four antioxidant enzymes-superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione
reductase (GR)-were examined. Two-thirds (4.26 units - m g protein-’) of the
SOD activity was found in the cytosol, and one-third (2.13 units - m g protein-’)
in the mitochondria. CAT activity was unusually high and not restricted t o
the microsomal fraction where peroxisomes are usually isolated. The activity
was distributed as follows: cytosol (163 units) mitochondria (125 units) and
microsomes (119 units). Similar t o CAT, the subcellular compartmentalization
of both GPOX and GR was unusual. N o activity was detected in the cytosol,
but in mitochondria and microsornes, GR levels were 5.49 and 3.09 units.
Although GPOX activity exhibited 14-16-fold enrichment in mitochondria and
microsornes, respectively, over the 850g crude homogenate, the level was
negligible (mitochondria = 1.4 x I O W 3 units; microsomes = 1.6 x l o p 3
units), indicating that this enzyme is absent. The,unusual distribution of CAT
has apparently evolved as an evolutionary answer to the absence of GR from
the cytosol, and the lack of GPOX activity.
Key words: antioxidant enzymes, catalase, detoxification of oxyradicals, glutathione
peroxidase, glutathione reductase, superoxide dismutase
INTRODUCTION
In all aerobic organisms the superoxide anion free radical is generated
through one-electron reduction of dioxygen from numerous biological sources
Received October 5,1987; accepted January6,1988.
Address reprint requests to Dr. Sami Ahmad, Department of Biochemistry, University of
Nevada, Reno, NV 89557-0014.
This work was supported by a USDA Science and Education competitive grant 86-CRCR-l2038 awarded to Ronald S. Pardini, Sami Ahmad, and Gary J. Blomquist, and is a contribution
of the Natural Products Laboratory and the Nevada Agricultural Experiment Station. We thank
Dr. Gregory J. Highison (Department of Anatomy) for electron microscopy of the subcellular
fractions.
0 1988 Alan R. Liss, Inc.
174
Ahmad et al.
which include small to large autoxidizable molecules (e.g., catecholamines,
hemoproteins), oxidoreductases (e.g., xanthine oxidase), and subcellular organelles such as nuclei, mitochondria, microsomes, and chloroplasts [l].
Damages from 02-*
generating systems have been recently reviewed [2].
Further, 0,- in its protonated form, - 0 O H radical, and the conversion of
0,- via the metal-catalyzed Haber-Weiss reaction (Fenton reaction) produces
in vivo H202 and -OH [3]. These products are more reactive than their
precursor, and in particular, -OH is very deleterious, as it causes lipid
peroxidation [4]. The potential is especially high for harmful consequences
of lipid peroxidation in insects in that lipids not only are essential components of cellular membranes but also have unique physiological functions in
insects [5].
Phytophagous insects are additionally subject to exogenous plant prooxidant sources of toxic oxygen compounds and free radicals. Plant pro-oxidants
consist of photodynamically active compounds such as furanocoumarins,
e.g., xanthotoxin [6], which mainly generates ' 0 2 and only secondarily some
0,: [q.Phenolic compounds such as flavonoids, e.g., quercetin, generate
0,- and H202apparently nonphotoactively upon enzymatic bioactivation [8]
and these products further generate -OH 191.
Among well-known defenses are antioxidant compounds such as a-tocopherol, ascorbate, P-carotene [lo], and urate [ll]. Additionally, a group of
enzymes consisting of SOD, CAT, GPOX and GR form a line of defense as
antioxidant enzymes in the destruction of 0,; through their sequential action, and prevent further 02:-dependent free-radical chain reaction [12].
Preliminary data were recently presented on activities of these antioxidant
enzymes in homogenates of larvae of the cabbage looper moth Tvichoplusiu ni
Hubner, southern armyworm Spodopteru eridaniu (Cramer), and black swallowtail butterfly Pupilio poZyxenes astevius Stoll [13]. Subsequently, details on
the ontogeny of these enzymes, as larvae advanced from third to fifth instar
and matured within each instar, were reported for cabbage loopers [14] and
southern armyworms [15].
We report now the subcellular distribution of the southern armyworm's
antioxidant enzymes and present evidence of unusual intracellular distribution of CAT and GR enzymes. A hypothesis is advanced that this unusual
distribution, especially of CAT, has evolved as an evolutionary answer to
lack of GPOX activity.
MATERIALS AND METHODS
Insects
Southern armyworms were reared according to the procedure recently
reported in detail elsewhere [15]. Regimes for rearing insects were photope-
*Abbreviations used: ANOVA = analysis of variance; CAT = catalase; Cu,Zn-SOD = copper
= dioxygen, ground-state oxygen, or triplet oxygen; GPOX =
and zinc-containing SOD; 302
glutathione peroxidase; GR = glutathione reductase (glutathione disulfide reductase); .OOH
lipid/organic peroxide; Mn-SOD
= hydroperoxy radical; . O H = hydroxyl radical; LOOH
= manganese-containing SOD; lo2= singlet oxygen; 02.= superoxide anion free radical;
SOD = superoxide dismutase.
Detoxification of Oxyradicals
175
riod, 14:lO (L:D); temperature, 25°C f 1°C: 22°C + 1°C (L:D), and relative
humidity, ca. 50-55%. The larval diet was essentially the same as described
by Rehr et al. [16], but contained foliage of lima bean, Phaseolus lirnensis,
instead of the red kidney bean, Phaseolus vulgavis.
Subcellular Fractionation
Subcellular fractions were obtained from homogenates of 40-80 g of midfifth-instar larvae of the southern armyworm by the method of Keeley [ l q ,
with modifications by Halarnkar et al. [IS].
The larvae were washed to remove adhering food and fecal particles, dried
on paper towels, and after immobilization by chilling (10 min at 3°C) were
crushed in a porcelain mortar with a pestle in 20 ml of an isotonic chilled
buffer, pH 7.4, of the following composition: 250 mM sucrose, 2.0 mM ethyleneglycol-bis-(0-aminoethylether) N,N’-tetraacetic acid, 3.4 mh4 (3-[N-morpholino] propanesulfonic acid) free acid, and 0.5% defatted bovine serum
albumin. The crude homogenate was passed through glass wool to remove
unmacerated tissues and chunks of cuticle. The debris from the glass wool
were rehomogenized in the mortar with the pestle and again filtered over
glass wool. The first and second filtrates of crude homogenates were combined, brought to ca. 120 ml with the homogenization buffer, and subjected
to differential centrifugation using rotors and centrifuges cooled to 2°C.
The homogenate was clarified by centrifugation at 5008 for 5 min and the
sediment discarded. The supernatant was spun by gradual acceleration to
8,OOOg for 60 s and held for 5 s before braking. The supernatant was separated. The pellet was gently stirred with a glass rod and resuspended by
adding, dropwise, the homogenization buffer. A washed fraction presumed
to contain ”nuclei” was obtained by a second spin at 8,OOOg. One-third of
the pellet was prepared for electron microscopy as described later, and the
remaining two-thirds were suspended in 4.0 ml of 50 mM potassium phosphate buffer, pH 7.8.
The 8,OOOg supernatant was centrifuged by rapid acceleration to 30,OOOg,
and after 15 s rapidly decelerated by braking. The supernatant was saved,
and the pellet was resuspended in the homogenization buffer and spun
again at 30,OOOg to obtain the washed mitochondrial pellet. A portion of the
pellet was reserved for electron microscopy, and the remainder resuspended
in 4.0 ml of the phosphate buffer, pH 7.8, for antioxidant enzyme assays.
The 30,OOOg supernatant was further centrifuged to obtain the microsomes
and cytosol. Centrifugation was at 100,900g for 45 min. The pellet was gently
stirred, resuspended in the homogenization buffer and spun again as above
to obtain the washed microsomal fraction. Portions of the microsomal pellet
were prepared for electron microscopy and assays of the antioxidant enzymes. The 100,900gsupernatant from the first spin was the cytosolic fraction.
Sonication
All particulate subcellular fractions, held on ice, were sonicated at the
”low” setting (Biosonick IV,Bronwill, Rochester, NY) twice for 10 s to release
contents of the membrane-bound organelles.
176
Ahmad et al.
Electron Microscopy
Pellets of the subcellular fractions were initially fixed for 1h at 4°C in a
1.5% gluteraldehyde solution, pH 7.38, containing 3 mM CaC12. After a
thorough washing in 0.144 M cacodylate buffer, the pellets were postfixed in
1.0% osmium tetroxide for 1 h at room temperature. The pellets were then
washed and suspended in a small volume of cacodylate buffer. An equal
volume of 5.0% agar was added to each pellet suspension and pipetted onto
a piece of parafilm. After the agar had set, small cubes were cut with a razor
blade and placed in a buffer solution. After dehydration in an ascending
series of ethanol solutions, the agar cubes were infiltrated and embedded in
an EponlAraldite resin. Ultrathin sections were stained by immersion in a
saturated lead citrate solution for 15 min. The preparations were then examined and photographed in a Hitachi 125E transmission electron microscope.
Assay for Non-Antioxidant Enzymatic Oxidation of NADPH
Endogenous NADPH oxidation in the microsomal and mitochondria1
preparations was followed at 340 nm, and quantified using the extinction
coefficient 6.22 x lo6 M-' cm-' for NADPH [19]. Carbon monoxide was
bubbled in some samples and its affect on suppressing NADPH oxidation
was recorded.
Assays for Antioxidant Enzymes
Enzymatic assays were performed with 20-30 pl aliquots of subcellular
fractions and only when a fraction exhibited marginal activity was the volume
of fractions increased to 60-90 pl. Proteins were determined by the method of
Lowry et al. [20].
SOD activity was determined indirectly by a sensitive colorimetric assay
described by Oberley and Spitz [21]. Units are defined by McCord and
Fridovich [22], and the SOD activity is expressed in units -mg protein-'mir-' at 25°C.
CAT activity was measured by the procedure of Aebi [23], in which
catalysis of H202 to H20 in an incubation mixture adjusted to pH 7.0 is
recorded at 254 nm for 1 min. The time (s) required for the decrease in A
from 0.45 to 0.40, representing the linear portion of the curve, was used for
calculation of the enzyme activity. One unit of CAT activity was defined as
causing the decomposition of 1pmol H202 -mg protein-' emin-' at pH 7.0
and 25°C.
GPOX assay was based on the oxidation of GSH to GSSG in the presence
of the substrate, H202,measuring indirectly by coupling this reaction to that
catalyzed by GR [24]. One unit of GPOX activity was defined as the oxidation
of 1nmol NADPH -100 pg protein-' -min-' at 25°C.
GR activity was assayed according to Racker [25]. GSSG was the substrate
which was reduced to GSH, and the reducing equivalents were provided by
NADPH. Consequently, the disappearance of NADPH due to oxidation to
NADP+ was monitored at 340 nm. One unit of GR activity was expressed as
the change of 0.001 A20 -mg protein-' emin-' at 25°C.
Detoxification of Oxyradicals
177
RESULTS
Basis for Selecting Subcellular Fractions for Enzyme Assays
In our preliminary experiments, activities were detected for all antioxidant
enzymes in all subcellular fractions, except for the absence of GR from the
cytosol. Surprisingly, the nuclear fraction exhibited SOD, CAT and GR
activities at levels remarkably similar to those reported for 850g supernatants
of whole-body homogenates of the southern armyworm [15]. Of concern was
the SOD level in the nuclei which was high and second only to the cytosolic
level. A CuZn-SOD which is normally present as a cytosolic enzyme in
eukaryotes was suggested to be present in nuclei, but was later discounted
as erroneous [26]. That the nuclear activity was indeed an artifact was
confirmed from electron microscopic studies which showed that the nuclear
fraction mostly contained intact whole cells. In other assays, we therefore
deleted the nuclear fraction, and concentrated on mitochondrial, microsomal
and cytosolic fractions.
Electron Microscopy of Mitochondria1 and Microsomal Preparations
Electron micrographs of a section of the mitochondrial and microsomal
pellets (Fig. 1)showed that the mitochondrial preparation contained 80+%
mitochondria, and the remainder was identified as fragments of the endoplasmic reticulum with and without ribosomes. Most of the mitochondria
were intact, with some showing partial to complete unalignment of the
cristae. In general, the preparation contained a higher proportion of intact
mitochondria than illustrated before [27. Figure 1also shows that the endoplasmic reticulum preparation consisted mainly of microsomes, both smooth
and ribosome-studded, and free ribosomes.
Possible Effects of Sonication on Subcellular Fractions Assayed
Destruction of the membrane integrity of the membrane-bound subcellular
fractions was not expected to affect assays of antioxidant enzymes in that
none of these enzymes are coupled andlor membrane bound. Sonication
was essential to facilitate interactions among the exogenous ingredients of
the incubation mixtures with the antioxidant enzymes released from membrane-bound organelles. Results presented later supported the premise that
sonication did not, at least significantly, inactivate the antioxidant enzymes.
For example, SOD activity was recorded in mitochondria, and levels of CAT
activity higher than in whole-body homogenates [15] were also recorded in
both microsomes and mitochondria.
Endogenous Oxidation of NADPH
Assays for antioxidant enzymes GPOX and GR are based on monitoring
the oxidation of NADPH to NADP+. In a cell there are many NADPHutilizing enzymes such as mitochondrial and microsomal transhydrogenase
(NADPH:NAD+ oxidoreductase), DT-diaphorase [NAD(P)H: (quinone-acceptor) oxidoreductase], and cytochrome P-450-dependent monooxygenases.
These enzymes oxidize NADPH, hence the possibility that endogenous oxi-
178
Ahmad et al.
Fig. 1. Electron micrographs of sections from the centrifugal fractions used as mitochondria1
(30,OOOg for 15-s pellet) and microsomal (100,900g for 45-min pellet) sources of the antioxidant
enzymes of S. eridania. Panel A shows that the mitochondria1 preparation consisted mainly
of unbroken mitochondria; in some mitochondria swelling was apparent. Slight contamination from vesiculated endoplasmic reticulum was evident; x 34,000. Panel B shows that the
microsomal preparation consisted entirely of spherical vesicles derived from both smooth
and rough endoplasmic reticulum, with some free ribosomes. Without histochemical analysis
the presence of peroxisomes that are formed from the Colgi-endoplasmic reticulum-lysosome complex was not confirmed; ~52,000. Panel C from the same fraction shown in B,
depicts intact rough endoplasmic reticulum as a constituent of the microsomal pellet; x 59,000.
Detoxification of Oxyradicals
179
dation leading to erroneous estimates of GPOX and GR levels needed clarification. Data presented in Table 1show rates of endogenous oxidation for
the sonicated mitochondrial and microsomal preparations. Since much of the
endogenous NADPH oxidation by these subcellular fractions has been attributed to high levels of cytochrome P-450 in insects [28], and CO strongly
inhibited the oxidation of NADPH (Table l), it logically follows that much of
the NADPH oxidation observed was from cytochrome P-450. However, rates
of the sonicated fractions were negligible, ranging from 1.4% to 2.7%, compared with the rates of cytochrome P-450-mediated endogenous NADPH
oxidation, without added substrate, reported for unsonicated microsomes of
the southern armyworm larvae 1191. Negligible levels of endogenous oxidation most likely resulted from much conversion of the labile cytochrome P450 to the inactive cytochrome P-420 presumably owing to sonication. Therefore, the levels recorded for GPOX and GR most probably are in the range
close to actual values.
Subcellular Distribution of SOD Activity
Data presented in Table 2 show that the southern armyworm's distinctive
subcellular compartmentalization and level of activity in the order cytosol >
mitochondria > microsomes, is statistically highly significant (P = 0.0001).
In the 8508 supernatant of whole-body homogenate of mid-fifth-instar southern armyworms, SOD activity was reported to be 3.1 units -mg protein-'
1151. Thus, some enrichment of SOD activity is evident in the cytosolic
fraction, where SOD level amounts to 4.26 units -mg protein-'. SOD activity
in the mitochondria is exactly one-half that of cytosol. This pattern of SOD'S
subcellular distribution in the southern armyworm is similar to other eukaryotes 1261. A Mn-SOD appears to be exclusively located in the mitochondrial
matrix, whereas a cyanide-sensitive CuZn-SOD is primarily located in the
cytosol. Further studies involving purification to enhance enrichment of
TABLE 1. Endogenous NADPH Oxidation (Without Added Substrate) in Microsomes and
Mitochondria of Mid-Fifth-Instar Larvae of S. eridania
Subcellular
fraction
Mitochondria
Microsomes
co
(-I+)
-
+
+
Sonicated
fraction
0.219
0
0.260
0.070
NADPH oxidationa
YO Of unsonicatedb
fraction
1.4-2.3'
1.7-2.7'
aData are averages of duplicate determinations, from values differing by less than 10%.
Sonicated fraction as obtained in the present study. Rates are given as nmol NADPH oxidized
. mg protein-' . min-'.
bPercent NADPH oxidation in sonicated vs. normal intact organelles was computed from
values of endogenous NADPH oxidation reported by Gunderson et al. [19] for the southern
armyworm midgut and fatbody subcellular preparations. For midguts they reported a rate of
24.95, whereas for fat bodies 39.71 nmol NADPH oxidized.min-l.nrno1 P-450-'. Cytochrome
P-450 level was 0.387 nmol-mg protein-'.
'Lower value in the range is relative to midgut, and the higher relative to fat body activity
I191'
180
Ahmad et al.
TABLE 2. SOD Activity in Subcellular Fractions
of Mid-Fifth-InstarLarvae of S. eridania*
Subcellular
fraction
Mitochondria
Microsomes
Cytosol
-
X & S.D. units
mg protein-’
.
2.13 & 0.13 b
0.78 f 0 . 0 2 ~
4.26 & 0.11 a
*Duplicate assays were conducted with two
replicates in each determination; n = 4. All
replicates were pooled for data analysis by
ANOVA; F(2,9) = 952.51, P > F = 0.0001. Means
not followed by the same letter are significantly
different (P < 0.05) by Duncan’s multiple range
test.
TABLE 3. CAT Activity in Subcellular Fractions
of Mid-Fifth-InstarLarvae of S. eridania*
Subcellular
fraction
Mitochondria
Microsomes
Cytosol
-
X f S.D. units
of enzyme
125 40 b
119 f 15b
163 f 30a
*Duplicate assays were conducted with four
replicates in each determination; n = 8. All
replicates were pooled for data analysis by
ANOVA; F(2,21)= 4.39, P > F = 0.0256. Means
not followed by the same letter are significantly
different (P < 0.05) by Duncan’s multiple range
test. A unit of CAT activity is equal to the
decomposition of 1 pmol H202- mg protein-’.
min-’ at pH 7.0 at 25°C.
activity and the use of cyanide and other appropriate inhibitors would more
firmly establish the nature of SOD’s in the cytosol and mitochondria1 matrix.
Not so readily explained is the SOD activity, even though small (0.78 units
‘mg protein-’, Table Z), in the microsomal fraction. As reviewed [26], there
is an abundance of literature on anomalies in the overall distribution pattern
of SOD’s, and often the minor activities are attributed to contaminating
organelles such as lysosomes which may contain CuZn-SOD. It has been
suggested that a reevaluation of the subcellular distribution of the two types
of SOD’s found in animals and an iron-containing SOD found in prokaryotes
and plants must be made from additional studies [26].
Subcellular Distribution of Catalase Activity
A most unusual distribution accompanied by enrichment of activity, relative to 8508 homogenate, was discerned for CAT (Table 3). The subcellular
distribution was in the order: cytosol > mitochondria 2 microsomes, and
the compartmentalization of enzyme activity in this manner was found to be
statistically valid (I’ = 0.0256). Moreover, Duncan’s multiple range test
showed that cytosolic CAT activity, 163 units was significantly different
Detoxification of Oxyradicals
181
(P < 0.05) from CAT levels, 125 units in mitochondria and 119 units mg-*
protein in microsomes (Table 3). There was no statistical difference at the 5%
level between the CAT levels of mitochondria and microsomes. The statistical
validation was essential to substantiate the unusual feature about the intracellular distribution of CAT in the southern armyworm. The levels of CAT
activity reflecting a twofold enrichment in the cytosol vs 8508 supernatant of
whole-body homogenate (80.5 units, [15]), and less than twofold but still
considerably higher CAT activity in mitochondria and microsomes, are
paradoxical.
Numerous studies have unequivocally shown that the peroxisomes or
microbodies of Rouiller is the site for CAT for the destruction of H202[29,30].
The peroxisomes which along with lysosomes are produced by the Golgiendoplasmic reticulum-lysosome complex, are usually isolated as spherical,
membrane-limited structures in the microsomal fraction and sometimes they
are seen attached to fragments of the endoplasmic reticulum. A number of
enzymatic processes in the peroxisomes generate H202 via two-electron
reduction of 3 0 2 without the intermediacy of 0 2 - [12]. The implication of the
unusual distribution of CAT in all cellular compartments of the southern
armyworm examined, unlike its normal restricted localization in other eukaryotes' peroxisomes, is discussed later in the text.
Subcellular Distribution of GR
Data presented in Table 4 show that the southern armyworm GR is present
in mitochondria and microsomes, but not in cytosol. Analysis of data by
ANOVA showed that this compartmentalization of GR is highly significant
(P = 0.0001). Further, the Duncan's multiple range test showed that the
higher GR level in the mitochondria is significantly (P Q 0.05) different from
that in the microsomes. The results also show some enrichment of activity
upon subcellular fractionation in that the level in the mitochondria, 5.49 units
mg protein-' (Table 4), is higher than 4.2 units mg protein-' reported for
850g supernatant of the whole-body homogenate of mid-fifth instar southern
armyworm [15].
TABLE 4. GR Activity in Subcellular Fractions of
Mid-Fifth-Instar Larvae of S. eriduniu*
Subcellular
fraction
Mitochondria
Microsomes
Cvtosol
-
X k S.D. units
of enzyme
5.49 k 0.90 a
3.03 & 0.39 b
0.00
C
*Duplicate assays were conducted with four
replicates in each determination; n = 8. All
replicates were pooled for data analysis by
ANOVA; F(2,21) = 162.26, P > F = 0.0001. Means
not followed by the same letter are significantly
different (P < 0.05) by Duncan's multiple range
test. One unit of GR activity is equal 0.001 A
340.mg protein-' .min-' at 25°C.
182
Ahmad et al.
As with CAT, the subcellular distribution of GR in the southern armyworm
is distinctly different from mammalian species, where two-thirds of the
enzyme occurs in the cytosol and one-third in the mitochondrial matrix [12].
The significance of the absence of GR from the southern armyworm cytosol,
and its presence in mitochondria and microsomes, is discussed later.
GPOX Activity
We had reported earlier that in the 850g supernatant of whole-body homogenates of mid-fifth-instar southern armyworms GPOX activity was on
the order of 1 x
units and nearly the same rate of NADPH oxidation
was observed by denaturing the enzyme by boiling for 1 h [15]. Hence, it
was concluded that the southern armyworm does not possess a GPOX-like
enzyme. In the present study, we observed an enrichment of GPOX activity
by a factor of 14 (mitochondria) and 16 times (microsomes) (Table 5). The
GPOX activity is still negligible and leads to the conclusion that GPOX is
absent in the armyworms.
DISCUSSION
SOD and CAT share the property of a sequential pathway involving
consecutive reactions in which two identical substrate molecules dismutate
to higher and lower oxidation states. SOD activity is apparently confined to
those cellular compartments where 02-formation mostly occurs. In the
vertebrate liver, 80435% of the SOD activity is in the cytosol with 15-20%
activity in the mitochondrial matrix [12]. In the southern armyworm the
compartmentalization of SOD activity is similar to that in vertebrates, with
67% activity in the cytosol and 33% in the mitochondria. This paper reports
for the first time subcellular distribution and activity ratios of SOD in an
insect species, even though SOD has been purified from two dipteran species. In Drosophila rnelanogustev only the Cu,Zn-SOD was found [31], whereas
in Muscu dornestica both Cu,Zn-SOD and Mn-SOD were found [32].
The dismutation product of 02:, H202, produced by SOD can be destroyed by CAT. However, the general consensus is that CAT is primarily
TABLE 5. GPOX Activity in Subcellular Fractions of
Mid-Fifth-Instar Larvae of S. eridania*
Subcellular
fraction
Mitochondria
Microsomes
Cytosol
-
X
1.4 x
1.6 x
0.0
S.D. units of enzyme
lop3 f 0.67
x 10-3a
a
b
f 0.51 x
*Duplicate assays were conducted with four replicates
in each determination; n = 8. All replicates were pooled
for data analysis by ANOVA; F(2,21)= 23.14,
P > F = 0.0001. Means not followed by the same letter
are significantly different (P < 0.05) by Duncan's
multiple range test. One unit of GPOX activity is equal
to 1nmol NADPH oxidized . 100 p g protein-' . min-'
at 25°C.
Detoxification of Oxyradicals
183
located in the peroxisomes, or smaller aggregates such as microperoxisomes
found in a variety of eukaryotic cells. In insects the subcellular distribution
of CAT has not been examined, except for its involvement in gluconeogenesis
during egg maturation of D. melunogustev [33]. The glycogen synthesis occurred in the membrane-bound organelles of the ooplasm which are continuous with the endoplasmic reticulum; therefore, these organelles were
presumed to be microperoxisomes and the site for CAT activity related to
gluconeogenesis. Peroxisomes contain flavin enzymes such as urate oxidase
and D-amino oxidase, which produce H202 by using 3 0 2 as an oxidizing
agent. Therefore, the main function of CAT is the destruction of peroxisomal
H202, and even though H202readily permeates through cell membranes,
the proposed "teamwork" or "sequential pathway" for SOD and CAT must
be evaluated with caution.
According to Chance et al. [12] in mammalian species other than the rat,
considerable CAT activity has been found in the liver cytosol, but unequivocal proof for the existence of extraperoxisomal CAT is at present unavailable
because of the fragility of the peroxisomes. CAT was suspected to occur in
mammalian mitochondrial matrix (Nohl and Hegner; in Nohl and Jordan
[34]), which was confirmed 2 years later [34]. Thus, the finding in the
southern armyworm that CAT is distributed throughout the cell is not entirely surprising, but the very high activity in the cytosol followed by high
levels in mitochondria and microsomes (where peroxisomes are isolated), is
intriguing and is discussed further in the form of a hypothesis, after a
discussion of GPOX-GR role(s) in this insect species.
Based on a previous study [15] and the results of this investigation that
revealed trivial activity, it seems more appropriate to conclude that at least
functionally, the enzyme GPOX is absent in the southern armyworm. A
similiar conclusion was reached for the larvae of the cabbage looper moth
[14]. On the other hand, the presence of GR was demonstrated for 850g
crude homogenate of cabbage loopers (1.17 units) [14], and in the southern
armyworm (4.20 units) [15]. In the latter there was evidence of an unusual
subcellular distribution and some enrichment of activity in the mitochondria.
These values for GR are considerably lower than the 23-27 units reported for
M. domesticu [35], and in general, for many vertebrate species. Therefore, we
reevaluate our earlier conclusion [14] that GPOX when present (so far demonstrated only in D. rnelunoguster [36]) and somewhat low levels of GR
present in cabbage loopers, serve in GSH-related pathways unrelated to their
widely accepted role in protection from oxidative stress (especially from
LOOH).
In M . domesticu GPOX activity was not demonstrated but the formation of
GSSG was recorded [35]. GSSG is apparently capable of scavenging 0,with the potential to scavenge other free oxyradicals [37]. Moreover, the thiol
status of mitochondria is critical to mitochondrial integrity in eukaryotes. For
example, if 12% of the mitochondrial thiols were oxidized, mitochondrial
swelling occurred [38]. Similarly, Bindoli et al. [39] showed that when 15%
of mitochondrial thiols were oxidized, lipid peroxidation occurred. Thus, the
GR may play an important role in insects such as the cabbage looper and the
armyworm, in recycling GSSG to GSH, to preserve mitochondrial integrity
184
Ahmad et al.
and function and also prevent lipid peroxidation. Furthermore, in the absence of GPOX (a Se-dependent enzyme) specific isozyme(s) of glutathione
transferase (non-Se-dependent enzyme) may substitute for Se-GPOX in destroying organic hydroperoxides but not H202. At this time, the evidence for
non-Se GPOX activity is only based on work with mammalian species [40].
We are currently investigating this possibility with our insect model. As
stated earlier in the text, lipid peroxidation is very deleterious [4] because
LOOH in a chain reaction breaks down into cytotoxic arene oxides or epoxides, aldehydes, and ketones. The enzyme glutathione transferase is of wide
occurrence in insects and its activity has been shown in the armyworm [28].
As reviewed, this enzyme detoxifies epoxides by forming a glutathione
conjugate while opening the epoxide ring [28]. While glutathione transferase
is a cytosolic enzyme, another enzyme, epoxide hydrolase, present in microsomes and cytosol, readily attacks reactive epoxides to form innocuous dihydrodiols [28]. Carbonyl reductases related to the alcohol dehydrogenase
(NADP+ dependent) may be important in converting the reactive aldehyde
and ketone products of LOOH during lipid peroxidation. These enzymes are
not well studied in insects [28]. The lepidopteran Monarch butterfly Danaus
plexippus converts the cardenolide uscharidin to the reduced enantiomers
calotropin and calactin possibly with a carbonyl reductase [41]. These enzymes have thus far been of interest in the metabolism of plant allelochemicals by phytophagous insects [28], and we are now examining their role in
alleviating oxidative stress especially as arising from formation of LOOH.
The foregoing discussion leads to the hypothesis that in the armyworm
protection from 0,- and H202 is afforded by SOD with CAT working sequentially, and the very high activity and broad intracellular distribution of
CAT almost guarantees complete destruction of both 0,- and H202.A freeradical chain reaction and the formation of -OH radical thus is prevented.
The GR’s endogenous role may be in regulating the balance of GSH and
GSSG, and further minimizes the threat of deleterious lipid peroxidation.
Many other enzymes discussed above may have ancillary but, nevertheless,
important functions in destroying cytotoxic products of LOOH, in the absence of GPOX which is the enzyme for normal site-specific destruction of
LOOH in the form of an alcohol.
The GPOX-GR role and the unique feature of their subcellular distribution
deserve further comments. The subcellular distribution of GPOX and GR in
mammalian species is considered complementary to that of CAT [12]. Normally, GPOX and GR are found in the cytosol and mitochondrial matrix.
Until now no information was available on the subcellular distribution of
these enzymes in insects. The finding that GPOX is absent in the southern
armyworm, and GR is conspicuously absent from the cytosol, located instead
in microsomes and mitochondria, is on the one hand unique, but on the
other hand raises the question of their role as a secondary line of defense
congruent to the SOD and CAT enzymes. The function of GPOX and GR in
mammalian species is small relative to that of CAT at low rates of H202
production in the peroxisomal fraction, but the contribution of GPOX-GR
system increases as the H202production is enhanced. This suggests that the
accumulation of a steady-state concentration of H202 in the nmol range in
Detoxification of Oxyradicals
185
the peroxisomes is sufficient to allow diffusion of H202in the cytosol [42].
Similarly, H202 produced in the endoplasmic reticulum by enzymes such as
cytochrome P-450 or from escape of 02:, and its conversion to H202especially in insects rich in this enzyme, the rate of H202 destruction by CAT
would be expected to rise. These concepts may explain why CAT is so widely
distributed in the southern armyworm.
In summary, the subcellular fractionation study revealed the presence of
both cytosolic and mitochondria1 SOD. CAT, which acts sequentially with
SOD to destroy H202 produced by dismutation of OZT,or by direct twowas found to be very highly active and distributed
electron reduction of 302,
in the cytosol, mitochondria, and microsomes. GR activity was found in the
mitochondria and microsomes, but GPOX activity was not detected, suggesting that GPOX-GR enzymes do not work sequentially in alleviating stress
from toxic oxyradicals.
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distributions, superoxide, glutathione, dismutase, eridani, spodoptera, reductase, subcellular, catalase, southern, activities, armyworm, peroxidase
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