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Plant Physiol. (1988) 87, 1-4
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Communication
Superoxide Free Radicals Are Produced in Glyoxysomes'
Received for publication November 18, 1987 and in revised form January 13, 1988
LuISA M. SANDALIO, VICTOR M. FERNANDEZ, FRANCIsco L. RUPEREZ, AND Luis A. DEL Rio*
Unidad de Bioqufmica Vegetal, Estacion Experimental del Zaidin, C.S. I.C., Aptdo. 419, 18080 Granada,
Spain (L.M.S., L.A.R.), and Unidad de Catalisis Homogenea y Biocatalisis, Instituto de Catdlisis,
C.S.I.C., Serrano 119, 28006 Madrid, Spain (V.M.F., F.L.R.)
ABSTRACT
The production of superoxide free radicals in pellet and supernatant
fractions of glyoxysomes, specialized plant peroxisomes from watermelon
(Citruius vulgaris Schrad.) cotyledons, was investigated. Upon inhibition
of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of 02- radicals and
this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated
the generation of superoxide radicals. Superoxide production by purines
was due to xanthine oxidase, which was found predominantly in the matrix
of glyoxysomes. The generation of 02- radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.
Glyoxysomes are a class of specialized peroxisomes occurring
in oilseeds that contain the fatty acid a-oxidation and glyloxylate
cycle enzymes in addition to the characteristic peroxisomal enzymes (17). Recently, we have demonstrated the presence of
SOD2 activity (EC 1.15.1.1) in glyoxysomes from watermelon
cotyledons (25) and in peroxisomes from pea leaves (9, 26). In
watermelon glyoxysomes, two SODs were detected, a preponderant Cu,Zn-containing SOD and, in a minor amount, a MnSOD (25), whereas in leaf peroxisomes only a Mn-containing
SOD was present (26). Considering that the metalloenzyme superoxide dismutase catalyze the disproportionation of superoxide
free radicals (14), the occurrence of SODs in glyoxysomes and
leaf peroxisomes strongly suggests that the production of superoxide free radicals takes place in these oxadative cell organelles.
In cellular and subcellular systems, the generation of 02- radicals has been demonstrated in neutrophils, monocytes, and macrophages (15), mitochondria (4), chloroplasts (2), microsomes
(20), and nuclei (21). However, there is no information regarding
the production of oxygen free radicals in purified peroxisomes
either of plant or animal origin, and this is important in order
to understand the physiological role of superoxide dismutases in
cell biology. But, more interestingly, it can contribute to the
knowledge of new alternative functions of peroxisomes in cellular
metabolism related to activated oxygen species (02 , OH), a
field unexplored so far and that could have a potential impact
' Supported by grant 2511/83 C02 from the CAICYT (Spain).
2Abbreviations: SOD, superoxide dismutase; Cu,Zn-SOD, cupro-zinccontaining superoxide dismutase; DETAPAC, diethylenetriaminepentaacetic acid; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; ESR, electron
spin resonance; Mn-SOD, manganese-containing superoxide dismutase;
XOD, xanthine oxidase.
in multiple areas of cell biology ranging from biomedicine (23,
28) to plant biochemistry (17).
In this work, using chemical methods and ESR spin trapping
techniques, we describe for the first time the generation of superoxide free radicals by endogenous metabolites in membrane
and matrix fractions of glyoxysomes.
MATERIALS AND METHODS
Reagents. Cyt c (type III), DETAPAC, xanthine, hypoxanthine, XOD, allopurinol, uric acid, glycolate, glycolate oxidase,
and D-alanine were from Sigma. Superoxide dismutase, urate
oxidase, D-amino acid oxidase, NADH, NAD +, FAD, and FMN
were purchased from Boehringer Mannheim. DMPO was from
Aldrich and was purified according to Buettner (7). All other
chemicals were of the highest quality available from Merck.
Isolation of Glyoxysomes. Glyoxysomes were purified from
watermelon cotyledons (Citrullus vulgaris Schrad.) by differential and sucrose density-gradient centrifugation as described in
Ref. (25). Purified glyoxysomes were pratically free of mitochondria and proplastids as checked by using appropriate marker
enzymes and by electron microscopy analysis (25). The sucrose
concentration in the purified glyoxysomal fraction was lowered
to about 0.5 M by careful and gradual dilution with 0.1 M sucrose,
and glyoxysomes were recovered by centrifugation at 12,000g
for 15 min. For the preparation of pellet and supernatant fractions, glyoxysomes were broken by hypotonic shock in 50 mm
phosphate buffer (pH 7.8), containing 0.02 mM FAD and 0.1
mM DETAPAC, and pellet was separated by centrifugation at
151,000g for 30 min. The supernatant was concentrated by precipitation with ammonium sulfate (70% saturation) followed by
centrifugation at 27,000g for 1 h, and the pellet obtained was
gently suspended in a small volume of the same buffer.
Superoxide Determination. For the chemical determination of
02- radicals, the superoxide dismutase-inhibitable reduction of
ferricytochrome c was followed (13) using acetylated Cyt c (4).
The assays were carried out at 25°C and the reaction mixture
(1.1 ml) contained: 50 mm phosphate buffer (pH 7.8), air-saturated, 10 mM NaCl, 0.1 mM DETAPAC; 23 ,uM acetylated
ferricytochrome c; and glyoxysomal fractions (5-74 Ag protein).
The reaction was started by adding the appropriate metabolic
inducer and was followed at 550 nm versus a mixture with identical composition but containing 1 p.m Cu,Zn-SOD. As an additional control, boil-denatured Cu,Zu-SOD was also added to
parallel reactions. The endogenous SOD of glyoxysomes was
inhibited by using either 0.1 to 1 mm KCN or 10 to 40 mM NaN3.
For the assay of pellet fractions, 0.02% Triton X-100 (v/v) was
included in the reaction buffer. The amount of 02- radicals
produced was calculated as described by Asada (2) using for Cyt
c an AE550 of 19 x 103 M-l cm-1 (19).
Spin Trapping. Superoxide formation in suborganellar frac-
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2
Plant Physiol. Vol. 87, 1988
SANDALIO ET AL.
was also detected by ESR spectroscopy using the spin trap DMPO
(7). The method used was essentially that of Rosen and Rauckman (24). ESR measurements were made at room temperature
using a Bruker ER 200D-SRC X-band spectrometer. The ESR
parameters used are described in the figure legend. The reaction
mixtures (0.5 ml) were placed in a 2 mm-thick quartz flat cell
and the reaction was initiated by adding the appropriate metabolic inducer and after about 20 s the spectrum was recorded.
Controls was identical composition but containing 1 A.M Cu,ZnSOD were used. All solutions were prepared in Milli-Q ultrapure
water (Millipore, Bedford, MA). Blanks with the complete reaction mixture were run in the absence of metabolic inducers.
Although DMPO was purified according to Buettner (7), occasionally a small ESR triplet signal (a, = 17.0 G) was present
in the blanks (signals in Fig. 1A) but this did not interfere with
the ESR signals of the oxygen free radicals. The spin trap adducts
DMPO-OOH and DMPO-OH were identified as in Ref. (12) in
the presence and in the absence of 1/uM Cu,Zn-SOD.
Assays. Xanthine oxidase (EC 1.1.3.22) was determined according to Rajagopalan (22). Urate oxidase (EC 1.7.3.3) was
assayed as described in Ref. (8). Proteins were determined by
the method of Bradford (5) using BSA as the standard.
RESULTS AND DISCUSSION
The ability to induce the generation of 02- radicals by different metabolites potentially present in glyoxysomes was tested
in subglyoxysomal fractions (pellet and supernatant) from Citrullus vulgaris. The pellet contained membranes and the supernatant comprised most of the matrix protein. The metabolites
studied included xanthine, hypoxanthine, NADH, NAD +, FAD,
FMN, acetyl-CoA, D-alanine, glycolic acid, and uric acid. As
shown in Table I, the production of superoxide radicals was
detected in the soluble fraction of glyoxysomes by the action of
xanthine and hypoxanthine (about 4 nmol 02- X mg l protein
x min- ), and this generation was completely suppressed by
allopurinol, a typical inhibitor of XOD. The glyoxysomal pellet
contained all of the NADH-dependent superoxide generation
activity but little of the purine induced activity. The xanthineinduced production of 02- in glyoxysomal supernatants was also
checked by ESR. The ESR signal of the spin trap adduct DMPOOH was detected 20 s after the addition of xanthine to the assay
mixture provided that 100 A.M CN- was present to inhibit the
endogenous Cu,Zn-SOD. A typical spectrum of the O2--derived
DMPO-OH adduct is illustrated in Figure 1C. The DMPO-OH
signal was completely abolished in the presence of exogenous 1
Table I. Production of Superoxide Radicals by Pellet and Supernatant
Fractions of Glyoxysomes
The detection of superoxide radicals was carried out with acetylated
Cyt c in the presence of 0.1 mm KCN, as described in "Materials and
Methods." All concentrations stated were final concentrations in reaction
mixtures.
Metabolic Inducer
None
50 /.M xanthine
+ 40 /LM allopurinol
50 ,uM hypoxanthine
+ 40,UM allopurinol
100,uM NADH
100,uMNAD+
+ 100,M acetylCoA
a
Not determined.
Pellet
Supernatant
(nmol 02 X mg-'
protein x min 1)
0
0
0.93
4.14
0
0
NDa
4.48
ND
0
5.08
0
0.25
0
0.42
0
1006
A
B
_
A
C
D
-A & -.A.
A
TV wl-- -W
A
A
-A
%pft*,V
E
F
FIG. 1. ESR spectra of DMPO spin-adducts formed during incubation
of glyoxysomal supernatants with xanthine. All spectra were recorded
at 9.89 GHz microwave frequency, 20 mW microwave power, 100 kHz
field modulation frequency, 0.8 G modulation amplitude, 1 s time constant, and using a scan time of 250 s. The receiver gain was 1 x 1i)"
(except E and F; 4 x 105). Samples in a final volume of 0.5 ml, contained
50 mm phosphate buffer (pH 7.8), 1 mm DETAPAC, and 0.1 M DMPO.
Except for A, reactions were started by adding 50 /iM xanthine. A, 14
,tg protein; B, 14 ,ug protein plus 50 LM xanthine; C, 14 jig protein plus
100 AUM CN- plus 50 ,AM xanthine; D, 14 ,ug protein plus 100 ,UM CN
plus 1 /LM SOD plus 50 J.M xanthine; E and F, 5 ,ug pure xanthine oxidase
plus 50 ,M xanthine after 30 s and 25 min reaction, respectively. All
spectra apart from E were recorded after 25 min of reaction.
,uM Cu,Zn-SOD (Fig. 1D) that clearly indicates that the DMPOOH adduct signal is derived from the unstable species DMPOOOH which rapidly decomposes into DMPO-OH even in the
presence of DETAPAC (7, 11). It is very well established that
the adduct DMPO-OH can arise from the superoxide spin adduct
(DMPO-OOH) without the participation of OH radicals (6).
The generation of 02- radicals by xanthine confirmed those
data obtained with the Cyt c method and suggested the existence
of a soluble xanthine oxidase in the matrix of glyoxysomes. Accordingly, pellet and supernatant fractions were assayed for XOD
activity and also for urate oxidase activity, a H,O-producing
oxidase whose location inside peroxisomes is very well known
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PRODUCTION OF SUPEROXIDE RADICALS IN GLYOXYSOMES
(17). The results obtained showed the presence of XOD in glyoxysomes, predominantly in the supernatant of these organelles,
whereas pellets were practically devoid of activity (Table II.)
Xanthine dehydrogenase or its interconvertible form xanthine
oxidase have been described to be located mainly in the cytosol
(18, 27) but more recently XOD has also been localized in peioxisomes from animals (1). The results described in this paper
show for the first time that xanthine oxidase is also present in
the matrix of glyoxysomes.
Several H202-producing oxidases known to be present in peroxisomes, including urate oxidase, D-amino acid oxidase, and
glycolate oxidase, were also assayed for O2 generation by spin
trapping ESR. Under the optimal conditions of each reaction,
glycolate oxidase (15 ,ug) and urate oxidase (4,g) were found
to produce SOD-inhibitable signals of the adduct DMPO-OH
when 5 mm glycolate or 50 ,uM uric acid were added to the
reaction mixtures. In the D-amino acid oxidase reaction (10 ,ug)
the addition of 100 ,uM D-alanine did not yield any signal of
oxygen free radicals (results not shown). This suggests that in
the enzymic reactions catalyzed by glycolate oxidase and urate
oxidase, superoxide free radicals are generated either as final
products, or as catalytic intermediates in the reduction of oxygen,
as has been described for certain oxidases and oxygenases (15).
The latter possibility seems more likely considering the weak
ESR signals obtained compared to those of pure XOD, a wellknown producer of 02- radicals (13, 14). Attempts were made
to study the generation of superoxide radicals in glyoxysomal
supernatants by uric acid but this had to be abandoned because
N3-, the inhibitor that had to be used for the endogenous SOD
due to the high CN- sensitivity of urate oxidase, was found to
generate oxygen free radicals in the presence of uric acid only.
The 02- -producing NADH-dependent system of glyoxysomal
pellets is reminiscent of that of the neutrophil membrane-bound
NADPH-oxidase where several redox compounds including cytochromes are involved (3), and this is supported by the existence
of an electron transport system in glyoxysomal membranes with
certain cytochromes playing an important role (10). According
to Hicks and Donaldson (16) Cyt b5 in the glyoxysomal membrane is probably responsible for its characteristic NADH-Cyt c
reductase activity. In our experiments, we found that when NADH
was used as metabolic inducer in glyoxysomal pellets, the Cyt c
reduction was inhibited by SOD. This suggests that Cyt b5 could
perhaps use O2 as electron-acceptor with the production of 02as an intermediate which would eventually bring about the final
reduction of the exogenous acceptor Cyt c.
The production rate of 02- radicals in glyoxysomes by potentially endogenous metabolites is of a similar order of magnitude
to that described for mitochondria (4), and suggests new active
oxygen-related roles for these H202-producing organelles in cellular metabolism, like the possibility that superoxide radicals could,
under appropriate conditions, generate the vastly reactive OH
species by reaction of 02- with H202 through a metal-catalyzed
Table II. Xanthine Oxidase and Urate Oxidase Activity in Pellet and
Supernatant Fractions of Glyoxysomes
Reaction mixtures contained 2 i,g protein of glyoxysomal supernatants
and 50,ug protein of glyoxysomal pellets.
Xanthine
Urate
Oxidase
Oxidase
(nmol x mg
protein x min-)
41.8
Supernatant
27.0
+ 20 uM allopurinol
8.20
NDa
Pellet
4.92
6.56
+ 20 tM allopurinol
0
ND
aNot determined.
3
Haber-Weiss reaction (14, 15). The glyoxysomal SOD could
modulate the formation of these lethal radicals so that they could
be used for reactions of these oxidative organelles requiring strong
oxidizing agents. On the other hand, the 0- -generating capacity
of xanthine and hypoxanthine in glyoxysomal matrices could be
indicative of the involvement of active oxygen species in the
metabolism of purines at the level of glyoxysomes.
Further experiments are still necessary on the biochemical
properties of the glyoxysomal XOD, the mechanism of the NADHdependent superoxide generation, and the kinetics of 02- production. Glyoxysomes can now be added to the list of plant cell
organelles, including chloroplasts and mitochondria, in which the
generation of superoxide free radicals has been demoilstrated.
The results here reported perhaps could be extended to other
peroxisomes from different eukaryotic organisms including animals.
Acknowledgments-We are grateful to Prof. Javier Soria, Instituto de Catalisis
y Petroleoquimica, CSIC, Madrid, for access to ESR facilities.
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SANDALIO ET AL.
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