Plant Physiol. (1998) 116: 1029-1036
NADH-Monodehydroascorbate Oxidoreductase Is One of the Redox
Enzymes in Spinach Leaf Plasma Membranes1
Alajos Bérczi and
Ian M. Møller*
Institute of Biophysics, Biological Research Centre, Hungarian
Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary (A.B.); and Department of Plant Physiology, Lund University, Box 117, S-221
00 Lund, Sweden (I.M.M.)
 |
ABSTRACT |
Amino acid analysis of internal
sequences of purified NADH-hexacyanoferrate(III) oxidoreductase
(NFORase), obtained from highly purified plasma membranes (PM) of
spinach (Spinacia oleracea L.) leaves, showed 90 to
100% homology to internal amino acid sequences of
monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different
plant species. Specificity, kinetics, inhibitor sensitivity, and
cross-reactivity with anti-MDA reductase antibodies were all consistent
with this identification. The right-side-out PM vesicles were subjected
to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for
NFORase and MDA reductase activities. Similar results were obtained
when the 300 mm sucrose in the homogenization buffer and in
all steps of the salt-washing and detergent treatments had been
replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for
removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by
salt-washing and/or CHAPS treatment. The PM-associated MDA reductase
may have a role in reduction of ascorbate in both the cytosol and the
apoplast.
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INTRODUCTION |
The plant PM contains intrinsic redox activities that have been
implicated in numerous biological processes such as ion uptake, Fe3+ reduction, hormonal growth control, perception of blue
light, and defense against pathogens (Møller and Crane, 1990
;
Rubinstein and Luster, 1993; Lüthje et al., 1997). These redox
activities have been characterized with regard to their substrates and
are known as oxidoreductases (Bérczi and Asard, 1995). The
highest activity can be measured with NADH as electron donor and
HCF(III) as electron acceptor, although redox activities can also be
detected with NADPH as electron donor and quinones (e.g. duroquinone
and ubiquinone-0) or other Fe(III) complexes (e.g. Fe-EDTA and Cyt c) as electron acceptors. In addition to the
NAD(P)H-dependent oxidoreductases, a high-potential b-type
Cyt has recently been shown to be involved in trans-PM electron
transport using ascorbic acid as the electron donor (Asard et al.,
1995).
Three different kinds of PM redox activity,
classified by their acceptor specificity, were separated 10 years ago
(Luster and Buckhout, 1988). However, in spite of this and in spite of the physiological importance of these enzymes, to date only a few of
them have been purified (mostly only partially) and characterized in
plants (Bérczi and Asard, 1995). Redox enzymes that have been purified to homogeneity and characterized are a 27-kD redox protein from maize root PM (Luster and Buckhout, 1989), two distinct NAD(P)H dehydrogenases from onion root PM (Serrano et al., 1994), and a 45-kD,
FAD-containing NFORase from spinach (Spinacia oleracea) leaf
PM (Bérczi et al., 1995). A PM-bound thioredoxin has recently been identified by immunoscreening in soybean (Shi and Bhattacharyya, 1996). This protein may play a key role in the regulation of the thiol-disulfide balance of PM proteins.
The production of reactive oxygen species is a rapid response of plant
cells to pathogens and can be considered to be the most general
mechanism in the plant defense system (Mehdy et al., 1996). Even under
optimal conditions, photooxidative stress in photosynthetic cells and
many metabolic processes produce reactive oxygen species (Foyer et al.,
1994). Ascorbate plays a central role in protecting plant cells against
the action of reactive oxygen species in excess. When ascorbate
scavenges reactive oxygen species (or other free radicals), it is
univalently oxidized to the MDA radical (Heber et al., 1996). Plants
have many systems for reducing MDA to regenerate the ascorbate pool,
and one of them is MDA reductase (Heber et al., 1996; Smirnoff, 1996).
The localization of an ascorbate-regenerating system in association with the PM would be as useful to the plant cell as it is for MDA
reductase in chloroplasts (Hossain et al., 1984),
glyoxysomes/peroxisomes (Bowditch and Donaldson, 1990; Mullen and
Trelease, 1996), and mitochondria (De Leonardis et al., 1995).
In the present paper we show that the physiological electron acceptor
for the previously purified NFORase is MDA and that the enzyme is
located on the inner (cytoplasmic) surface of the PM. The importance of
the localization of an MDA reductase on the cytoplasmic surface of the
PM is also discussed.
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MATERIALS AND METHODS |
Plant Material and PM Purification
Spinach (Spinacia oleracea L.) was grown under
controlled conditions as described by Askerlund et al. (1991). PM
vesicles were isolated from 4-week-old leaves by aqueous polymer
two-phase partitioning using the batch procedure (Larsson et al.,
1987). PM vesicles were isolated both in the presence of 300 mm Suc without added monovalent salt (physiological osmotic
conditions) and in the presence of 150 mm KCl without added
Suc (physiological osmotic and ionic strength conditions) in the
homogenization medium. Purified PM vesicles were stored at 
80°C in
25 mm Mops-KOH buffer, pH 7.0, supplemented with either 300 mm Suc or with 100 mm KCl, 100 mm
Suc, and 1% (v/v) glycerol.
Enzyme Purification
Frozen PM vesicles, purified after homogenization with 300 mm Suc-containing homogenization buffer, were thawed,
diluted 10-fold with 10 mm His-HCl buffer, pH 5.8, and then pelleted by high-speed centrifugation (45-Ti rotor, Beckman)
at 100,000g at 4°C for 30 min to change the storage buffer
to the proper solubilization buffer. NFORase from the pellet was
purified as described by Bérczi et al. (1995) and will be
referred to as enzyme 1 (the CHAPS-solubilized enzyme). The supernatant
was concentrated using a pressure cell concentrator (Amicon, Beverly,
MA) with a membrane filter (YM10, Amicon), and the chromatographic
steps given by Bérczi et al. (1995) to purify NFORase were
applied to the supernatant, except that CHAPS was omitted from the
solutions used. This enzyme will be referred to as enzyme 2 (the
osmotically released enzyme).
Fractionation of the MDA Reductase Activity
A fractionation procedure was performed to localize the MDA
reductase activity in the PM preparation (Fig.
1). The storage buffer used throughout
the fractionation procedure contained either 25 mm Mops-KOH
buffer, pH 7.0, 300 mm Suc, and 1% (v/v) glycerol, or 25 mm Mops-KOH buffer, pH 7.0, 100 mm KCl, 100 mm Suc, and 1% (v/v) glycerol.
Frozen PM vesicles were thawed, diluted 10-fold with their storage
buffer, and pelleted by high-speed centrifugation at
200,000g at 4°C for 45 min to separate membrane vesicles
(both sealed and unsealed) from soluble proteins released from leaky
vesicles during thawing. The supernatant was concentrated using the
pressure cell with the membrane filter (S1 fraction). The pellet was
resuspended in the proper storage buffer (P1 fraction) and then diluted
10-fold with 25 mm Mops-KOH buffer, pH 7.0, supplemented
with 0.6 m KCl. After a 5-min incubation, membrane vesicles
were pelleted as above. The pellet was resuspended in the proper
storage buffer (P2 fraction) and supplemented with Brij 58 to obtain a
final detergent concentration of 0.05% (w/v) and a detergent-to-lipid
ratio of about 1:1 (w/w) in the suspension. The supernatant (S2
fraction) was concentrated as above.
The Brij-58 treatment converts right-side-out PM vesicles into
inside-out vesicles (Johansson et al., 1995). After a 5-min incubation,
the Brij 58-treated vesicles were pelleted and the supernatant (S3
fraction) was concentrated as above. The pellet was resuspended in the
proper storage buffer (P3 fraction) and then diluted 10-fold with 25 mm Mops-KOH buffer, pH 7.0, supplemented with 0.6 m KCl. After a 5-min incubation, the salt-washed membrane vesicles were pelleted and the supernatant (S4 fraction) was
concentrated as described above. The pellet was resuspended in the
proper storage buffer supplemented with Brij 58 to a detergent
concentration of 0.05% (w/v) (P4 fraction) and then diluted 5-fold
with 10 mm His-HCl buffer, pH 5.8. This suspension was
combined with the same volume of 10 mm His-HCl, pH 5.8, 2 mm EDTA, 2 mm EGTA, and 30 mm CHAPS
(the final CHAPS-to-protein ratio was about 15:1 [w/w]). After a
15-min incubation at room temperature, the unsolubilized membrane
proteins were pelleted as described above, the supernatant was again
concentrated (S5 fraction), and the pellet was resuspended in 25 mm Mops-KOH buffer, pH 7.0, supplemented with 1% (v/v)
glycerol (P5 fraction). Redox activities were determined as soon as the fractions were obtained (i.e. before freezing and storing them at
80°C), and the protein content of the fractions was determined on
the next day.
Enzyme Assays and Protein Determination
NAD(P)H-utilizing redox activity with HCF(III) and MDA was
measured as given by Bérczi et al. (1991) and Hossain et al.
(1984), respectively. MDA was generated by enzymatic oxidation of
ascorbic acid as given by Hossain et al. (1984). The MDA
concentration was determined by measuring the
A360 according to the method of Bielski et
al. (1971). Protein concentration was estimated according to the
method of Markwell et al. (1978) using BSA as a standard.
Electrophoresis and Immunoblotting
Samples for SDS-PAGE separation and enhanced chemiluminescence
visualization of MDA reductase were prepared by the method of Wessel
and Flügge (1984), which uses an organic solvent method for
precipitating soluble as well as hydrophobic proteins. This was done to
remove lipids, detergents, and salts from the different samples and to
permit the loading of equal MDA reductase activity from each fraction.
SDS-PAGE was run on a 10 to 15% gradient gel in the buffer system of
Laemmli (1970) with a Protean TM II apparatus (Bio-Rad). The proteins
were electroblotted onto cellulose nitrate membrane (0.45 µm, BA85,
Schleicher & Schuell), reacted with polyclonal antibody raised against
the MDA reductase, which was purified from cucumber fruits (a kind gift
from Prof. K. Asada, Kyoto University, Uji, Kyoto, Japan), and
visualized by the enhanced chemiluminescence method according to the
manufacturer's instructions (Amersham). The primary and secondary
antibodies were diluted 1:3000 and 1:5000, respectively.
Amino Acid Sequencing
The purified NFORase from both the CHAPS-solubilized (enzyme 1)
and the osmotically released (enzyme 2) protein mixture was separated
on gradient SDS-polyacrylamide gel. The 45-kD bands were cut out and
sequenced by Dr. Bo Ek (Department of Cell Research, Swedish University
of Agricultural Sciences, Uppsala, Sweden). For internal sequencing,
the gel pieces were incubated with LysC from Achromobacter
lyticus (Waco, Osaka, Japan) essentially as described by Rosenfeld
et al. (1992). The eluted peptides were separated using a SMART
chromatography station equipped with a µRPC SC C2/C18 2.1/10 column
(Pharmacia). Amino acid sequencing was performed on a sequencer
according to the manufacturer's instructions (model 476A, Applied
Biosystems/Perkin-Elmer).
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RESULTS |
Identification of the NFORase as MDA Reductase
The NFORase purified and characterized from spinach leaf PM by
Bérczi et al. (1995) was obtained from frozen/thawed PM that were
diluted in a low-salt medium and pelleted before the solubilization and
purification of the membrane-associated NFORase. About one-half of the
NFORase activity found in the frozen PM was released into the
supernatant by this procedure and was discarded. However, we decided to
continue the search for the natural substrate for this enzyme and
therefore purified the NFORase both from the pellet (enzyme 1) and from
the supernatant (enzyme 2) using essentially the same purification
procedure, except that CHAPS was omitted in all steps involving enzyme
2. No significant difference was observed between the two enzymes
either during the purification or in their catalytic properties. They
had similar substrate specificity, specific activity, inhibitor
sensitivity (not shown), and apparent molecular size on SDS-PAGE.
N-Terminal sequencing of the two enzymes failed, but after LysC
digestion and HPLC separation, two internal sequences were analyzed for
each of the enzymes. One of the sequences was identical in both
preparations (Fig. 2). Both this common
sequence and the two other sequences were identical or very similar
(more than 90% identical) to amino acid sequences of MDA reductase
obtained from pea (Pisum sativum) (Murthy and Zilinskas,
1994), cucumber (Cucumis sativus) (Sano and Asada, 1994), or
tomato (Lycopersicon esculentum) (Grantz et al., 1995). MDA
reductase is an FAD-containing enzyme of 47 kD (Sano and Asada, 1994),
which is consistent with the results of Bérczi et al. (1995).
Thus, it is highly probable that both enzymes 1 and 2 are MDA reductase
and that the physiological substrate of the PM NFORase purified by
Bérczi et al. (1995) is MDA.
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| Figure 2.
Internal amino acid (aa) sequences of the NFORase
purified from spinach leaf PM vesicles. Frozen PM vesicles were thawed
and diluted with buffer with low ionic strength and low osmotic
potential and then pelleted by centrifugation. Enzyme 1 was purified
from the pellet after CHAPS solubilization, and enzyme 2 was purified from the supernatant containing proteins released by the
freezing-thawing procedure and hypo-osmotic shock. Search for
homologous internal sequences in the Swiss-Prot databank identified
highly homologous sequences in monodehydroascorbate radical reductases
in three different species. Numbers on the right are the amino acid
positions of the highly homologous sequences in the known MDA
reductases. Bold letters indicate identity.
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When the MDA reductase activity of the PM and purified enzyme 1 was
tested, it showed the same donor specificity and almost the same degree
of purification as the NFORase activity (Table I). The effect of three inhibitors of
NFORase activity, mersalyl, PCMB, and dicumarol, was very similar with
MDA as electron acceptor when both the PM and the purified enzyme
(enzyme 1) were tested (Table II; also in
Bérczi et al., 1995). The following
Km values were obtained by varying the MDA,
NADH, or NADPH concentrations (Fig. 3):
Km(MDA) = 2.2 ± 0.3 µm
(with 50 µm NADH, n = 3);
Km(NADH) = 2.5 ± 0.8 µm; and Km(NADPH) = 13.2 ± 3.6 µm (with 2.0 ± 0.2 µm MDA and n = 3 in both cases). These
values are in good agreement with earlier results published for other
MDA reductases (Hossain et al., 1984; Hossain and Asada, 1985;
Borraccino et al., 1986, 1989; Bowditch and Donaldson, 1990).
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| Figure 3.
Redox activity of the NFORase (enzyme 1) when the
concentration of MDA ( ), NADH ( ), and NADPH ( ) was varied and
the concentrations of NADH and MDA, respectively, were held constant.
Results presented are from one enzyme preparation; two other
preparations gave similar results (for Km
values, see ``Results'').
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Fractionation of the Redox Activity in the PM Vesicles
A very detailed fractionation procedure was carried out for
two reasons: (a) Since MDA reductase is found in the cytoplasm, we
wanted to ascertain whether the activity in our PM vesicles could be
due to an artifactual association of cytoplasmic MDA reductase with the
vesicles. (b) If MDA reductase is strongly associated with the PM, we
wanted to know its orientation, i.e. whether it is associated with the
outer, apoplastic surface or with the inner, cytoplasmic surface.
Traditionally, plant tissues are homogenized in the presence of a 0.3 to 0.5 m concentration of an osmoticum such as Suc or mannitol to avoid rupturing organelles such as mitochondria and chloroplasts. Therefore, our standard PM preparation included 0.3 m Suc in all media. However, the cytosol contains salts
rather than sugars as osmotica, and at a much lower ionic strength in the homogenization medium there is a risk that soluble proteins adhere
electrostatically and unspecifically to membranes, thereby creating
artifactual associations. To avoid this, we also purified our PM
vesicles in the presence of 150 mm KCl instead of 300 mm Suc.
The frozen/thawed PM vesicles, purified with Suc or KCl, were both
right-side-out and tightly sealed, since the latency of the NFORase was
more than 80% (Table III). The two kinds
of PM preparation contained the same specific activity of both NFORase and MDA reductase (Table IV).
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Table III.
Latency of NFORase and MDA reductase activities in
the pellets of the fractionation experiments
PM vesicles were purified in the presence of either 300 mm
Suc or 150 mm KCl (see ``Materials and Methods''). The
origin of the fractions is explained in Figure 1. Latency of enzyme
activity was measured with Triton X-100 (± 0.015% [w/v]) and
calculated as a percentage (Larsson et al., 1984). Values are averages
of three independent series of PM preparations both with Suc and with
KCl.
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Table IV.
Fractionation of the NFORase and MDA reductase
activity of spinach leaf PM vesicles
PM vesicles were purified in the presence of either 300 mm
Suc or 150 mm KCl (see ``Materials and Methods''). The
origin of the fractions is explained in Figure 1. Values are the
averages of three independent series of PM preparations both with Suc
and with KCl and are given as percentages of the PM (100%).
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Very little protein, NFORase, and some MDA reductase activity were
released into the supernatant (S1; Table IV) when these PM vesicles
were diluted with an iso-osmotic medium. This is different from results
reported previously (Bérczi et al., 1995), in which the dilution
was with a hypo-osmotic medium known to cause rupture and resealing of
the vesicles (Bérczi and Møller, 1986) and to lead to the loss
of some enzymatic activity from the vesicle lumen (S1; Table IV).
When the right-side-out vesicles were washed with 0.6 m
KCl, which will release loosely attached proteins, little protein or
redox activity was released (S2; Table IV). The majority of the NFORase
activity and MDA activity was inside the permeability barrier of the
right-side-out vesicles at this point.
The addition of Brij 58 converts sealed right-side-out PM vesicles into
sealed inside-out vesicles (Johansson et al., 1995). This would release
any proteins merely enclosed within the right-side-out vesicles but not
proteins firmly attached to their inner surface. With our vesicles,
Brij-58 treatment caused the release of 17 to 31% of the NFORase and
30 to 54% of the MDA activity (S3; Table IV). The percentage released
from the KCl-prepared PM was higher, showing that more enzyme activity
was bound to the cytoplasmic side of the PM in the Suc-prepared
vesicles and was not released by the inversion. Consistent with the
results of Johansson et al. (1995), the latency in the pellet after
Brij treatment (P3; Table III) was very low for both NFORase and MDA
reductase. This indicates that the residual enzyme activity (about
one-third to one-half) was bound to the cytoplasmic surface of the PM
vesicles now exposed to the medium.
Washing these inside-out PM vesicles with 0.6 m KCl
released a substantial part of the residual activity (S4; Table IV),
and washing them with 1.0 m KCl released virtually all of
the MDA activity (results not shown). After the 0.6 m KCl
treatment, a further gentle solubilization with CHAPS removed the
remaining MDA reductase activity, and left 10 to 15% of the NFORase
activity and about one-half of the protein in the final pellet (S5, P5; Table IV).
The recovery of protein and MDA reductase was 90 to 110%, and the
recovery of NFORase was approximately 70% in this multistep fractionation procedure (Table IV), indicating that all of the enzyme activities were accounted for.
Samples from the various subfractions containing the same amount of MDA
reductase activity were separated by SDS-PAGE, blotted, and
immunoreacted with antibodies raised against cytosolic MDA reductase
from cucumber (Fig. 4). Only a band at 45 kD reacted with the antibodies and was seen in all fractions. The broad
band at high molecular mass in fractions S5 (Fig. 4, lanes F and O) is
artifactual, since (a) it was not seen in the preceding fractions and
(b) hardly any protein was seen at that position after silver or
Coomassie blue staining. The band at 45 kD, and, thus, the antigenicity
per activity unit, was stronger in fractions S1 and S3 (Fig. 4, lanes
B, D, K, and M), where enzyme 2 is recovered, than in fractions S4 and
S5 (Fig. 4, lanes E, F, N, and O), which contain enzyme 1, the bound
form.
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| Figure 4.
Immunoblotting of the PM and supernatant fractions
of the MDA reductase activity fractionation experiment using anti-MDA
reductase antibodies. Lanes A to F, Fractions from PM preparation with
Suc in the homogenization buffer; lanes J to O, fractions from PM preparation with KCl in the homogenization buffer. A and J, PM; B and
K, S1 fraction; C and L, S2 fraction; D and M, S3 fraction; E and N, S4
fraction; F and O, S5 fraction; G and H, Mono-Q fraction Q27 and
affinity fraction A40 (Bérczi et al., 1995); and I, standard proteins. About the same total MDA reductase activity (approximately 5 nmol min 1) was applied in each lane (except lane I). The
positions of standard proteins of known molecular mass are shown on the
left. All fractions were from the same experiment. Three independent
fractionation experiments gave similar results. The high-molecular-mass
band in lanes F and O is an artifact probably caused by CHAPS.
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DISCUSSION |
The 45-kD FAD-containing NFORase from spinach leaf PM
(Bérczi et al., 1995) is here identified as MDA reductase
based on a high degree of sequence identity (Fig. 1), substrate
specificity (Table I), inhibitor sensitivity (Table II), kinetic
constants (Fig. 3), and immunological cross-reactivity (Fig. 4).
Very little MDA reductase was associated with the outer, apoplastic
surface of the PM, as shown by the high latency of MDA reductase in P1
and the low MDA activity in the first two supernatants (S1 and S2,
Tables III and IV). Between one-third and one-half of the MDA activity
seemed to be soluble inside the vesicles or weakly bound to the inner,
cytoplasmic surface, since it was released by Brij-58 treatment (S3,
Table IV). This would be equivalent to enzyme 2 as described in
``Materials and Methods''.
However, even when the PM vesicles had been isolated under high-salt
conditions (150 mm KCl), approximately 40% of the MDA reductase activity remained bound to the PM vesicles after Brij-58 treatment (P3 = S4 plus S5; Table IV). A wash with 0.6 m KCl removed only part of this activity (Table IV); a wash
with 1.0 m KCl (not shown) or CHAPS treatment was necessary
to remove the last 20% (S5, Table IV). These observations indicate
that a large part of the MDA reductase, copurifying with spinach leaf
PM (enzyme 1; see ``Materials and Methods''), was tightly bound by
mainly electrostatic forces to the inner cytoplasmic surface. It is
unlikely that the enzyme has membrane-spanning helices or is anchored
to the PM via a fatty acid, as was reported for a subpopulation of
nitrate reductase (Stöhr et al., 1995), but there may well be a
docking protein in the PM. The presence of MDA reductase (often called
ascorbate free radical reductase in the literature) activity in the
plant PM preparations confirms previous reports (Morré et al.,
1986; Luster and Buckhout, 1988; Gonzalez-Reyes et al., 1992), but in none of these studies was it shown how or where MDA reductase was
associated with the membrane.
Apart from the difference in the binding to the PM, we have detected
only one difference between enzyme 2, the soluble form of MDA reductase
released from the lumen of the PM vesicles by hypo-osmotic rupture, and
enzyme 1, the form bound to the PM surface. This difference relates to
the immuno-cross-reactivity (Fig. 4): enzyme 2 (S1 and S3) reacted
stronger than enzyme 1 (fractions S4 and S5), although the same MDA
reductase activity had been loaded into all lanes. However, this
difference could be an artifact caused by the sample preparation method
(see ``Materials and Methods'') and caution should be shown in
the interpretation. With our present knowledge it is not possible to
determine whether enzymes 1 and 2 are encoded by different genes or
whether the two populations are formed by posttranslational
modification of one MDA reductase gene product.
The Km(NADH) for the purified MDA reductase
with MDA as electron acceptor was 2.5 µm (Fig. 3;
``Results''), whereas it has been reported to be 25 to 77 µm for the enzyme in PM vesicles from various sources with ferricyanide as acceptor (Askerlund et al., 1988; Møller and
Crane, 1990, and refs. therein). This large difference may partly be
due to the difference in electron acceptor or in species. However, it
may also be a product of electrostatic interactions: Both the outer and
inner surface of the plant PM has a pI below pH 4.0 (Larsson et al.,
1990, and refs. therein). This means that the net charge is negative at
neutral pH, giving rise to a negative surface potential. This will
repel the likewise negatively charged NADH (Edman et al., 1985, and
refs. therein). Thus, the concentration of NADH at the active site
would be less than that in the bulk solution, leading to an
overestimation of the Km when the enzyme is
attached to the PM surface. On the other hand, Askerlund et al. (1988)
determined Km(NADH) in the presence of 7.5 mm Mg2+, which will reduce the size
of the surface potential to near 0, so at least in that case the
electrostatic component was probably small.
The majority of the NFORase activity in spinach leaf PM is caused by
MDA reductase. However, there was always a significant part (10-15%)
of the NFORase activity left in the PM after the CHAPS treatment (P5),
which removed the last MDA reductase (Table IV). This indicates that
the spinach leaf PM contains at least one more redox enzyme for which
the natural substrates are still unknown, in addition to one or two
b-type Cyts (Askerlund et al., 1989; Møller et al., 1991).
Dicotyledons and nongrass monocotyledons reduce
Fe3+ to Fe2+ before the
uptake of Fe2+. This reduction is probably
carried out by a transmembrane reductase in the PM of root cells
(Møller and Crane, 1990; Lüthje et al., 1997). Once
Fe2+ has been taken up, it is reoxidized to
Fe3+, which is transported up to the shoot, where
it is thought to require another reduction to
Fe2+ before it can be taken up into the leaf
cells. The PM of leaf cells would therefore also be expected to contain
an Fe-reducing enzyme, although perhaps at a fairly low level. It is
possible that the residual NFORase activity in spinach leaf PM after
salt-washing and mild-detergent treatment is the Fe-reducing enzyme.
Experiments are in progress to investigate this possibility.
MDA reductase has been found in both particulate and soluble
(cytosolic?) fractions (Arrigoni et al., 1981; Borraccino et al.,
1986), in spinach chloroplasts (Hossain et al., 1984), potato tubers
(Borraccino et al., 1986), cucumber fruit (Hossain and Asada, 1985;
Sano et al., 1995), leaves of various plants (Heber et al., 1996),
soybean root nodules (Dalton et al., 1992), and castor bean endosperm
glyoxysomes (Bowditch and Donaldson, 1990; Mullen and Trelease, 1996).
In glyoxysomes the enzyme was located in the outer, cytoplasmic surface
of the membrane, where it was suggested to be part of a transmembrane
transfer of reducing equivalents very similar to that depicted in
Figure 5 (Bowditch and Donaldson, 1990;
Mullen and Trelease, 1996). It is quite possible that MDA reductase is
associated with the surface of other intracellular membranes. This
would explain why NFORase activity can be measured in many purified
membrane fractions, including the ER and the tonoplast (Fredlund et
al., 1994). It would also explain why antibodies raised against a 44-kD
NFORase from potato tuber microsomes (Galle et al., 1984) cross-react
with one or several polypeptides of 45 to 55 kD in the same membrane
fractions (Fredlund et al., 1994). Perhaps Galle et al. (1984) isolated
MDA reductase from these membranes rather than the NADH-Cyt
b5 reductase they reported. The latter is
smaller: 36 kD in erythrocyte membranes (Kitajima et al., 1981), 34.5 kD in pea microsomes (Jollie et al., 1987), and 32 kD in rat liver PM
(Kim et al., 1995).
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| Figure 5.
Schematic representation of the location of
PM-bound MDA reductase and of the functional interaction between the
trans-PM b-type Cyt and the PM-bound MDA reductase.
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There are at least two known membrane processes in which MDA
reductase placed on the inner surface of the PM can be useful. Ascorbate has a key role in scavenging oxidative radicals because of
its ability to reduce tocopheryl free radicals appearing when tocopherol quenches lipid hydroperoxyl radicals (Packer et al., 1979;
Scarpa et al., 1984). Also, there is a high-potential b-type Cyt present in the plant PM (Askerlund et al., 1989; Møller et al.,
1991) that transports electrons from ascorbate in the cytoplasm to MDA
in the apoplast (Horemans et al., 1994; Asard et al., 1995). In both
cases, ascorbate is oxidized to MDA and the presence of an MDA
reductase in situ would facilitate the recovery of the ascorbate pool
in the vicinity of the membrane surface. In the latter case, apoplastic
ascorbate, which has important functions in cell wall synthesis and in
host-pathogen interactions (Smirnoff, 1996), can be kept reduced as
illustrated in Figure 5. It would be interesting to see whether
pathogen infection or oxidative stress changes the degree to which MDA
reductase associates with the PM. The study plants with a changed
expression of MDA reductase (e.g. by transformation with antisense RNA)
with respect to resistance to pathogens and oxidative stress would also
be illustrative.
| |
FOOTNOTES |
1
This work was supported by grants to A.B. from
the Hungarian National Science Foundation (nos. OTKA T012747 and
T019863), the Hungarian Academy of Sciences, the Swedish Royal Academy
of Sciences, and Kungliga Fysiografiska Saellskapet and to I.M.M. from
the Swedish Natural Science Research Council and the Wenner-Gren Stiftelserna.
*
Corresponding author; e-mail ian_max.moller{at}fysbot.lu.se; fax
46-46-222-4113.
Received June 26, 1997;
accepted November 13, 1997.
| |
ABBREVIATIONS |
Abbreviations:
Brij 58, polyoxyethylene 20 dodecylether or
C12E20.
CHAPS, 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane
sulfonate.
dicumarol, 3,3
-methylene-bis-(4-hydroxy-coumarin).
HCF(III), potassium hexacyanoferrate (III) or ferricyanide.
MDA, monodehydroascorbate radical.
NFORase, NADH-HCF(III) oxidoreductase.
PCMB, p-chloromercurobenzoate.
PM, plasma membrane(s).
| |
ACKNOWLEDGMENTS |
We are grateful to Prof. Christer Larsson, Dr. Kenneth M. Fredlund, and Dr. Per Askerlund for practical advice and many helpful discussions, to Dr. Miguel A. Quinones for comments about the manuscript, and to Mrs. Lena Carlsson and Mrs. Christina Nilsson for
excellent technical assistance.
| |
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Abstract
Introduction
