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Plant Physiol. (1999) 119: 1305-1314
Identification, Separation, and Characterization of
Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The
existence in higher plants of an additional 
-oxidation system in
mitochondria, besides the well-characterized peroxisomal system, is
often considered controversial. Unequivocal demonstration of
-oxidation activity in mitochondria should rely on identification of
the enzymes specific to mitochondrial -oxidation. Acyl-coenzyme A
dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified
mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus
annuus L.) seeds, using as the enzyme assay the reduction of
2,6-dichlorophenolindophenol, with phenazine methosulfate as the
intermediate electron carrier. Subcellular fractionation showed that
this ACAD activity was associated with mitochondrial fractions.
Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase
activity in peroxisomes showed differences of substrate specificities.
Embryonic axes of sunflower seeds were used as starting material for
the purification of ACADs. Two distinct ACADs, with medium-chain and
long-chain substrate specificities, respectively, were separated by
their chromatographic behavior, which was similar to that of mammalian
ACADs. The characterization of these ACADs is discussed in relation to
the identification of expressed sequenced tags corresponding to ACADs
in cDNA sequence analysis projects and with the potential roles of
mitochondrial -oxidation in higher plants.
All of the tissues of higher plants, even nonfatty and
nonsenescent, appear to possess the capacity for fatty acid
We obtained direct evidence of acetyl-CoA production from substrate
fatty acids by mitochondria purified from carbohydrate-starved maize
(Zea mays L.) root tips (Dieuaide et al., 1993 The first step of Here we present the characterization of the substrate specificity of
ACAD activity in purified mitochondria and in partially purified
preparations of ACAD from maize root tips and from embryos of
early-germinating sunflower seeds. The existence of true ACAD activities that are distinct from the well-described ACOX activity is
thus confirmed. Furthermore, the typical activities that have been
described in animal cells, with straight short-chain, medium-chain, and
long-chain substrates, and with branched-chain substrates, such as
isovaleryl-CoA, are shown to exist in higher plants. The partial
purification of distinct ACAD of the medium-chain and long-chain types
(Ikeda et al., 1983 Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-oxidation (Gerhardt, 1983
, 1985; Kindl, 1987). This catabolism
in higher plants proceeds primarily by peroxisomal -oxidation
(Cooper and Beevers, 1969a, 1969b; Gerhardt, 1985), in contrast with
mammalian tissues where -oxidation takes place in both peroxisomes
and mitochondria. Thus, the existence in higher plants of an additional mitochondrial -oxidation system is often considered controversial (Gerhardt et al., 1995; Hoppe and Theimer, 1997). It is well
established that the massive degradation of fatty acids during early
growth of fatty seeds proceeds through glyoxysomal -oxidation
(Cooper and Beevers, 1969a, 1969b; Hoppe and Theimer, 1997). However, studies of the catabolism of branched-chain amino acids, in which the
isobutyryl-CoA, 2-methyl-butyryl-CoA, and isovaleryl-CoA catabolites of
Val, Ile, and Leu, respectively, undergo -oxidation, have led
Gerbling and Gerhardt (1989) to hypothesize the existence of
extra-peroxisomal -oxidation for Leu and Val degradation. The
localization of -methyl-crotonyl-CoA carboxylase, which catalyzes a
subsequent step of Leu catabolism, in mitochondria of sycamore cells
(Aubert et al., 1996) may be an indication of the mitochondrial location of this extra-peroxisomal -oxidation. Furthermore,
mitochondria from pea cotyledons (Wood et al., 1986) were shown to
contain at least some of the enzymes of -oxidation. In the case of
enoyl-CoA hydratase, an isoenzyme immunologically distinct from the
peroxisomal enzyme was partially purified from mitochondria
(Miernyk et al., 1991). However, the existence of enzymes that are
known to be specific to mitochondrial -oxidation in mammalian
tissues remains to be fully demonstrated in higher plants (Hoppe and
Theimer, 1997).
).
The inhibition of this acetyl-CoA production by respiratory-chain
inhibitors further showed that, like in mammalian cells, mitochondrial
-oxidation in higher plants was dependent on the respiratory chain.
In this case, mitochondrial -oxidation, in contrast with peroxisomal activity, was found to be strictly dependent on carbohydrate starvation (Dieuaide et al., 1993). However, Gerhardt et al. (1995) showed that
pea cotyledon mitochondria could catalyze the formation of acid-soluble
[14C] products from
[1-14C]palmitoyl-L-carnitine.
This activity was significant and showed inhibition by cyanide, thus
indicating a limited level of mitochondrial fatty acid -oxidation in
nonfatty and nonstarved plant tissues. Moreover, Salon (1988) has shown
that the oxidation of hexanoate in early-germinating embryos of lettuce
(Salon et al., 1988) was inhibited by mercaptopropionate, which is an
inhibitor of mitochondrial -oxidation in mammals (Sabbagh et al.,
1985). Early-germinating embryos of sunflower (Helianthus
annuus L.) seeds appeared to have a similar fatty acid metabolism
(Salon, 1988).
-oxidation consists of the desaturation of
acyl-CoA to 2-trans-enoyl-CoA. In animal tissues this is
catalyzed by mitochondrial ACAD, transferring electrons to an
electron-transferring flavoprotein, which feeds reducing equivalents to
the respiratory chain (Engel, 1992), and by peroxisomal ACOX, the
flavin moiety of which is reoxidized directly by
O2 (Osmundsen et al., 1991). Inhibition of
mitochondrial -oxidation by respiratory-chain inhibitors (Dieuaide
et al., 1993; Gerhardt et al., 1995) thus strongly suggested that
higher-plant mitochondria possessed ACAD activity, which was
identified in mitochondria from carbohydrate-starved maize root
tips (Dieuaide et al., 1993). The peroxisomal ACOX activity of higher
plants was described more than 25 years ago (Cooper and Beevers, 1969a,
1969b). Long-chain ACOX from cucumber (Kirsch et al., 1986) and pumpkin
(Hayashi et al., 1998) have been characterized, and we showed that
higher plants also possess distinct short-chain and medium-chain ACOX
(Hooks et al., 1996), which appear to be differentially expressed,
depending on developmental and metabolic status (Eccleston et al.,
1995; Hooks et al., 1995). In animal systems the ACOX- and
ACAD-catalyzed steps exert strong control on the discrimination of
substrates and on the overall flux of -oxidation (Aoyama et al.,
1994a, 1994b). Furthermore, in mammalian tissues the comparison of ACOX
(Vanhove et al., 1993b) and ACAD (Nagao and Tanaka, 1992) has greatly
clarified the respective functions of peroxisomal and mitochondrial
-oxidation. This is why the systematic study of higher-plant ACOX
and ACAD is likely to provide new information concerning the
functions of peroxisomal -oxidation and to determine the functions
of mitochondrial -oxidation.
, 1985) further demonstrates that these ACAD
activities are due to a family of enzymes.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
. Three-millimeter-long tips of seminal roots were excised and
either immediately used for the preparation of organelles or incubated for carbohydrate starvation treatment. In this latter case, the excised
root tips were incubated at 25°C in the mineral nutrient medium
supplemented with 1% (v/v) of the antibiotic and antimycotic mixture
A7292 from Sigma and 0.1 M Mes-KOH, pH 6.0. A gas
mixture containing 50% (v/v) O2 and 50% (v/v)
N2 was continuously bubbled through the
incubation medium to maintain a partial O2
pressure above 35 kPa, which is the critical O2
pressure for maize roots in aqueous solutions (Saglio et al., 1984).
Sunflower (Helianthus annuus L. cv Frankasol) seeds were
obtained from the Centre Technique Interprofessionnel des
Oléagineux Métropolitains (Paris, France). Seeds were
soaked in sterile distilled water for 6 h at 25°C in the dark.
At this stage the seminal root did not emerge from the testa. Embryonic
axes were excised from early-germinating seeds and either immediately
used for organelle separation or frozen and stored at 
80°C until
protein extraction.
Preparation of Organellar Fractions from Maize Root Tips and from Embryonic Axes of Early-Germinating Sunflower Seeds
The isolation of low- and high-density mitochondria from freshly excised maize root tips or from maize root tips that had been subjected to 48 h of carbohydrate starvation treatment was carried out by differential and Percoll (Pharmacia) gradient isopyknic centrifugations as previously described (Couée et al., 1992). In the present work
only the mitochondria of high density, which are more differentiated,
with many cristae and a dense matrix, were used. Mitochondrial
preparations from nonstarved and carbohydrate-starved tissues were
resuspended at final protein concentrations of 6 ± 2 and 14 ± 3 mg mL
1, respectively (means ± SE for at least five separate experiments), in 0.1 mL of 10 mM KH2PO4-KOH
buffer, pH 7.2, containing 1 mM sodium EDTA, 300 mM mannitol, and 0.1% (w/v) fatty acid-free BSA. The
latency of matrix enzyme markers was 95%, thus showing the integrity
of the purified mitochondria. Peroxisome-enriched fractions were
obtained by centrifugation of a crude cellular extract on a one-step
(35% and 60%, w/w) Suc gradient, as described by Dieuaide et al.
(1992). Both peroxisomes and mitochondria were enriched in this
preparation, with a 2.5-fold enrichment factor. Separation of
organelles from the embryonic axes of early-germinating sunflower seeds
was carried out by a modification of the protocol of Attucci et al.
(1991). Differential centrifugations consisted of a low-speed centrifugation at 120g for 15 min, which was followed by a
medium-speed centrifugation at 12,000g for 20 min, thus
yielding a crude organellar preparation. The main modification
consisted of using a 20% (v/v), instead of 12% (Attucci et al.,
1991), Percoll gradient for the subsequent isopyknic centrifugation of
this crude organellar fraction. The resulting mitochondrial and
peroxisomal preparations were diluted in 10 mM
KH2PO4-KOH buffer, pH 7.2, containing 1 mM sodium EDTA, 700 mM sorbitol, and 1% (w/v) fatty acid-free BSA,
and then centrifuged at 12,000g for 20 min to eliminate
Percoll. The pellets were finally resuspended in 0.1 mL of the same
medium.
Analysis of Proteins
Protein was determined by the method of Bradford (1976) using
-globulin (Calbiochem) as the standard. SDS-PAGE analysis of protein
was carried out in an electrophoresis unit (Mighty Small II, Hoefer,
San Francisco, CA), essentially as described by O'Farrell (1975).
Enzyme Activities
All enzyme activities were assayed spectrophotometrically at 30°C, unless otherwise specified, according to previously published methods. All assays were first performed on blanks containing all of the constituents of the assay except the substrate, which was added to initiate the reaction. Activities were linear with respect to time for at least 2 min and were proportional to the amounts of sample protein added to the assay. The activities of fumarase (EC 4.2.1.2) and catalase (EC 1.11.1.6) were assayed as described by Hill and Bradshaw (1969) and Aebi (1987), respectively. The activity of Glc-6-P
dehydrogenase-6-phosphogluconate dehydrogenase was assayed as described
by Brouquisse et al. (1991). ACOX (EC 1.3.3.6) was assayed as described
by Gerhardt (1987). ACAD (EC 1.3.99.2,3) activity was assayed in terms
of the reduction of DCPIP as an electron acceptor and PMS as an
intermediate electron carrier (Izai et al., 1992). The decrease in
A600 was followed in a reaction mixture
containing 50 mM Hepes-KOH buffer, pH 8.0, 1 mM KCN, 1 mM
salicylhydroxamic acid, 50 µM FAD, 100 µg
mL1 DCPIP, 100 µg mL1
PMS, the enzyme sample, and 50 µM acyl-CoA
substrate in a final volume of 1.1 mL. The reaction was started by the
addition of the acyl-CoA substrate. Blanks in the absence of enzyme
sample were carried out to assess the rate of reduction by
contaminating CoA-SH. This activity was assayed at 25°C to minimize
the background rate. Purified ACOX from maize plantlets showed no
apparent activity of DCPIP reduction (Hooks et al., 1996), thus
confirming the specificity of the assay. The activity of acyl-CoA
thioesterase (EC 3.1.2.2) was assayed in the presence of 0.15 mM 5,5
-dithiobis-(2-nitrobenzoate) under the
same conditions, except that DCPIP and PMS were omitted, by following
the appearance of 2-nitro-5-thiobenzoate at 412 nm. The extinction
coefficient of 2-nitro-5-thiobenzoate was taken to be 14,150 M1
cm1 (Riddles et al., 1983). This activity was
also assayed at 25°C, which served as a control of the PMS-DCPIP
assay.
Partial Purification of ACAD
Proteins from carbohydrate-starved maize root tips, maize whole seminal roots, and embryonic axes or cotyledons from early-germinating sunflower seeds were extracted by homogenization in a Waring blender in 10 mM KH2PO4-KOH buffer, pH 7.5, containing 0.1% (w/v) Triton X-100, 0.2% (w/v) polyvinylpolypyrrolidone, 5 mM Cys, 0.5 mM EDTA, and 0.1 mM PMSF. Particulate material was eliminated by squeezing the sample through cheesecloth and subsequent centrifugation at 5,000g for 30 min. Sunflower extracts were further centrifuged at 5,000g for 10 min to remove the superficial lipid layer. For purification, these crude extracts were sequentially fractionated at different saturations of ammonium sulfate. Fractionation was performed by adding ammonium sulfate over a period of 30 min to ice-cold protein extracts at a concentration of approximately 10 mg mL1. Solutions were stirred for another
30 min and then centrifuged at 12,000g for 15 min. The
different precipitated fractions were then assayed for ACAD activity.
The 40% to 60% ammonium sulfate fraction from embryonic axes of
early-germinating sunflower seeds was purified further according to the
method of Ikeda et al. (1983), which was developed for the separation
of the different ACAD from rat liver.
1 with a
linear gradient of NaCl from 0 to 600 mM in 10 mM KH2PO4-KOH buffer, pH 8.0, containing 0.5 mM EDTA and 10% (w/v)
glycerol.
1 with a linear gradient of phosphate from
10 to 500 mM, pH 7.0, in the presence of 10% (w/v)
glycerol. All chromatographic steps were carried out at 0°C to 4°C
and driven by an Econo System (Bio-Rad).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Characterization of ACAD Activities Associated with Mitochondria in Maize Root Tips
The purification of mitochondria from maize root tips yields two main populations of mitochondria, low density and high density, corresponding to the meristematic and differentiating regions of the tip, respectively (Couée et al., 1992). Low-density mitochondria are not present in carbohydrate-starved maize root tips and show poor
integrity of the outer membrane and low respiration rates with weak
respiratory controls (Couée et al., 1992). Furthermore, these
low-density mitochondria showed low levels of ACAD activity (data not
shown). Therefore, for the present study only the high-density mitochondria, which are more differentiated, with many cristae and a
dense matrix, were used for the characterization of ACAD activity. The
ratios of ACAD-specific activities in purified high-density mitochondria to that in the crude extract were 13 (±1, SE)
and 4.5 (±0.5, SE) during purification from nonstarved and
carbohydrate-starved maize root tips, respectively. These enrichment
factors were therefore identical to those of typical enzyme markers of
mitochondria such as fumarase and NAD-specific isocitrate dehydrogenase
(Couée et al., 1992). In contrast, the activities of the
peroxisomal enzyme markers catalase, urate oxidase, and ACOX were
decreased 7- to 30-fold in the course of the purification from the
crude organellar extract to Percoll-purified mitochondria (Dieuaide et
al., 1993). The removal of peroxisomal enzyme markers during this
purification was comparable with that obtained for highly purified
mitochondria from potato tuber (Neuburger et al., 1982).
) that DCPIP-reducing
activity in purified maize root tip mitochondria was not due to
acyl-CoA thioesterase activities. Furthermore, the dye-reduction assay
with the C5 acyl-CoA valeryl-CoA gave no
detectable activity whether with crude protein extracts or with
purified mitochondria, whereas the acyl-CoA thioesterase activity with
the same substrate using the 5,5-dithiobis-(2-nitrobenzoate) assay was
significant, thus indicating that the dye-reduction system was specific
to ACAD activity. Therefore, it was clear that ACAD activity in maize root tips was associated with high-density mitochondria.
Characterization of ACAD Activities Associated with Mitochondria in
Embryonic Axes of Early-Germinating Sunflower Seeds
Partial Purification and Characterization of Medium-Chain and
Long-Chain ACAD Activities from Embryonic Axes of Early-Germinating
Sunflower Seeds
Previous (Dieuaide et al., 1993 Received September 18, 1998;
accepted December 18, 1998.
Abbreviations:
ACAD, acyl-CoA dehydrogenase.
ACOX, acyl-CoA
oxidase.
DCPIP, 2,6-dichlorophenolindophenol.
EST, expressed sequence
tag.
PMS, phenazine methosulfate.
We thank Dr. Ian A. Graham for critical reading of
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The dual location of ) to estimate the levels of ACAD activity in
the high-density mitochondrial pool of maize root tips prior to and
after carbohydrate starvation. Table I shows that carbohydrate starvation resulted in an increase of ACAD
activity associated with high-density mitochondria of maize root tips.
Carbohydrate starvation was previously shown to result in a 5- to
10-fold increase of ACOX activities in the peroxisomal pool of maize
root tips (Dieuaide et al., 1993; Hooks et al., 1995). Table
II shows the levels of ACOX and ACAD
activities in peroxisomes and mitochondria from carbohydrate-starved
maize root tips. The absence of ACOX activity in purified mitochondria
(Dieuaide et al., 1993) implied that ACOX activity in partially
purified peroxisomes was not due to contaminating mitochondrial
enzymes. The main feature of ACOX substrate specificities in
peroxisomes was the significantly lower level of activity with
isobutyryl-CoA and isovaleryl-CoA relative to the activity with
straight-chain substrates. The partial purification of peroxisomes also
implied that ACOX-specific activities were underestimated. Activities with butyryl-CoA, hexanoyl-CoA, and octanoyl-CoA substrates were therefore genuinely higher for peroxisomal ACOX than for mitochondrial ACAD. In contrast, the main feature of ACAD substrate specificities in
mitochondria was the significant level of activity with isobutyryl-CoA and isovaleryl-CoA relative to that with straight-chain substrates.
View this table:
Table I.
Effects of carbohydrate starvation on total ACAD
activity in maize root tip high-density mitochondria
ACAD activity was measured as described in ``Materials and Methods''.
High-density mitochondria were purified from nonstarved or 48-h
carbohydrate-starved maize root tips, as previously described
(Couée et al., 1992 ). The size of the high-density mitochondrial
pool in 1000 root tips was estimated from the yields of mitochondrial
protein and mitochondrial enzyme markers (Couée et al., 1992).
Results are the means ± SE of at least three experiments.
View this table:
Table II.
Substrate specificity of ACOX and ACAD activities
in peroxisomes and high-density mitochondria from carbohydrate-starved
maize root tips
ACOX and ACAD activities were measured as described in ``Materials and Methods''. Partially purified peroxisomes and purified high-density
mitochondria were isolated from 48-h carbohydrate-starved maize root
tips, as previously described (Couée et al., 1992 ; Dieuaide et
al., 1993). Results are the means ± SE of at least three
experiments.
had shown that functional mitochondria
could be isolated from dry embryonic axes of sunflower seeds. Differential centrifugation of organellar extracts from embryonic axes of 6-h-germinating sunflower seeds yielded a medium-speed pellet
showing enrichment in both mitochondrial and peroxisomal enzyme
markers. There was no detectable activity of the cytosolic and plastid
enzyme marker Glc-6-P dehydrogenase-6-phosphogluconate dehydrogenase (data not shown), thus indicating that this pellet was
free of cytosolic and plastid contaminants. Furthermore, the medium-speed pellet did not contain any detectable acyl-CoA
thioesterase activity. This organellar preparation was separated by
isopyknic centrifugation on a 20% (v/v) Percoll gradient. Separation
of mitochondria from 6-h-germinating sunflower seeds thus necessitated higher densities of Percoll for isopyknic centrifugation, which was in
line with the differentiation and densification of mitochondria during
imbibition (Attucci et al., 1991). Figure
1 shows that the mitochondrial enzyme
marker fumarase and the peroxisomal enzyme markers catalase and ACOX
showed distinct patterns of distribution along the Percoll gradient.
Catalase and ACOX activities were recovered mainly in the high-density
fractions of the gradient, which was in accordance with the high
density of intact peroxisomes (Schwitzguebel and Siegenthaler, 1984).
In contrast, fumarase activity was recovered in two peaks, in the
low-density and in the high-density fractions. The distribution of
palmitoyl-CoA-dependent ACAD activity was clearly different from that
of ACOX and was identical to that of fumarase activity in the upper
fractions of the gradient. However, its low level in the high-density
fractions of the gradient contrasted with the significant level of
fumarase.
View larger version (25K):
[in a new window]
Figure 1.
Subcellular localization of
palmitoyl-CoA-dependent ACAD activity in embryonic axes from
early-germinating sunflower seeds. Crude mitochondria from embryonic
axes of early-germinating sunflower seeds were further separated by
isopyknic centrifugation on a 20% Percoll gradient. Fractions of 2 mL
were collected and assayed for the activity of mitochondrial fumarase
(A), palmitoyl-CoA-dependent ACAD (B), peroxisomal ACOX (C), and
peroxisomal catalase (D). The scale of volumes from 0% to 100% ranges
from the top to the bottom of the Percoll gradient. Separation of
organelles and enzyme activity measurements were carried out as
described in ``Materials and Methods''.
and showed a latency of 95% with
mitochondrial enzyme markers such as NAD-specific isocitrate
dehydrogenase. However, it showed variable levels of contamination with
ACOX activity ranging from 0 to 2 nmol min1
mg1. Isopyknic centrifugation of the
mitochondrial preparation on a 12% (v/v) Percoll gradient resulted in
the separation of catalase and ACOX activities in the upper part of the
gradient and of ACAD and fumarase activities in the lower part of the
gradient, thus showing that these ACOX and catalase activities were due
to the presence of soluble contaminants rather than to contaminating peroxisomes. The peroxisomal preparation was contaminated with mitochondria, in accordance with the results of Attucci et al. (1991).
View this table:
Table III.
Substrate specificity of ACOX and ACAD activities
in peroxisomes and mitochondria from embryonic axes of
earlygerminating sunflower seeds
ACOX and ACAD activities were measured as described in ``Materials and Methods''. Partially purified peroxisomes and purified mitochondria
were isolated from embryonic axes of 6-h-soaked sunflower seeds, as
described in ``Materials and Methods'' and in ``Results''. Results
are the means ± SE of at least three experiments.
and Ikeda and Tanaka (1983a, 1983b) described
in detail the optimal strategy for separation and purification of all
of the different ACAD from rat liver. This strategy was therefore
attempted for the purification of distinct higher-plant ACAD. A number
of maize and sunflower tissues were tested as starting material for
purification. Table IV shows that
embryonic axes and cotyledons from early-germinating sunflower seeds as
well as carbohydrate-starved maize root tips showed high levels of palmitoyl-CoA-dependent ACAD activity in crude protein extracts. However, protein extracts of sunflower embryonic axes, in contrast with
extracts of sunflower cotyledons or carbohydrate-starved maize root
tips, did not present any detectable acyl-CoA thioesterase activity.
View this table:
Table IV.
Total palmitoyl-CoA-dependent ACAD activity in
crude protein extracts from maize and sunflower tissues
ACAD and acyl-CoA thioesterase activities were measured as described in
``Materials and Methods'' in the presence of 50 µM
palmitoyl-CoA. Maize and sunflower tissues were obtained and protein
extraction was carried out as described in ``Materials and Methods''.
Results are the means ± range or ± SE from two or
three experiments.
). However, isobutyryl-CoA-dependent ACAD activity
(Table III) was not recovered in any of the ammonium sulfate fractions.
1 mg1 and
1.1 nmol min1 mg1 with
palmitoyl-CoA and myristoyl-CoA, respectively, as the substrates. After
the sample was dialyzed extensively against 10 mM
KH2PO4-KOH buffer, pH 8.0, containing 0.5 mM EDTA and 10% (w/v) glycerol, the 40% to
60% fraction was applied to a DEAE-Sepharose column as described in
``Materials and Methods''. Approximately 80% of total
palmitoyl-CoA-dehydrogenating activity using the dye-reduction assay
was retained on the column. Elution with a 0 to 0.6 M
gradient of NaCl yielded a large peak of palmitoyl-CoA-dehydrogenating
activity (Fig. 2A). Fractions 25 to 48 were pooled, concentrated by dehydration against Suc, and extensively
dialyzed against 10 mM
KH2PO4-KOH buffer, pH 7.0, containing 10% (w/v) glycerol. The resulting fraction was applied to a
hydroxylapatite column. Nearly 100% of
palmitoyl-CoA-dehydrogenating activity was retained on the column.
Figure 2B shows that elution with increasing concentrations
of phosphate from 0 to 0.5 M resulted in the resolution of
two distinct peaks of palmitoyl-CoA-dehydrogenating activity eluting at
0.25 and 0.4 M phosphate. Fractions 15 and 32, which showed
highest activity, had specific activities, with palmitoyl-CoA as the
substrate, of 20 and 140 nmol min1
mg1, respectively, which corresponded to
apparent purification factors of 13- and 100-fold relative to the 40%
to 60% ammonium sulfate fraction.
View larger version (29K):
[in a new window]
Figure 2.
Separation of distinct ACAD by column
chromatography on DEAE-Sepharose (A) and hydroxylapatite-HT (B).
Proteins from embryonic axes of early-germinating sunflower seeds were
fractionated by ammonium sulfate precipitation as described in
``Materials and Methods''. Column chromatography and ACAD activity
measurements with 50 µM palmitoyl-CoA as the substrate
were carried out as described in ``Materials and Methods''. A, The
resuspended 40% to 60% ammonium sulfate fraction was dialyzed and
then loaded onto a DEAE-Sepharose column. Bound proteins were eluted by
a linear NaCl gradient from 0 to 0.6 M. B, The pooled
fractions from nos. 25 to 48 were concentrated and dialyzed before
application to a hydroxylapatite-HT column. Bound proteins were eluted
by a linear gradient of phosphate from 0 to 0.5 M. The two
peaks of active fractions were pooled separately to give ACAD1 and
ACAD2 preparations.
1
mg1 for this latter substrate, which
corresponded to an apparent purification factor of 770-fold relative to
the 40% to 60% ammonium sulfate fraction. Neither ACAD1 nor ACAD2
showed activity with isobutyryl-CoA or isovaleryl-CoA. Finally, the
dependency on FAD was tested. Thus, ACAD1 and ACAD2, when assayed with
the substrates giving highest activity, showed a 2- to 3-fold decrease
of activity in the absence of FAD, which is in line with previous
results on mammalian ACAD (Ikeda et al., 1985). Attempts to purify
ACAD1 and ACAD2 to homogeneity by affinity chromatography on
palmitoyl-CoA-agarose or Cibacron blue 3GA (Ikeda et al., 1983) were
unsuccessful as a result of instability of enzyme activity during these
chromatographic steps.
View larger version (53K):
[in a new window]
Figure 3.
SDS-PAGE analysis of protein fractions in the
course of partial purification of ACAD from embryonic axes of
early-germinating sunflower seeds. The different protein fractions were
obtained as described in ``Materials and Methods'' and in the legend
of Figure 2. Aliquots of the 0% to 40% (lane a, 100 µg of protein)
and 40% to 60% (lane b, 100 µg of protein) ammonium sulfate
fractions, of the pooled fractions from the DEAE-Sepharose step (lanes
c, 100 µg of protein), and of ACAD1 (lanes d, 50 µg of protein) and
ACAD2 (lanes e, 12.5 µg of protein) preparations were separated by
SDS-PAGE. Proteins were visualized by Coomassie blue staining. The
migration of molecular mass markers is given on the right.
View this table:
Table V.
Chain-length substrate specificity of ACAD
activities in partially purified ACAD preparations from embryonic axes
of early germinating sunflower seeds
ACAD was purified as described in ``Materials and Methods''.
Partially purified ACAD1 and ACAD2 preparations were obtained as
described in the legend for Figure 2. ACAD activities in ACAD1 and
ACAD2, which were measured as described in "Materials and Methods,"
are given as activities relative to palmitoyl-CoA-dependent (20 nmol
min 1 mg1) and myristoyl-CoA-dependent (850 nmol min1 mg1) specific activities,
respectively.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) and present data show that the
PMS-DCPIP dye-reduction assay is functional with protein preparations from higher plants and specific to ACAD activity. Thus, ACOX enzymes do
not drive this assay, as shown by subcellular fractionation experiments
and more definitely by the inability of purified ACOX to show any
apparent ACAD activity (Hooks et al., 1996). However, one must bear in
mind that at least some ACAD enzymes, such as the human short-chain
ACAD, can show ACOX activity (Vanhove et al., 1993a), but it did not
seem to be the case for ACAD activities in maize root or sunflower seed
mitochondria (Dieuaide et al., 1993; this work). Unexpectedly, acyl-CoA
thioesterase activity did not seem to be able to drive the PMS-DCPIP
dye-reduction assay, whether in purified mitochondria or in protein
extracts from maize (Dieuaide et al., 1993; this work). The PMS-DCPIP
dye-reduction assay was therefore found to be useful to measure ACAD
activity in organelle purification or protein purification from higher plants.
-oxidation (Schulz, 1991). However, the differences of
distribution between fumarase activity and palmitoyl-CoA-dependent ACAD
activity in mitochondria from embryonic axes of early-germinating sunflower seeds (Fig. 1) also indicated that this ACAD activity was
associated with a particular subset of mitochondria. This was in
agreement with the heterogeneity of mitochondrial subpopulations during
seed imbibition (Attucci et al., 1991) and would further suggest that
the different mitochondrial subpopulations may show metabolic
specialization. In both maize and sunflower differences of substrate
specificities were observed between ACOX activities of peroxisomes and
ACAD activities of mitochondria. Thus, isobutyryl-CoA and
isovaleryl-CoA generally gave significant activities with mitochondrial
ACAD and low, or undetectable, activities with peroxisomal ACOX, which
would be in line with the possible extra-peroxisomal location of
catabolism of Leu and Val (Gerbling and Gerhardt, 1989).
). Chromatographic
behavior and substrate specificities of ACAD1 and ACAD2 were similar to
those of long-chain ACAD and medium-chain ACAD, respectively (Ikeda et
al., 1983). However, apparent substrate specificities were somewhat
different. Mammalian long-chain ACAD shows no activity with short-chain
acyl-CoAs, whereas ACAD1 showed some activity with hexanoyl-CoA, and
mammalian medium-chain ACAD shows no activity with myristoyl-CoA,
whereas ACAD2 showed significant activity with this
C14 substrate.
) with the protein sequence
of rat mitochondrial long-chain ACAD (Tanaka et al., 1990) through
TBLASTN (Altschul et al., 1990) resulted in the identification of rice
EST no. D24729 (K. Yamamoto and T. Sasaki, unpublished data). After
complete sequencing of this clone (I. Couée, unpublished data),
the resulting sequence gave its best BLASTX homology score (373)
with mammalian mitochondrial isovaleryl-CoA dehydrogenase. This
sequence and the derived protein sequence were also used to interrogate
further dbEST through BLASTN and TBLASTN. This interrogation resulted
in the identification of EST no. AA231888 from oat (A.E. VanDeynze,
M.E. Sorrells, W.D. Park, N.M. Ayres, H. Fu, S.W. Cartinhour, and S.R.
McCouch, unpublished data) and EST nos. H77217 and AA650785 from
Arabidopsis, which correspond to EST no. U72505 (F. Grellet, P. Gaubier, H.-J. Wu, M. Laudie, C. Berger, and M. Delseny, unpublished
data). Whereas EST no. AA231888 from oat gave its best BLASTX homology score (193) with a putative isovaleryl-CoA dehydrogenase from Caenorhabditis elegans, EST no. U72505 from Arabidopsis gave its best BLASTX homology score (605) with glutaryl-CoA dehydrogenase from the hyperthermophilic, strictly anaerobic, sulfate-reducing (Aalen
et al., 1997) archaeon Archaeoglobus fulgidus. Thus, all of
these clones show the best homologies with ACADs involved in the
metabolism of amino acids, which would also be in line with the
possible extra-peroxisomal catabolism of Leu and Val (Gerbling and
Gerhardt, 1989) and with the activity measurements of Tables II and
III. It must also be noted that only the clones from rice and oat show
best homologies with the sequences of well-characterized mitochondrial
enzymes from eukaryotic organisms.
), enzyme
purification (this work), and large-scale sequencing projects (Newman
et al., 1994) reveal the existence of ACAD in higher plants. The
identification of ESTs and the cloning of the corresponding full-length
cDNAs should greatly facilitate the precise characterization of
higher-plant ACAD. Knowledge of their substrate specificities will
provide direct insight into the physiological functions of these
enzymes, as to whether they are involved in massive degradation of
quantitatively important compounds, such as fatty acids or branched-chain amino acids, or in the synthesis or removal of specialized molecules, such as hormones or growth regulators. Thus, for
a number of -oxidation pathways, where the substrates would be
specialized molecules such as 12-oxophytodienoate (Mueller, 1997) or
cinnamate (Klessig and Malamy, 1994), the enzymes involved are not yet
known. For instance, conventional -oxidation of the C18 precursor to jasmonate would involve 15 steps, including the initial activation of the carboxyl group and the
final release of jasmonate by acyl-CoA thioesterase activity (Mueller,
1997). The compartment in which these transformations take place is not known (Mueller, 1997; Parchmann et al., 1997). It would obviously be of
great interest to determine whether the enzymes involved are
mitochondrial or peroxisomal.
1
This work was partly funded by the Aquitaine
(France) Regional Council.
FOOTNOTES
2
Present address: Centre National de la Recherche
Scientifique UMR 6553, Université de Rennes I, Campus
Scientifique de Beaulieu, Bâtiment 14, 263 Avenue du
Général Leclerc, 35042 Rennes cedex, France.
*
Corresponding author; e-mail ivan.couee{at}univ-rennes1.fr; fax
33-299286915.
ABBREVIATIONS
ACKNOWLEDGMENT
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-oxidation system.
Biochem Biophys Res Commun
201:
1541-1547
[CrossRef][Medline]-oxidation system.
J Biol Chem
269:
19088-19094
-methyl-crotonyl-coenzyme A carboxylase in higher plant cells during carbohydrate starvation: evidence for a role of MCCase in leucine catabolism.
FEBS Lett
383:
175-180
[CrossRef][Medline]-Oxidation in glyoxysomes from castor bean endosperm.
J Biol Chem
244:
3514-3520
-oxidation after glucose starvation in maize root tips.
Plant Physiol
99:
595-600
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Biochem J
296:
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-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues.
Planta
159:
238-246
[CrossRef]-oxidation.
Biochem J
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-oxidation enzymes in rat liver mitochondria.
J Biol Chem
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1027-1033
-Oxidation of fatty acids by specific organelles.
In
PK Stumpf,
EE Conn,
eds, The Biochemistry of Plants, Vol 9.
Academic Press, London, pp 31-52-oxidation.
Biochim Biophys Acta
1085:
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[Medline]-oxidation enzymes in germinating pea cotyledons.
Planta
167:
54-57
Copyright Clearance Center: 0032-0889/99/119//10
© 1999 American Society of Plant Physiologists
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