From the Department of Cell Biology, National
Institute for Basic Biology, Okazaki, 444 Japan,
§ Department of Molecular Biomechanics, The Graduate
University for Advanced Studies, Okazaki 444, Japan, and the
¶ Dipartimento di Biologia delle Piante Agrarie, Sezione di
Fisiologia Vegetale, via Mariscoglio 34, I-56124 Pisa, Italy
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ABSTRACT |
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A cDNA clone for pumpkin acyl-CoA oxidase (EC
1.3.3.6; ACOX) was isolated from a gt11 cDNA library constructed
from poly(A)+ RNA extracted from etiolated
cotyledons. The inserted cDNA clone contains 2313 nucleotides and
encodes a polypeptide of 690 amino acids. Analysis of the
amino-terminal sequence of the protein indicates that the pumpkin
acyl-CoA oxidase protein is synthesized as a larger precursor
containing a cleavable amino-terminal presequence of 45 amino acids.
This presequence shows high similarity to the typical peroxisomal
targeting signal (PTS2). Western blot analysis following cell
fractionation in a sucrose gradient revealed that ACOX is localized in
glyoxysomes. A partial purification of ACOX from etiolated pumpkin
cotyledons indicated that the ACOX cDNA codes for a long chain
acyl-CoA oxidase. The amount of ACOX increased and reached to the
maximum activity by day 5 of germination but decreased about 4-fold on
the following days during the subsequent microbody transition from
glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA
was already high at day 1 of germination, increased by about 30% at
day 3, and faded completely by day 7. These data indicated that the
expression pattern of ACOX was very similar to that of the glyoxysomal
enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the
-oxidation spiral, during germination and suggested that the
expression of each enzyme of
-oxidation is coordinately
regulated.
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INTRODUCTION |
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There are at least three types of microbodies in higher plants
(glyoxysomes, leaf peroxisomes, and unspecialized microbodies) that are
distinguishable by their enzyme complements (1, 2). During the
postgerminative growth of pumpkin seedlings and upon exposure to light,
etiolated cotyledons turn green; at the same time, a functional
transition from glyoxysomes to leaf peroxisomes occurs (3, 4). In
fat-storing seeds of plants such as pumpkin, lipid bodies are present
in seed cells that store triacylglycerols, which are subsequently
converted to fatty acids by lipase. Fatty acids represent the main
energy and carbon sources for germinating seedlings. In glyoxysomes,
fatty acids are degraded to acetyl-CoA via the -oxidation pathway,
and acetyl-CoA is metabolized by the glyoxylate cycle bypassing the
decarboxylating steps of the Krebs cycle. We have shown previously that
the expression of glyoxysomal enzymes and leaf peroxisomal enzymes are
regulated not only at the transcriptional level but also at the
posttranscriptional level during the microbody transition (5, 6). The
gene expressions of the enzymes of the
-oxidation and glyoxylate
cycles seem to be coordinately regulated. In a recent paper, we
reported the nucleotide and deduced amino acid sequences of the
cDNA for 3-ketoacyl-CoA thiolase (7). The time course for thiolase
mRNA and thiolase levels during germination and postgerminative
growth implied that the regulation of expression of this enzyme is
similar to that of glyoxylate cycle enzymes, e.g. malate
synthase (8) and citrate synthase (9). The glyoxysomal
-oxidation
spiral consists of three different proteins: acyl-CoA oxidase
(ACOX),1 enoyl-CoA
hydratase/3-hydroxy acyl-CoA dehydrogenase (bifunctional protein), and
3-ketoacyl-CoA thiolase (thiolase). ACOX converts acyl-CoA into
trans-2-enoyl-CoA in the first step of the
-oxidation spiral and corresponds to the acyl-CoA dehydrogenase present in mitochondria of mammalian cells. Both enzymes are flavoproteins. Some
plant ACOXs have been purified and characterized (10, 11) and have been
shown to have different substrate specificities (for long, medium, and
short chain acyl-CoAs, respectively). To further investigate the
-oxidation enzymes at the molecular level, we cloned a cDNA
coding for a long chain ACOX, which is localized in glyoxysomes. Here,
we report the nucleotide and deduced amino acid sequences of the
cDNA. Developmental changes in the level of mRNA and protein
were also determined in pumpkin cotyledons during seed germination and
subsequent postgerminative growth.
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EXPERIMENTAL PROCEDURES |
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Plant Materials-- Pumpkin (Cucurbita sp. Kurokawa Amakuri) seeds were purchased from Aisan Seed Co. (Aichi, Japan). Seeds were soaked in running tap water overnight and germinated in Rock-Fiber soil (66R; Nitto Boseki, Chiba, Japan) at 25 °C in darkness. Some seedlings were transferred to light after 5 days.
Construction of the gt11 cDNA Library--
Total RNA was
extracted from etiolated cotyledons of 4-day-old dark-grown seedlings
by the SDS-phenol method. Poly(A)+ RNA was prepared by
column chromatography on oligo(dT) cellulose (Becton Dickinson).
cDNA transcribed from the poly(A)+ RNA was
constructed using the
gt11 system (Amersham, Tokyo, Japan).
Amino Acid Sequence Analysis-- Determination of the amino-terminal sequence of glyoxysomal acyl-CoA oxidase was performed essentially as described by Matsudaira (12). Isolated glyoxysomes were subjected to SDS-PAGE and proteins were transferred to a polyvinylidene difluoride membrane (Problot, Applied Biosystems, Chiba, Japan). The membrane was stained with Coomassie Blue, and the band corresponding to acyl-CoA oxidase was cut out with a razor blade. Protein sequencing was performed by automated Edman degradation in a protein sequencer (model 473A, Applied Biosystems).
Screening and Sequencing of cDNA--
A full sequence of
pumpkin acyl-CoA oxidase was obtained by polymerase chain reaction
employing 5'-CACAGGGAGATTCAAGA-3' and 5'-TCGGATCGAATGTAGCT-3' as the
sense and antisense primers, respectively. The amplified DNA fragment
was used as probe for the following experiment. Screening and plaque
hybridization were performed by the standard techniques. The insert of
the isolated phage clone was subcloned into the plasmid vector
Bluescript II SK() (Stratagene). A series of unidirectional deletion
clones was constructed with a deletion kit (Takara Shuzo, Kyoto,
Japan), and DNA sequencing was performed by the method of Sanger
et al. (13). DNA sequences were analyzed with
GeneWorks Release 2.2 computer software (IntelliGenetics, Mountain View, CA). The BLAST server was utilized for the analysis of
homologies among proteins. Alignment of several acyl-CoA oxidases was
performed using Clustal W software (14).
Preparation of a Specific Antiserum-- The pumpkin acyl-CoA oxidase cDNA was inserted into pET32b vector (Novagen, Madison, WI). A fusion protein between acyl-CoA oxidase and a histidine tag was synthesized in Escherichia coli cells and purified by column chromatography on Ni2+-resin. The purified protein in 1 ml of sterilized water was emulsified with an equal volume of Freund's complete adjuvant (DIFCO, Detroit, MI). The emulsion was injected subcutaneously on the back of a rabbit. Four weeks later, a booster injection (about 0.25 mg of protein) was similarly given to the first injection. Blood was taken from a vein in the ear 7 days after the second booster injection. The serum was used for immunoblotting.
Hydrophobic Interaction Chromatography-- Five-day-old etiolated pumpkin cotyledons were homogenized at 4 °C with 3 volumes of 150 mM Tris-HCl, pH 7.8, 10 mM KCl, 1 mM dithiothreitol, 10 µM FAD, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol. The homogenate was centrifuged at 15,000 × g for 20 min. To the resulting supernatant, an equal volume of 50 mM sodium phosphate, pH 7.0, containing 3.4 M (NH4)2SO4 was added and loaded onto a 1-ml Pharmacia (Uppsala, Sweden) phenyl-Sepharose high performance HiTrap column. The column was washed with 50 mM sodium phosphate, pH 7.0, containing 1.7 M (NH4)2SO4. Bound proteins were eluted by increasing the concentration of 50 mM sodium phosphate, pH 7.0, containing 60% ethylene glycol.
Subcellular Fractionation-- Four-day-old etiolated cotyledons were homogenized in a Petri dish by chopping with a razor blade for 5 min in 10 ml of a medium that contained 150 mM Tricine-KOH, pH 7.5, 1 mM EDTA, and 0.5 M sucrose. The homogenate was passed through four layers of cheesecloth. 3 ml of the filtrate was layered onto a sucrose gradient that consisted of a 1-ml cushion of 60% (w/w) sucrose and 11 ml of a linear sucrose gradient from 60 to 30% without buffer. The gradient was centrifuged at 25,000 × g for 3 h in an SW 28-2 rotor in an ultracentrifuge (XL-90; Beckman, Fullerton, CA). After centrifugation, fractions (0.5 ml each) were collected with an automatic liquid fractionation (ALC-2L; Advantec, Tokyo). All procedures were carried out at 4 °C.
Enzyme Assay-- Enzyme activities were measured at 25 °C in a 1-ml reaction mixture and monitored with a Hitachi (Tokyo) U-2000 spectrophotometer as follows: acyl-CoA oxidase (EC 1.3.3.6) according to Gerhardt (15), with the concentration of acyl-CoA substrates reduced to 25 µM; catalase (EC 1.11.1.6) according to Aebi (16); cytochrome c oxidase (EC 1.9.3.1) according to Hodges and Leonard (17).
Northern Blot Hybridization--
10 µg of total RNA was
extracted from etiolated pumpkin cotyledons and subjected to
electrophoresis on an agarose gel that contained 0.66 M
formaldehyde and 10 mM MOPS (pH 7.5). RNA was transferred
onto a Hybond N+ membrane (Amersham, Tokyo) in 50 mM NaOH and cross-linked by exposure to UV light
(Funa-UV-Linker, FS-800; Funakoshi, Tokyo). The ACOX insert was excised
by digestion with KpnI and labeled with
[-32P]dCTP (Amersham) using a Megaprime DNA labeling
kit (Amersham). The membrane was hybridized in 0.5 M sodium
phosphate (pH 7.2), 1 mM EDTA, 7% SDS, and 1% bovine
serum albumin with 1.0 × 106 cpm ml
1 of
radiolabeled DNA for 18 h at 42 °C. The membrane was washed in
SSC buffer/0.1% SDS for 15 min, in 0.1× SSC/0.1% SDS for 15 min, and
in SSC buffer at 60 °C twice for 15 min each. X-ray film was exposed
to the washed membrane, and radioactivity was measured on the imaging
plate of a BioImaging analyzer (FUJIX BAS 2000; Fuji Photo Film, Tokyo)
after an 18-h exposure.
Western Blot Hybridization-- Pumpkin cotyledons were homogenized in extraction buffer (0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1% SDS), the homogenate was centrifuged at 15,000 × g for 20 min, and the supernatant was subjected to SDS-PAGE. Then, an immunoblot analysis was performed by the method of Towbin et al. (18). Immunologic reactions were detected by monitoring by activity of horseradish peroxidase (ECL system; Amersham) or of alkaline phosphatase (Organon Teknika, West Chester, PA). The intensity of the signal was quantitated with a densitometer. The antiserum against thiolase was prepared as described previously (5). Protein was quantitated with a protein assay kit (Nippon Bio-Rad Laboratories, Tokyo).
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RESULTS |
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Cloning and Characterization of a cDNA for acyl-CoA
Oxidase--
Initially, we isolated glyoxysomal membranes from pumpkin
cotyledons from seedlings grown in the dark for 5 days. The membranes were treated with 0.1 M NaCO3. The soluble
proteins were separated by SDS-PAGE and blotted electrophoretically
onto a polyvinylidene difluoride membrane. Polypeptides were stained
with Coomassie Brilliant Blue, and a protein band of approximately 73 kDa was cut out and subjected to protein sequencing by automated Edman degradation. The amino-terminal amino acid sequence
(AAGKAKAKIEVDMGSLSLYMRGKHREIQERVFEYFN) was used to design degenerate
primers; a 1.6-kilobase pair DNA fragment was obtained by polymerase
chain reaction using a cDNA library produced from 5-day-old
etiolated pumpkin cotyledons as a template. The DNA fragment showed a
high similarity with ACOX from animals. Then, the fragment was used as
a probe to screen a gt11 cDNA library from 4-day-old etiolated
pumpkin cotyledons. Several positive recombinant phages were recovered,
including one containing the longest insert of approximately 2.4 kilobase pairs.
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Comparison of Pumpkin ACOX with Related Proteins-- The polypeptide encoded by the pumpkin ACOX cDNA shows the highest sequence identity (76%), with a putative Phalaenopsis ACOX. The cDNA clone of Phalaenopsis was isolated as one of senescence-related genes in Phalaenopsis petals (21) and was not characterized in detail. The identity with ACOXs from animal sources is about 30%, e.g. 30% with rat pristanoyl-CoA oxidase, 29% with rat trihydroxycoprostanoyl-CoA oxidase, and 28% with rat palmitoyl-CoA oxidase. This led Do and Huang (21) to postulate that the Phalaenopsis cDNA codes for an ACOX.
The sequences of pumpkin ACOX, Phalaenopsis ACOX, and rat pristanoyl-CoA oxidase (PRISCOX) are aligned for comparison in Fig. 2. PRISCOX is a peroxisomal protein that oxidizes the CoA-esters of 2-methyl-branched fatty acids, e.g. pristanic acid, and straight long chain acyl-CoAs (22). Pumpkin ACOX and Phalaenopsis ACOX are represented in their mature forms (putative for Phalaenopsis ACOX). The overall identity of the amino acid sequences looks low, but it is possible to recognize a stretch of identical amino acids from amino acids 444 to 488. This portion also shows high identity with the amino acid sequences of rat trihydroxycoprostanoyl-CoA oxidase, rat palmitoyl-CoA oxidase, and Caenorhabditis elegans acyl-CoA oxidase (data not shown). In addition, this region includes the putative flavin mononucleotide binding site (19) and six of the eight amino acids that represent the acyl-CoA dehydrogenase protein signature 2 (PS2: [QE]-x(2)-G-[GS]-x-G-[LIVMFY]-x(2)-[DEN]-x(4)-[KR]-x(3)-[DE]) (20). In respect to the acyl-CoA dehydrogenase protein signature 1 (PS1: [GAC]-[LIVM]-[ST]-E-x(2)-[GSAN]-G-S-D-x(2)-[GSA]) (20), it is possible to find seven of the nine amino acids in all three sequences of Fig. 2. This confirms the low homology between acyl-CoA oxidases and acyl-CoA dehydrogenases and suggests that the two protein stretches are involved in the FAD binding and/or in the binding with substrate.
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Pumpkin ACOX Is Synthesized as a Larger Precursor-- Some peroxisomal proteins are synthesized as larger precursors such as malate dehydrogenase (23, 24), thiolase (7, 25), and citrate synthase (9). These precursors are cleaved at a site near the amino-terminal end of the protein. An amino-terminal sequence comparison of the precursor protein with the mature protein, determined by protein sequencing following SDS-PAGE, indicates that pumpkin ACOX precursor protein is cleaved at the carboxyl side of amino acid 45 to give into the mature protein size. In recent studies, it was shown that the amino-terminal presequences of peroxisomal proteins also act as peroxisomal targeting signals (PTS2) (26, 27). Fig. 3 shows the alignment of the amino-terminal regions of pumpkin glyoxysomal ACOX, Phalaenopsis ACOX, and pumpkin glyoxysomal proteins that are synthesized as precursors of higher molecular mass (malate dehydrogenase (23, 24), citrate synthase (9), and thiolase (7, 25)). However, the cleavage site for the Phalaenopsis ACOX has not yet been confirmed. The targeting consensus sequence, R-[I/L/Q]-x5-H-L, is highly conserved. Thus, it is suggested that pumpkin ACOX and, by analogy, Phalaenopsis ACOX contain a PTS2-type targeting signal.
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Activity of ACOX in Etiolated Pumpkin Cotyledons-- To confirm that the ACOX cDNA actually codes for an ACOX, a crude extract from 5-day-old etiolated pumpkin cotyledons was subjected to hydrophobic interaction chromatography on a phenyl-Sepharose column (Fig. 4). Three peaks of ACOX activity were detected. The first peak (circles) was obtained using hexanoyl-CoA (C6) as a substrate and thus indicates the presence of a short chain ACOX. Two overlapping peaks were obtained with palmitoyl-CoA (C16) (squares) and decanoyl-CoA (C10) (triangles) as substrates and thus indicate a long/medium chain ACOX. Western blotting with polyclonal antiserum raised against the pumpkin ACOX expressed in E. coli (Fig. 4B) shows clearly that the antiserum recognizes a protein of approximately 73 kDa only in fractions showing long/medium chain ACOX activity. Because it was reported that plant long chain ACOX has a subunit molecular mass of approximately 72 kDa (10), and the medium chain ACOX has a subunit molecular mass of 62 kDa (11), we conclude that the isolated pumpkin cDNA encodes for a long chain ACOX.
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Subcellular Localization of ACOX in Etiolated Pumpkin
Cotyledons--
To investigate the subcellular localization of the
ACOX protein, enzyme activity and immunoblotting analyses were
performed after fractionation by sucrose density gradient
centrifugation of a pumpkin organelle homogenate (Fig.
5). Catalase and thiolase were used as
glyoxysomal markers, and cytochrome c oxidase was used as a
mitochondrial marker. ACOX activities were present in the supernatant
and the glyoxysomal fractions. A small peak of activity was also
detected in the mitochondrial fractions (namely fraction 11) but did
not overlap with the activity of cytochrome c oxidase. As a
small catalase activity was present in the fraction, these activities
might be due to the contamination of glyoxysomes. The immunoblotting
analysis confirmed that a protein of 73 kDa, corresponding to ACOX, is
mainly present in the supernatant and in the glyoxysomal fractions
(Fig. 5B). Interestingly, a similar pattern was also
obtained for thiolase, another -oxidation enzyme.
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Time Course of mRNA and Protein Levels during Germination in
Pumpkin Cotyledons--
During greening of pumpkin cotyledons, the
composition of matrix enzymes in microbodies changes dramatically,
glyoxysomal enzymes decrease, and leaf peroxisomal enzymes are
synthesized. Like thiolase, ACOX is a part of the fatty acid
-oxidation spiral. Therefore, we followed the changes in the level
of ACOX and thiolase mRNA and protein during the postgerminative
growth of seedlings (Figs. 6 and
7). The relative levels of ACOX mRNA
in dark-grown seedlings during the 9-day period after germination are
indicated by Northern blot in Fig. 6A (top panel)
and are quantified by densitometry (bottom panel). ACOX
mRNA levels reached a maximum after 3 days and thereafter gradually
disappeared. When some of the seedlings were transferred to light after
5 days (middle panel), the pattern did not change. Similar
results were obtained for thiolase mRNA (Fig. 6B).
Notably, the total amount of ACOX mRNA was much lower than that of
thiolase mRNA. The ACOX protein levels are shown in Fig.
7A, in which the three panels correspond to the three
mRNA panels in Fig. 6A. The curve for the dark-grown seedlings (closed circles, bottom panel) shows
that the peak in the ACOX protein was delayed with respect to the
mRNA peak, reaching a maximum level at day 5 after germination and
subsequently decreasing. Similar results were obtained for thiolase
(Fig. 7). Moreover, following illumination (open circles,
bottom panels), ACOX disappeared more rapidly than thiolase.
In general, however, the two patterns seem to be very similar.
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DISCUSSION |
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In the present study, we report the cDNA sequence of a pumpkin glyoxysomal long chain ACOX in addition to the cDNA sequence of a previously reported Phalaenopsis ACOX (21).
The present results clearly show that the protein encoded by this gene
is a plant long chain ACOX. The deduced amino acid sequences of pumpkin
and Phalaenopsis cDNA sequences have an identity of
76%, indicating that the Phalaenopsis cDNA also codes
for a long chain ACOX. Comparing other ACOXs, the best identity (30%) is obtained for the rat pristanoyl-CoA oxidase, which acts on 2-methyl-branched CoA-esters and straight long chain acyl-CoAs (22).
Mammalian peroxisomes contain three ACOX isozymes that are not capable
of oxidizing acyl-chain CoA esters of less than 8 carbons. In mammalian
cells, the -oxidation of short chain fatty acids is accomplished in
mitochondria, in which acyl-CoA dehydrogenases act instead of ACOXs.
Three peroxisomal mammalian ACOXs have been identified: PRISCOX (30%
identity), palmitoyl-CoA oxidase (28% identity), which reacts with CoA
esters of very long, long, and medium chain fatty acids (28), and
trihydroxycoprostanoyl-CoA oxidase (29% identity), which oxidizes the
CoA esters of the bile acid intermediates dihydroxycoprostanic acid and
trihydroxycoprostanic acid (29). On the contrary, plant peroxisomes
seem to contain ACOXs that are active on short, medium, and long chain
acyl-CoAs (11) and are able to perform a complete
-oxidation of
fatty acids to acetyl-CoA (2). Three plant ACOX isozymes have
previously been purified and characterized. One is from cucumber
cotyledons that is active on long and medium chain acyl-CoAs and that
is a homodimer with subunits of 72 kDa (10). The other two are from
maize and are active on medium and short chain acyl-CoAs, respectively
(11). The former is a monomeric enzyme of 62 kDa, and the latter is a
homotetrameric enzyme of 15 kDa. Three different genes seem to code for
the three ACOX isoforms, as they have different subunit molecular
weights (11). The report by Hooks et al. (11) was the first
to imply the presence of a short chain ACOX in eukaryotic cells.
Mammalian ACOX isoforms, nevertheless, show slightly different substrate preferences and seem to have very similar subunit molecular weights of about 75 kDa. Therefore, only the plant long chain ACOX
should share common ancestral genes with the mammalian ACOXs.
In the present study, we were able to correlate the sequence of the isolated ACOX clone with a long chain specific ACOX by applying an antiserum against the expressed ACOX/histidine-tagged fusion protein. This antiserum recognized only long chain and medium chain ACOX activity and not short chain ACOX activity when pumpkin enzymes were separated by hydrophobic interaction chromatography (Fig. 4). The immunoreactive band corresponded to a molecular mass of 73 kDa in accordance with the calculated molecular mass of mature pumpkin long chain ACOX (72,414 Da) and with the previous report of 72 kDa for the cucumber long chain ACOX (10).
The levels of ACOX mRNA do not seem to be greatly controlled by light. The ACOX protein that built up during the initial 5 days of germination disappeared during the transition from glyoxysomes to leaf peroxisomes upon exposure of the seedlings to light. Similar patterns have previously been observed for malate synthase (8) and citrate synthase (9). The appearance and disappearance of the mRNAs preceded the change in the ACOX protein during the microbody transition. Thus, the ACOX levels seem to be determined at both the translational and posttranslational levels.
It is worth noting that pumpkin glyoxysomal long chain ACOX proteins
are synthesized as larger precursors containing a cleavable amino-terminal presequence, namely PTS2 (27, 30), as in the case for
some other plant peroxisomal proteins, such as malate dehydrogenase
(23, 24), citrate synthase (9), and thiolase (7, 25). In all cloned
mammalian ACOXs, a carboxyl-terminal signal (PTS1) is present, but
there is no PTS2 signal (31). This indicates that the plant ACOX import
mechanism differs from the mammalian one. It has been suggested that
ACOX is a key enzyme of -oxidation because it can control and
regulate the flux of acyl-CoAs at the first step of the
-oxidation
spiral (32). Particularly, the long chain acyl-CoA oxidase may
represent a regulatory point considering the fact that most fatty acids
of plant storage lipids are long chain molecules. In conclusion, this
type of control mechanism could tightly regulate the long chain ACOX
(as the first step of the
-oxidation cascade), or it could be
involved in a coordinate or differential regulation of the expression
of the three ACOX enzymes in plant tissues (11). To verify such a
hypothesis, the cloning and an expression analysis of the two other
ACOXs will be necessary.
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ACKNOWLEDGEMENTS |
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We thank Prof. Dr. Claus Schnarrenberger (Free University of Berlin) for stimulating discussions and helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by "Research for the Future" Program Grant JSPS-RFTF 96L00407, the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research (09440271, 09274101, and 09274103), the Ministry of Education, Science and Culture, Japan, a grant from the Nissan Science Foundation (Tokyo), and the NIBB program for Molecular Mechanisms of Stress Response.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF002016.
To whom correspondence should be addressed. Tel.:
81-564-55-7500; Fax: 81-564-55-7505; E-mail:
mikosome{at}nibb.ac.jp.
1 The abbreviations used are: ACOX, acyl-CoA oxidase; PTS, peroxisomal targeting signal; thiolase, 3-ketoacyl-CoA thiolase; PRISCOX, pristanoyl-CoA oxidase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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