From the Biocenter Oulu and Department of
Biochemistry, University of Oulu, Linnanmaa, FIN-90570 Oulu, Finland,
§ Department of Pathology, University of Oulu, Kajaaninitie
52 A, FIN-90220 Oulu, Finland, ¶ Human Genetics Unit, Molecular
Medicine Centre, University of Edinburgh EH4 2XU, United Kingdom,
Department of Biosciences, Division of Biochemistry, P. O. Box
56, FIN-00014 University of Helsinki, Finland, and ** Howard Hughes
Medical Institute, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
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rECH1, a recently identified rat cDNA
(FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995)
Genomics 27, 457-466) encodes a polypeptide belonging to
the hydratase/isomerase superfamily. We modeled the structure of rECH1
based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts
that rECH1p has the hydratase fold in the core domain and two domains
for interaction with other subunits. When we incubated
3,5,8,11,14-eicosapentaenoyl-CoA with purified rECH1p, the spectral
data suggested a switching of the double bonds from the
3-
5 to the
2-
4 positions. This was confirmed by
demonstrating that the product was a valid substrate for
2,4-dienoyl-CoA reductase. These results indicate that rECH1p is
3,5-
2,4-dienoyl-CoA isomerase.
Subcellular fractionation and immunoelectron microscopy using
antibodies to a synthetic polypeptide derived from the C terminus of
rECH1p showed that rECH1p is located in the matrix of both mitochondria
and peroxisomes in rat liver. Consistent with these observations, the
36,000-Da rECH1p has a potential N-terminal mitochondrial targeting
signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the
targeting signal results in a mature mitochondrial form with a
molecular mass of 32,000 Da; transport to peroxisomes yields a protein
of 36,000 Da.
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INTRODUCTION |
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An increasing body of evidence indicates that the metabolism of
unsaturated fatty acids can occur in both mitochondria and in
peroxisomes (1). Unsaturated fatty acids, which have only a few double
bonds and not very long carbon chains, are effectively degraded by
mitochondrial -oxidation. Conversely, very long chain (poly)unsaturated fatty acids and their derivatives, some of which are
inhibitors of mitochondrial
-oxidation, undergo rapid chain shortening in mammalian peroxisomes with the probable exception of
fatty enoyl-CoA esters that have
5 double bonds (2).
Since trans-2-enoyl-CoA is the only unsaturated intermediate
in both peroxisomal and mitochondrial -oxidation and since the double bonds of natural unsaturated fatty acids are mostly in the
cis configuration at either odd- or even-numbered positions, auxiliary enzymes are required to link them to the
-oxidation pathway. These auxiliary activities include 2,4-enoyl-CoA reductase (EC
1.3.1.34),
3-
2-enoyl-CoA isomerase (EC
5.3.3.8), and
3,5-
2,4-dienoyl-CoA
isomerase. In mammalian tissues two mitochondrial and one peroxisomal
isoform of 2,4-dienoyl-CoA reductase have been described (3, 4).
cDNAs encoding the 120-kDa mitochondrial isoform from rat (5) and
human (6) and the 69-kDa peroxisomal enzyme from Saccharomyces
cerevisiae have been cloned (7). Additionally, cDNAs encoding
the mitochondrial short-chain
3-
2-enoyl-CoA isomerase (8, 9) and the
peroxisomal form of the same enzyme (10) have been cloned and well
characterized. The peroxisomal form is an integral part of the
peroxisomal multifunctional hydratase/dehydrogenase enzyme type 1 (MFE1).1
3,5-
2,4-Dienoyl-CoA isomerase activity
has been detected both in mitochondria and peroxisomes in rat liver,
and several of these enzymes have been purified (11-14), but none have
been cloned.
Using a subtractive strategy, FitzPatrick et al. (15)
recently isolated a peroxisome proliferator-induced rat liver cDNA that encodes a novel member of the hydratase/isomerase protein family
with a type 1 peroxisomal targeting signal (SKL) at its C terminus. The
gene was named rat enoyl-CoA hydratase (rECH1), referring to its
possible activity. Here we show that the rECH1 protein (rECH1p)
actually is a 3,5-
2,4-dienoyl-CoA
isomerase participating in the metabolism of unsaturated fatty acids
with double bonds at the
5 position. This is the first
characterization of a dienoyl-CoA isomerase at the molecular level.
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EXPERIMENTAL PROCEDURES |
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Molecular Modeling-- We collected amino acid sequences related to rECH1p from data bases by using Blast (16) and Fasta (17) algorithms and aligned them using the Clustal W program (18). Secondary structure predictions were performed with SOPM (19), Gibrat (20), Levin (21), DPM (22), and PhD methods (23, 24). Finally, all the data were combined and aligned with the sequences of 2-enoyl-CoA hydratase 1 (25) and 4-chlorobenzoyl-CoA dehalogenase (26), for which the three-dimensional structures have been determined.
rECH1p was modeled based on the structure of the 2-enoyl-CoA hydratase 1 at 2.5 Å resolution (25) (Protein Data Bank (27)), ID number 1DUB. The sequence alignment was performed with the GCG (28) and Clustal W program packages. The final alignment was performed using information from multiple sequence alignments and secondary structural information from the three-dimensional structures. The model was built with the program Insight II (Biosym Technologies, Inc., San Diego, CA). A side chain rotamer library was used to model amino acid substitutions. Deletions and insertions were modeled by searching a data base of high resolution structures. The search was performed for fragments of the required length and end point separation by using two residues at each end of the loop as anchor points. The fragments obtained were evaluated on the basis of three criteria: root mean square deviation from the anchor points, sequence similarity, and interference with the core region. There were three insertions and no deletions with respect to hydratase 1. The model was refined by energy minimization with the program Discover using Amber force field in a step-wise manner. First, hydrogen atoms were relaxed, then the borders of insertions and deletions were harmonically restrained, and the rest of the molecule was fixed. In the next step, the borders of insertions and deletions and the CAntibodies--
We immunized rabbits with a synthetic peptide
(CAMEKKDSKSITFSKL-COOH) from the C-terminal end of rECH1p. An
N-terminal cysteine residue was added to link the peptide to Imject
maleimide-activated ovalbumin (Pierce). We also raised rabbit
antibodies against porcine fumarase (EC 4.2.1.2) (Boehringer Mannheim)
purified by reverse phase chromatography on a microreverse phase column
C2/C18 PC 3.2/3 column (Pharmacia Biotech Inc.). The IgG fraction from
these antisera was purified by ammonium sulfate precipitation to a
saturation of 45% followed by size exclusion chromatography on
Superdex 200 high resolution 10/30 column (Pharmacia) in equilibrium
with 10 mM sodium phosphate, 2 mM potassium
phosphate, 140 mM NaCl, 3 mM KCl, 5 mM -mercaptoethanol, pH 7.4, (PBS) containing 0.02% sodium azide. Rabbit anti-peroxisomal multifunctional enzyme 1 antibody
(10) and anti-rat
3-
2-enoyl-CoA isomerase
antibody (29) were obtained as described. Antibodies against the rat
catalase were a gift from Dr. Stefan Alexson (Karolinska Institute,
Huddinge, Sweden). The use of animals for immunization was approved by
the university committee on animal experimentation.
Expression of Truncated rECH1 in Escherichia coli--
We
utilized rECH1 cDNA in Bluescript KS (15) as a template for
polymerase chain reaction amplification. The forward primer (5
-GACATATGGCATACGAGTCTATTCAAGTAACATCTGC) corresponds to nucleotides 170-196 with an additional 11 5
nucleotides providing an
NdeI restriction site, an ATG initiation methionine codon,
and a GCA (alanine) codon. 3
of this, the sequence continues with TAC
encoding Tyr-54 of the full-length protein. The reverse primer
(5
-CAGGATCCCACTCAGAGCTTGGAGAAGGTGAT) corresponds to nucleotides
974-997 and contains an additional BamHI restriction site
on its 5
end. We cloned the amplified polymerase chain reaction
fragment into pUC 18 plasmid with the Sure Clone ligation kit
(Pharmacia). After digestion with NdeI and BamHI,
the cDNA was ligated into a pET 3a expression vector (30). The
protein was expressed in the BL21(DE3)LysS E. coli strain
using a pET expression system (Novagen, Madison, WI) according to
manufacturer instructions. Induction was carried out for 3 h at
35 °C, and the cells were harvested by centrifugation, washed with
PBS, and suspended in 0.01 volume of the same buffer. The cells were
lysed by freezing in liquid nitrogen and thawing at 37 °C. RNase,
DNase, and lysozyme were added to final concentrations of 2, 20, and
100 µg/ml, respectively, and the mixture was incubated at 35 °C
for 45 min. The lysate was centrifuged at 15,000 × g for 15 min, and the supernatant was used for further experiments.
Subcellular Fractionation-- Rat liver was homogenized in 0.25 M sucrose, 0.1% (v/v) ethanol, 1 mM EDTA, pH 7.2, and fractionated by differential centrifugation into nuclear, heavy mitochondrial, light mitochondrial, microsomal, and cytosolic fractions as described by Verheyden et al. (31). Peroxisomes were purified from the light mitochondrial fraction by centrifugation in a Nycodenz step gradient as described by Vanhove et al. (32). Mitochondria were isolated by centrifugation in a self-generating Percoll gradient (33). Thirty µg of crude homogenate and 10 µg of mitochondrial and peroxisomal fractions were separated in an SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose filter. Filters were used for immunodetection with a secondary antibody, goat anti-rabbit IgG-conjugated with horseradish peroxidase (Bio-Rad).
Purification of rECH1p-- Fourteen ml of the lysate containing 52 mg of soluble proteins was applied to a DEAE-Sephacel column (2.5 × 11 cm) in equilibrium with 20 mM potassium phosphate, pH 6.5, and 100 mM NaCl. The flow-through fraction containing rECH1p was dialyzed against 3 × 500 ml of 20 mM MES, pH 6.0, and applied to a 1-ml Resource S column (Pharmacia) in equilibrium with the same buffer. The bound rECH1p was eluted with a linear 20-ml gradient from 0 to 0.3 M NaCl in 20 mM MES, pH 6.0. During the purification procedure, the elution of rECH1p was detected by dot-blotting aliquots of fractions on a nitrocellulose filter and immunolabeling with anti-rECH1. The native molecular weight of rECH1p (20 µg) was determined by size exclusion chromatography on a Superdex 200 high resolution 10/30 column (Pharmacia) in 200 mM phosphate buffer, pH 7.4, containing 1 mM EDTA.
Enzyme Assays--
3-
2-Enoyl-CoA
isomerase and 2-enoyl-CoA hydratase 1 (EC 4.2.1.17) activities
were assayed as described by Palosaari and Hiltunen (10).
2-Enoyl-CoA hydratase 2 (EC 4.2.1.-) activity hydrating
trans-2-enoyl-CoAs to D-3-hydroxyacyl-CoAs was
assayed by monitoring the hydration of trans-2-decenoyl-CoA
to D-3-hydroxydecanoyl-CoA (34) coupled to the further
oxidation in a reaction by recombinant D-specific
3-hydroxyacyl-CoA dehydrogenase (a gift from Yongmei Qin, University of
Oulu, Oulu, Finland). Substrate concentrations of 60 µM
were used for activity measurements. For the activity measurement of
3,5-
2,4-dienoyl-CoA isomerase, the
substrate was generated by incubating 0.2 unit of yeast acyl-CoA
oxidase (Sigma) with 60 µM of arachidonoyl-CoA in 1 ml of
20 mM potassium phosphate, 3 mM EDTA, pH 7.4, at 23 °C for 10 min. Because the yeast acyl-CoA oxidase has
intrinsic
3-
2-enoyl-CoA isomerase
activity (11), 3,5,8,11,14-eicosapentenoyl-CoA accumulated in the
reaction, as indicated by the increase of absorbance at 240 nm, and
thus the addition of
3-
2-enoyl-CoA
isomerase was dispensable. The dienoyl-CoA isomerase activity was
monitored by following the appearance of a conjugated
2-
4 double bond at 300 nm (35). An
extinction coefficient of 27,800 M
1
cm
1 for trans-2,4-dienoyl-CoA was used for the
calculations (36). 2,4-Dienoyl-CoA reductase purified from
Candida tropicalis was kindly provided by Kari Koivuranta,
University of Oulu, Oulu, Finland.
Immunoelectron Microscopy-- Livers were harvested from control and clofibrate-treated (fed with 0.5% (w/w) clofibrate-supplemented chow for two weeks) Sprague-Dawley rats after vascular perfusion fixation. For this procedure, the rats were heparinized (15 units of heparin/g of body weight), euthanized, and perfused through the left ventricle and ascending aorta at a hydrostatic pressure of 120 cm (37) for 2 min with PBS containing 2 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. A prefixative solution containing 8% paraformaldehyde and 20 mM ethylacetimidate in PBS was then administered for 3 min. After this, the main fixative solution containing 4% paraformaldehyde and 0.25% (v/v) glutaraldehyde in PBS was infused for 15 min. Ethylacetimidate was included in the prefixative solution for its described capacity for maintaining antigen accessibility and preservation of antigenicity (38). After perfusion fixation, 1-2-mm blocks of liver were further fixed for 2 h at room temperature by immersion in the same fixative solution. Finally the tissue blocks were washed three times in 7.5% (w/v) sucrose in PBS, where they were kept at 4 °C until processed.
For thin sections, tissues were embedded in Micro-Bed resin (Electron Microscopy Sciences, Fort Washington, PA) according to manufacturer instructions, except that the tissues were infiltrated from 70% ethanol instead of completion of the dehydration protocol. The resin was polymerized at 4 °C under UV light (365 nm) for 60 h. For immunogold labeling, grids were incubated for 5 min with 50 mM glycine in PBS and for 15 min in blocking medium containing 1% bovine serum albumin and 1% fish skin gelatin in PBS (PBS-BSA-FSG). Primary labeling of the sections was made by incubating the grids for 2 h at room temperature with the primary antibody. Similar grids were incubated with the IgG fraction of rabbit serum containing antibodies to the rat peroxisomal peroxisomal MFE1 and mitochondrial ![]() |
RESULTS |
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Molecular Modeling and Structure Prediction of rECH1--
The
sequence identity between rECH1p and enoyl-CoA hydratase 1 is modest
(25%), but the residues and elements that are functionally and
structurally important in hydratase 1 are conserved (Fig. 1) (25). We modeled the rECH1p structure
using the coordinates of hydratase 1 as a template. The alignment of
rECH1 with the mature form of hydratase 1 (starting at Gly-30) (39)
shows 48 additional N-terminal amino acid residues, three insertions,
and three amino acid residues in the C terminus (SKL). The C-terminal extension could not be modeled. Regions containing a single amino acid
residue insertion appear in the beginning and in the end of the
trimerization domain 1, but the insertions do not affect the general
scaffolding of the protein. There is a large insertion in the helical
portion of the second part of the hydratase spiral (Fig.
2). This section was modeled as an
extended -helix based on structural requirements and sequence
analysis. The corresponding region in hydratase 1 has a flexible loop,
which might indicate allowance of greater variability in this region.
Insertions were modeled using a library of loops generated with the
Insight II program from known high resolution three-dimensional
structures in the Protein Data Bank. The loop library was scanned for
structural elements giving the best matches with the predicted
secondary structure element in the inserted region after positioning
the insert in the model. The final model has a typical globular
structure according to several structural tests (40).
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Expression of rECH1 and Purification--
We expressed rECH1p in
E. coli as an N-terminal-truncated version beginning at
Tyr-54. An additional N-terminal Met-Ala sequence was contributed by
the 5 primer. The truncation site was chosen based on the following
observations. The amino acid sequence similarity of rECH1p with the
mature rat mitochondrial enoyl-CoA hydratase 1 (39),
Pseudomonas sp. 4-chlorobenzoate dehalogenase (41) (Fig. 1),
and the mature rat mitochondrial short-chain
3-
2-enoyl-CoA isomerase (9) (data not
shown) started near Tyr-54 of rECH1p. Furthermore, the model of rECH1p
indicates that the N-terminal residues (1-48) are not involved in the
folding of the isomerase/hydratase fold or the T1 and T2 domains and
apparently do not contribute to the architecture of the substrate
binding site.
Search for Function of rECH1p--
The functionally characterized
members of the hydratase/isomerase superfamily utilize coenzyme A
thioesters of carboxylic acids as substrates, and therefore, we
hypothesized that rECH1p could also bind acyl-CoA esters. Some members
of the family act on 2 or
3 double bonds
of enoyl-CoA esters or catalyze hydration reactions. Because
trans-2-eicosenoyl-CoA and 3,5,8,11,14-eicosapentenoyl-CoA fulfill the substrate criteria mentioned above and because they have
known spectral characteristics in the near UV region (13, 14) we chose
them for screening substrates.
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Subcellular Localization-- Two bands corresponding to molecular masses of 36,000 and 32,000 Da were detected by immunoblot analysis with anti-rECH1p of rat liver homogenate subjected to SDS-polyacrylamide gel electrophoresis (Fig. 5). When purified peroxisomes and mitochondria were immunoblotted, the 36,000-Da protein was associated with peroxisomes, whereas the 32,000-Da protein was enriched in the mitochondrial preparation. These data suggest that rECH1p in rat liver has dual subcellular location. To study this further, we performed immunoelectron microscopy utilizing the protein A-colloid gold-labeling technique. Gold particles localized the rECH1p to peroxisomes and mitochondria in both control and clofibrate-treated rats. To determine the relative amount of antigen in both organelles, we calculated the gold/area ratio (number of gold particles/µm2, mean ± S.D. in electron microscopy frames measured) for both organelles. The area outside the organelles was taken as background. In control (clofibrate-treated) rats, the labeling density was 4.17 ± 1.47 (31.2 ± 13.0; p < 0.003 control versus clofibrate-treated) in mitochondria, 7.0 ± 4.29 (25.7 ± 9.2; p < 0.006) in peroxisomes, and 1.75 ± 0.96 (0.75 ± 0.96; difference not significant) in background (Fig. 6).
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DISCUSSION |
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Published studies on the distribution of the
3,5-
2,4-dienoyl-CoA isomerase in the rat
including subcellular fractionation, immunoblotting, immunoelectron
microscopy, enzyme activity measurements, and protein purification
provide evidence that the dienoyl-CoA isomerase activity in liver is
associated with both peroxisomes and mitochondria (11-14, 44).
However, the amino acid sequences or molecular characteristics of the
enzyme(s) are not known. The results of our present experiments indicate unambiguously that rECH1 encodes dienoyl-CoA isomerase. Overexpression of rECH1 in E. coli gives an enzymatically
active protein, and purified recombinant protein transfers the
conjugated
3-
5 double bond of
3,5,8,11,14-eicosapentenoyl-CoA to the
2-
4 position. We verified this reaction
spectroscopically by following the changes of absorbance in the near UV
region and by demonstrating that the product of the double-bond
transfer was a substrate for 2,4-dienoyl-CoA reductase. Identification
of rECH1p as a
3,5-
2,4-dienoyl-CoA
isomerase adds a new activity among the reactions catalyzed by the
members of the hydratase/isomerase family.
At present, the only members of the hydratase/isomerase enzyme family with known structures are 2-enoyl-CoA hydratase 1 (25) and 4-chlorobenzoyl dehalogenase (26). The modeled subunit of rECH1 shows the presence of both trimerization domains similar to hydratase 1 and 4-chlorobenzoyl dehalogenase, suggesting that the enzyme forms a trimer. In line with this proposal, size exclusion chromatography gave a molecular mass of 170,000 Da. This indicates that in the native state rECH1p may be a hexamer similar to the rat mitochondrial 2-enoyl-CoA hydratase 1, which is a dimer of two trimers (25).
Prediction of the secondary structure elements of the rECH1p indicates the presence of the hydratase spiral (25) forming the CoA binding site and the pocket where catalysis takes place. It is also logical to assume that the basic folding of rECH1 would be similar to the other members of the same family. Interestingly, in the hydratase 1, the E144 (25), which acts as the catalytic base for activation of a water molecule in the hydratase, is replaced with Asp-127 in rECH1.
The origin of the two immunodetected bands of molecular masses of 36,000 and 32,000 Da in the liver homogenates is intriguing. A logical explanation is provided by the results of immunoblot experiments with peroxisomes and mitochondria from rat liver. Because the band of 36,000 Da was detected in isolated peroxisomes and rECH1p has the known peroxisomal targeting signal type 1 (SKL) in its C terminus, it is possible that the product encoded by the entire open reading frame is targeted to the peroxisomal matrix and remains uncleaved. However, the first 40 amino acid residues of rECH1p resemble a mitochondrial matrix targeting signal as shown by Mitoprot II analysis (45). Thus, the band of 32,000 Da detected in tissue homogenates and in isolated mitochondria likely represents a processed mitochondrial isoform. The size difference between the 36,000 and 32,000 Da bands is consistent with the length of the predicted mitochondrial targeting signal. Thus, rECH1p is a new example of dual-organelle distribution of the same protein in mammalian cells. The immunoelectron microscopy of liver sections also provides further support for the dual distribution of rECH1p. An earlier example of this kind of intracellular targeting is the mammalian hydroxymethylglutaryl-CoA lyase (46), which also has a single mRNA translated into a polypeptide with both targeting signals. As a consequence, in mammalian tissues, a larger peroxisomal and a smaller mitochondrial hydroxymethylglutaryl-CoA lyase isoform is found. Similarly, in some species like rat and mouse, alanine:glyoxylate aminotransferase is located both in mitochondria and peroxisomes, whereas in humans it is mostly peroxisomal. In some patients with inherited deficiency of alanine:glyoxylate aminotransferase (primary hyperoxaluria type I), mutations alter the targeting of the protein so that most of it localizes to mitochondria rather than to peroxisomes (47). Interestingly, expression in Pichia pastoris of full-length rECH1 gave two bands with molecular weights corresponding to those observed in rat liver homogenates (data not shown).
The 32,000-Da size of the form of rECH1p that we localized to mitochondria is consistent with the subunit sizes previously reported for mitochondrial dienoyl-CoA isomerase (13). Furthermore, our observation that mitochondrial and peroxisomal dienoyl-CoA isomerases are actually isoforms of the same protein explains their similar kinetic properties (12). The relationship of rECH1p to the rat liver dienoyl-CoA isomerase activities of 55,000 Da (11) and 66,000 Da (12) is uncertain.
Recently Tserng et al. (48) carried out experiments applying
a stable isotope-labeled substrate technique to assess the contribution
of dienoyl-CoA isomerase-/dienoyl-CoA reductase-dependent pathway versus direct -oxidation of
cis-5-enoyl-CoA esters. C10 substrate was
metabolized completely via dienoyl-CoA isomerase-reductase pathway in
both rat liver and heart mitochondria. When the chain length of the
substrate was extended to C14, the isomerase/reductase pathway remained the major metabolic route, although its overall contribution to the metabolism decreased to 86 and 65% in liver and
heart mitochondria, respectively. This experiment verified the role of
isomerase/reductase pathway in rat mitochondria, and the authors also
suggest that 2,4-dienoyl-CoA reductase is the rate-limiting enzyme in
this pathway.
Thus far only one inborn error of the auxiliary enzymes of
-oxidation has been identified in humans, namely a single patient with 2,4-dienoyl-CoA reductase deficiency (49). This paucity of
clinical material might simply reflect the fact that until recently
this enzyme system has been poorly appreciated, or it may be that
affected individuals are not recognized.
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ACKNOWLEDGEMENTS |
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We are grateful to Tanja Kokko, Irma Vuoti, and Marika Yppärilä for their skillful technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Sigrid Juselius Foundation and from the Academy of Finland. A portion of this work was supported by an award from National Institute of Child Health and Human Development, National Institutes of Health Grant HD10981 to the Kennedy Kreiger Institute.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.
An Investigator in the Howard Hughes Medical Institute.
§§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Oulu, Linnanmaa, FIN-90570 Oulu, Finland. Tel.: 358-8-553 1150; Fax: 358-8-553 1141.
1 The abbreviations used are: MFE1, multifunctional hydratase/dehydrogenase enzyme type 1; rECH1, rat enoyl-CoA hydratase; rECH1p, rECH1 protein; PBS, phosophate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
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