From the Department of Pharmaco-Biology, Laboratory
of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy, the § Department of
Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology,
University of Calabria, Arcavacata di Rende, 87030 Cosenza,
Italy, the ¶ Institute of Genetics, University of Bari, 70125 Bari, Italy, and
The Medical Research Council-Dunn Human
Nutrition Unit, Hills Road, Cambridge CB2 2XY, United Kingdom
Received for publication, October 20, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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In Saccharomyces
cerevisiae, the genes ODC1 and ODC2 encode isoforms of the
oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates
across the inner membranes of mitochondria and are members of the
family of mitochondrial carrier proteins. Orthologs are encoded in the
genomes of Caenorhabditis elegans and Drosophila
melanogaster, and a human expressed sequence tag (EST)
encodes part of a closely related protein. Information from the EST has
been used to complete the human cDNA sequence. This sequence has
been used to map the gene to chromosome 14q11.2 and to show that the
gene is expressed in all tissues that were examined. The human protein
was produced by overexpression in Escherichia coli,
purified, and reconstituted into phospholipid vesicles. It has similar
transport characteristics to the yeast oxodicarboxylate carrier
proteins (ODCs). Both the human and yeast ODCs catalyzed the transport
of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a
counter-exchange mechanism. Adipate, glutarate, and to a lesser extent,
pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were
also transported by the human ODC. The main differences between the
human and yeast ODCs are that 2-aminoadipate is transported by the
former but not by the latter, whereas malate is transported by the
yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a
common intermediate in the catabolism of lysine, tryptophan, and
hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely
to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism
of lysine, tryptophan, and hydroxylysine.
In mammals, 2-oxoadipate is produced from lysine in the cytosol of
cells via the saccharopine and the pipecolic acid pathways. Catabolites
of hydroxylysine and tryptophan enter these pathways as
2-aminoadipic- Over the years, several patients with 2-oxoadipate acidemia, an in-born
error of metabolism of lysine, tryptophan, and hydroxylysine, have been
reported, most of them slightly to deeply mentally retarded with
hypotonia or seizures (1-7). It was speculated without experimental verification that their abnormal levels of serum and urinary
2-oxoadipate might arise from a defect in 2-oxoadipate dehydrogenase.
As this enzyme is found in the matrix of mitochondria, and 2-oxoadipate is produced in the cytosol (8), impaired transport of 2-oxoadipate into
the organelle provides an alternative explanation, but until the
present work no such transport function had been demonstrated in man.
In this paper, the identification of the human 2-oxoadipate
mitochondrial carrier (ODC)1
is described. It is based on two isoforms, ODC1 and ODC2 encoded in the
genome of Saccharomyces cerevisiae, that transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate across the inner membranes of mitochondria (9). However, yeast and man are too distant
phylogenetically for these isoforms to provide a basis for direct
cloning of the mammalian counterpart. Therefore, orthologs were sought
and detected in Caenorhabditis elegans and Drosophila melanogaster and used to bridge between yeast and man. In this way, a human EST was identified that encodes a fragment of a related protein. It provided information to complete the human cDNA
sequence. The encoded protein is 299 amino acids long and has the
characteristic features of the family of mitochondrial carrier proteins
(10-13). It was overexpressed in Escherichia coli,
reconstituted into phospholipid vesicles, and shown to have transport
specificity and other biochemical properties similar to those of the
recombinant yeast ODC isoforms, including transport of
2-oxoadipate.
Sequence Search and Analysis--
Data bases of the genomes of
C. elegans and D. melanogaster at the Sanger
Center (Hinxton, United Kingdom) and at the National Center for
Biotechnology Information (Washington, D. C.) were screened with the
sequences of the S. cerevisiae ODC proteins (9) using the
program BLASTP. The NCBI nonredundant EST human data base was probed
with the program TBLASTN. Amino acid sequences were aligned with
ClustalW (version 1.7).
Human and Rat cDNAs for the ODC--
Touchdown PCRs (14)
were performed with adaptor-ligated double-stranded human liver
cDNA (1 ng, CLONTECH), as described previously (15, 16). The full-length cDNA sequence was
obtained in two PCR reactions of 5'- and 3'-extension using the adaptor primers AP1 and AP2 (CLONTECH) and a primer set 1F,
2F, 1R, and 2R (Fig. 1) designed from the nucleotide sequence of the
human EST, R29313. The PCR products were identified, recovered from agarose gels, cloned into the pCR2.1 topo-vector (Invitrogen), and
sequenced. The cDNA for rat ODC was amplified similarly with oligonucleotides based on the human cDNA sequence.
Cytogenetics--
Human metaphase chromosome spreads were
obtained from phytohemagglutinin-stimulated peripheral
lymphocytes. A probe for FISH analysis was made by PCR amplification of
human genomic DNA using primers corresponding to nt 916-939 (forward
sense) and nt 1191-1212 (reverse sense) of the human cDNA for ODC
(see Fig. 1). The resulting 4.2-kb fragment was cloned in the pCR2.1
topo-vector and sequenced to confirm its identity. The FISH experiments
were performed as described previously (17).
Expression Analysis by RT-PCR--
Total RNAs (2 µg) were
extracted and reverse-transcribed with the Gene Amp RNA PCR Core kit
(PerkinElmer Life Sciences) using either random hexamers or
oligo(dT)16 as primers (final volume, 40 µl). A 230-bp
fragment of the ODC cDNA was then amplified from the reverse
transcription reaction products (20 µl) by 35 cycles of PCR using
oligonucleotides RT1F (nt 662-682) and RT1R (nt 869-892) as forward
and reverse primers, respectively (Fig. 1). The products were probed
with the radiolabeled oligonucleotide RT1P (nt 804-825; Fig. 1). As a
control, a 384-bp Bacterial Overexpression of the Human ODC--
The coding
regions for the human and rat ODCs were amplified from human and rat
liver cDNAs (1 ng) by 35 cycles of PCR. The forward and reverse
primers in these reactions corresponded to nt 311-331 and 1188-1210
of the human ODC cDNA (see Fig. 1) and to nt 100-120 and 976-996
of the rat ODC cDNA (deposited as GenBankTM
accession number AJ289714). The forward and reverse primers carried an
NdeI and a XhoI site, respectively, at their
5'-ends. The 0.9-kb products were gel-purified and cloned into the pRUN expression vector. Transformants of E. coli DH5 Transport Assays--
The recombinant protein in Sarkosyl was
reconstituted into liposomes in the presence of substrates, as
described previously (9). External substrate was removed from
proteoliposomes on a Sephadex G-75 columns. The transport activity at
25 °C was determined by measuring the uptake (forward exchange) or
the efflux (backward exchange) of [14C]oxoglutarate in
exchange for unlabeled counter-substrates (22). For backward exchange
measurements the proteoliposomes containing 1 mM internal
oxoglutarate were prelabeled, after reconstitution, by carrier-mediated
exchange equilibration by adding 10 µM
[14C]oxoglutarate (22). After 30 min, the residual
external radioactivity was removed by passing the proteoliposomes
through a column of Sephadex G-75. In forward exchange reactions,
transport was started by adding [14C]oxoglutarate to the
proteoliposomes and in the backward exchanges by adding nonradioactive
substrate. In both cases, transport was stopped after 1 min (in the
initial linear range of substrate exchange) by addition of 10 mM pyridoxal 5'-phosphate and 10 mM bathophenanthroline (the "inibitor-stop" method (22)). In controls, the inhibitors were added at the beginning together with the external substrate. Finally, the external substrate was removed, and the radioactivity in the liposomes was measured (22). In forward exchange
measurements, the experimental values were corrected by subtracting
control values, and the rate was calculated in millimoles/min/gram of
protein. In the case of backward exchanges, the rate in Other Methods--
Proteins were analyzed by SDS-PAGE and
stained with Coomassie blue dye. N-terminal sequencing was carried out
as described previously (21). The amount of pure ODC was
estimated by laser densitometry of stained samples (21). The amount of
ODC incorporated into liposomes was measured as described
previously (23) and varied between 29 and 37% of the protein
added to the reconstitution mixture. Western blotting was carried out
as described previously (24) with a rabbit antiserum raised
against the bacterially expressed rat ODC protein.
Sequences of the Human and Rat ODCs--
By screening data bases,
nematode and fruit fly clones (R11.1 and AAF45544.1) were found with 36 and 38% of the residues of their encoded proteins identical,
respectively, to the yeast ODC protein sequences. A human EST data base
was interrogated with these sequences, and a clone of 286 bp (R29313)
was identified that encoded a protein sequence that was 62 and 68%
identical, respectively, to regions of the C. elegans and
the D. melanogaster proteins.
The human EST was extended in the 5'- and 3'-directions by two PCR
experiments. Among the products of the 5'-extension reaction, one band
of about 1200 bp hybridized with primer 2F. Its sequence overlapped the
EST. The 3'-extension product contained a single band of about 950 bp.
Its sequence overlapped the 5'-extension. The final human cDNA
sequence of 2024 nucleotides (Fig. 1)
consisted of a 310-bp 5'-untranslated region, followed by an open
reading frame of 900 bp, a 814-bp 3'-untranslated region containing a polyadenylation signal at nt 1974-1979 (25), and a
poly(A)+ tail (see Fig. 1). The ATG codon at nt 311-313 is
preceded by an in-frame stop codon 102-bp upstream and is likely to be
the translational initiation codon. The N terminus of the human protein is at approximately the same position as in the rat, yeast, nematode, and D. melanogaster orthologs, confirming this view. The
open reading frame encoded a polypeptide of 299 amino acids with a calculated isoelectric point of 9.6 and a molecular mass of 33,300 Da.
The cDNA for the rat ODC was cloned in a similar way. This sequence
(GenBankTM accession number AJ289714) consists of
1456 nucleotides with a 99-bp 5'-untranslated region, followed by an
open reading frame of 897 bp, and a 460-bp 3'-untranslated region,
containing neither a polyadenylation signal nor a poly(A)+
tail. The open reading frame encoded a polypeptide of 298 amino acids
with a molecular mass of 33,276. The human ODC is 82, 56, 55, 32, and
33% identical to the R. norvegicus, C. elegans,
D. melanogaster, and S. cerevisiae ODC1 and ODC2
proteins, respectively.
Chromosomal Location of the Gene for Human ODC--
The gene for
human ODC was found on chromosome 14 at 14q11.2 by FISH experiments
conducted on normal human metaphase chromosomes (Fig.
2). There was no evidence in this
experiment for a second gene encoding an isoform, and no isoforms were
detected in searches of data bases of human, nematode, and fruit fly
sequences.
Expression of ODC in Various Tissues--
The tissue distribution
of mRNAs for the human and rat ODCs was studied by RT-PCR performed
on total RNA populations, using primers and probes from regions of
identity between the human and the rat nucleotide sequences. The ODC
was detected in all tissues that were examined (Fig.
3A, panel a). The
relatively weak signal from heart arises because less total RNA was
employed than in other tissues, as the control demonstrates (see
panel b in Fig. 3A). A similar pattern of
expression was observed by Western blot analysis of rat mitochondria
(see Fig. 3B, panel a).
Bacterial Overexpression of the Human ODC--
The human ODC was
overexpressed in E. coli C0214(DE3) (see Fig.
4, lane 4) in the form of
inclusion bodies. The purified protein gave a single band by SDS-PAGE
(Fig. 4, lane 5) with an apparent molecular mass of
32 kDa. The protein was not detected in bacteria harvested immediately
before induction of expression (Fig. 4, lanes 1 and
2), nor in cells harvested after induction but lacking the
coding sequence in the expression vector (lane 3). The
N-terminal sequence (SAKPEVSLVR) of residues 1-10 of the purified
protein was identical to that predicted for residues 2-11 of the human ODC (Fig. 1). About 35 mg of purified protein were obtained per liter
of culture.
Functional Characterization of Human ODC--
Proteoliposomes
reconstituted with recombinant ODC catalyzed a counter-exchange of
external [14C]oxoglutarate for internal oxoglutarate
with first order kinetics (rate constant 0.08 min Substrate Specificity and Inhibitor Sensitivity--
The substrate
specificity of human ODC was investigated in greater detail by
measuring the uptake of [14C]oxoglutarate into
proteoliposomes that had been preloaded with a variety of substrates
(Fig. 5A) or the efflux of
[14C]oxoglutarate from proteoliposomes in the presence of
external nonradioactive substrates (Fig. 5B). High
[14C]oxoglutarate transport activities were observed when
oxoglutarate, oxoadipate, glutarate, and adipate were used as
counter-substrates on both sides of the liposomal membrane. High
activities were also found with external pimelate and oxopimelate. To a
lesser extent, internal pimelate, oxopimelate, and 2-aminopimelate, as well as both internal and external 2-aminoadipate, citrate, and oxaloacetate, also exchanged for labeled oxoglutarate. Low exchange, if
any, was found with aspartate, fumarate, glutamate, glutathione, isocitrate, malate, maleate, malonate, oxalate, pyruvate, suberate, and
succinate (Fig. 5, A and B).
The uptake of 0.2 mM [14C]oxoglutarate by
proteoliposomes containing 20 mM oxoglutarate (reaction
time, 1 min) was inhibited almost completely by 2 mM
pyridoxal 5'-phosphate (97%) and partly by 2 mM
bathophenanthroline and 2 mM 2-cyanocynnamate (73 and 69%
inhibition, respectively). Low concentrations of organic mercurials (10 µM) also inhibited markedly the ODC activity
(p-chloromercuribenzene sulfonate, 58%; HgCl2,
64%; and p-hydroxymercuribenzoate, 47%). No significant
inhibition was observed with 2 mM butylmalonate, phenylsuccinate, 1,2,3-benzenetricarboxylate, and
N-ethylmaleimide (inhibitors of other characterized
mitochondrial carriers) and 10 µM carboxyatractyloside (a
powerful inhibitor of the ADP/ATP carrier).
Kinetic Characteristics of Human ODC--
To obtain kinetic
information about the [14C]oxoglutarate/oxoglutarate
exchange, the dependence of the exchange rate on substrate concentration was investigated at different concentrations of externally added [14C]oxoglutarate (0.05-2
mM) at a constant internal concentration of 20 mM. The transport affinity (Km) and the
specific activity (Vmax) values for oxoglutarate
exchange at 25 °C, calculated from a standard double-reciprocal set
of 35 experiments, were 0.22 ± 0.02 mM and 7.5 ± 1.2 mmol/min/g of protein, respectively. The activity was calculated
by taking into account the amount of ODC recovered in the
proteoliposomes after reconstitution.
The inhibition constants (Ki) of several externally
added substrates are summarized in Table
I. All of them increased the apparent
Km without changing the Vmax
of [14C]oxoglutarate/oxoglutarate exchange (not shown)
acting as competitive inhibitors. In general, the
oxoglutarate/oxoglutarate exchange was prevented by external addition
of each of the substrates that are transported by human ODC (Fig. 5,
A and B), and it was not affected by substrates
of other mitochondrial carriers such as phosphate, ADP, ornithine, and
carnitine. However, suberate, which is not transported by the ODC
protein, inhibited the exchange activity significantly (Table I),
suggesting that it binds to the substrate binding site of ODC without
being transported.
The phylogenetic distance between S. cerevisiae and man
frequently precludes the use of yeast sequences to identify human orthologs directly with certainty. The problem is even more severe when
the protein in question is a member of a family with different functions but related sequences, as are the two isoforms of the yeast
oxodicarboxylate carrier (9), which is a recently identified member of
the family of mitochondrial transport proteins. Known family members
are involved in the traffic of various substrates and metabolites
across the inner membranes of the organelle. One possible solution,
employed successfully in the cloning the rat dicarboxylate carrier
(16), is to use the yeast sequence to identify orthologs in
phylogenetically intermediate species where the genome sequence is
known, such as C. elegans and D. melanogaster, and then to
use these orthologs to identify potential orthologs in man. Thus, the
yeast ODC isoforms were used to find clones in the C. elegans and D. melanogaster genomes, and they were used to identify a short related protein sequence in a human EST. This sequence proved to be part of the human ODC and provided the route to
the complete human and rat ODC sequences. Both have the tripartite structure and the sequence motif that are characteristic of the mitochondrial carrier family (10-13).
The transport characteristics of the human ODC are similar to the yeast
ODC isoforms. The main differences are the ability of the human protein
to transport 2-aminoadipate, although rather poorly, and its incapacity
to transport L-malate. The properties of the human ODC
differ markedly from those of the oxoglutarate-malate carrier, which
has greatest affinities for C4 and C5 oxodicarboxylates and
dicarboxylates (19, 26, 27), whereas the human ODC prefers the C5-C7
homologs (Fig. 5 and Table I). In contrast to the bovine oxoglutarate-malate carrier, the human ODC does not transport malate,
succinate, and maleate, but it does transport citrate, albeit at low
efficiency. The low sequence identity between the ODC and
oxoglutarate-malate carrier is also consistent with their different
properties and functions.
The best substrates for the human ODC, both on the external and the
internal membrane surfaces, are 2-oxoadipate and 2-oxoglutarate. Therefore, the physiological role of the human ODC is most likely to be
to catalyze the uptake of 2-oxoadipate into the mitochondrial matrix in
exchange for internal 2-oxoglutarate, thus performing a central role in
catabolism of lysine, hydroxylysine, and tryptophan. Since ODC
functions by a counter-exchange mechanism, the carrier-mediated uptake
of 2-oxoadipate requires the efflux of a counter-substrate. On the
basis of transport measurements, 2-oxoglutarate may serve as the
counter-substrate of ODC for 2-oxoadipate. The efflux of 2-oxoglutarate
is required by the lysine-oxoglutarate reductase, which, in the first
step of lysine catabolism, converts lysine and 2-oxoglutarate into
saccharopine. In agreement with its central role in cell metabolism,
the human ODC is expressed in all tissues that were analyzed.
Another possible role for the human ODC may be to catalyze the uptake
of 2-aminoadipate into the mitochondrial matrix when this amino acid is
not rapidly transaminated to 2-oxoadipate in the cytosol, for
example after diets rich in amino acids that cause a decrease in
2-oxoglutarate content in the cytosol and consequently an inhibition of
the 2-aminoadipate aminotransferase. In this respect it is worth
mentioning that transaminases interconverting oxoadipate and
aminoadipate are present both in the cytosol and inside the
mitochondria (28, 29). The physiological role of ODC also suggests its
possible involvement in 2-oxoadipate acidemia, which is accompanied by
accumulation and excretion of large amounts of 2-oxoadipate,
2-aminoadipate and 2-hydroxyadipate in urine, and by the clinical
symptoms of mental retardation, hypotonia, motor and developmental
delay, cerebellar ataxia, and learning disability (1-7). The molecular
defect(s) responsible for this disease have not been characterized.
Fibroblasts of patients with this condition are unable to oxidize
2-amino[1-14C]adipic and 2-oxo[1-14C]adipic
acid to 14CO2 (2, 6) to any significant extent.
Therefore, it was suggested that the disease may be due to defective
2-oxoadipate dehydrogenase, but no such defect has ever been
demonstrated. The alternative possibility that defective ODC might
provide the basis for this human metabolic disease can now be investigated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-semialdehyde and 2-oxoadipate, respectively. In
the matrix of mitochondria, 2-oxoadipate is decarboxylated to
glutaryl-CoA by the 2-oxoadipate dehydrogenase complex and then
converted to acetyl-CoA.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin fragment was amplified from the remainder
of the reverse transcription products (18).
cells
were selected on ampicillin (100 µg/ml) and screened by direct colony
PCR and restriction digestion of plasmids. The sequences of inserts
were verified. The overproduction of the ODC as inclusion bodies in the
cytosol of E. coli was accomplished as described first for the bovine oxoglutarate-malate carrier (19), except that the host cells
were E. coli CO214(DE3) (20). Control cultures with the
empty vector were processed in parallel. Inclusion bodies were
isolated, and ODC was purified by centrifugation and washing steps as
described previously (19, 21).
cpm/min was
obtained from the decrease of internal radioactivity in 1 min. The
reconstituted protein was assayed for other exchange activities by the
inhibitor-stop method (22).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Sequences of the cDNA for the human ODC
and of the encoded protein. Amino acids are numbered from 1 to
299. The stop codon is denoted by an asterisk. The
boldface sequence is a potential polyadenylation signal. The
underlined sequence in the 5'-region is a stop codon
in-frame with the initiator methionine. The partial cDNA sequence
was extended in 3'- and 5'-directions with primers AP1 and AP2 and
nested oligonucleotides 1F/2F or 1R/2R, respectively. Primers RT1F and
RT1R and probe RT1P were employed in reverse transcription polymerase
chain reaction experiments. Primer sequences are boxed;
horizontal arrows pointing to the right or
left indicate that the primers were synthesized as either
the sequence shown or its complement, respectively.
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Fig. 2.
Chromosomal location of the gene for the
human ODC. A human metaphase spread was hybridized in
situ with a human ODC probe generated by PCR. The red
probe is a 4.2-kb genomic fragment of human ODC. The arrow
indicates the location of ODC at 14q11.2. A partial metaphase spread,
from a different metaphase, is shown in the inset.
Pseudocoloring and merging of images were performed with Adobe
PhotoshopTM.
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Fig. 3.
Expression of the ODC in various
tissues. A, analysis of total RNA from human
(h) and rat (r) tissues. Panel a,
hybridization with probe RT1P of the ODC cDNA fragments obtained by
RT-PCR. panel b, ethidium bromide staining of the -actin
cDNA fragments obtained by RT-PCR. B, immunodetection of
the ODC protein in mitochondria isolated from rat tissues. In
panels a and b, mitochondria (150 µg of
protein) and recombinant rat ODC (75 ng) were exposed to antisera to
ODC and subunit IV of the cytochrome c oxidase,
respectively.
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Fig. 4.
Overexpression of the human ODC in E. coli. Proteins were separated by SDS-PAGE and stained with
Coomassie Blue. Lane M, markers (bovine serum albumin,
carbonic anhydrase, and cytochrome c); lanes
1-4, E. coli C0214(DE3) containing the expression
vector, without (lanes 1 and 3) and with the
coding sequence of ODC (lanes 2 and 4). Samples
were taken at the time of induction (lanes 1 and
2) and 5 h later (lanes 3 and 4).
The same number of bacteria was analyzed in each sample. Lane
5, purified ODC protein (1.2 µg) originated from bacteria shown
in lane 4.
1), isotopic equilibrium being approached
exponentially (data not shown). The exchange reaction was inhibited
completely by a mixture of pyridoxal 5'-phosphate and
bathophenanthroline. In the absence of substrate in the
proteoliposomes, or if the solubilized protein was boiled before the
incorporation into liposomes, there was no uptake of labeled external
substrate. Similarly, no oxoglutarate/oxoglutarate exchange was
detected by reconstitution of Sarkosyl-solubilized material from
bacterial cells either lacking the expression vector for ODC or
harvested immediately before induction of expression. Furthermore, the
proteoliposomes did not catalyze homoexchange activities for phosphate,
carnitine, glutamate, aspartate, glutamine, ornithine,
L-malate, ADP, and ATP (internal concentration, 10 mM; external concentration, 1 mM).
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Fig. 5.
Substrate specificity of human ODC.
A, dependence of ODC activity on internal substrate.
Proteoliposomes were preloaded internally with the indicated substrates
(concentration, 20 mM). Transport was started by the
addition of 0.2 mM [14C]oxoglutarate and
stopped after 1 min. B, dependence of ODC activity on
external substrate. Proteoliposomes were preloaded internally with 1 mM oxoglutarate and then the internal substrate pool was
labeled by carrier-mediated exchange equilibration. After removal of
external substrate by Sephadex G-75 chromatography, the efflux of
[14C]oxoglutarate was started by adding the indicated
substrates (concentration, 1 mM) and terminated after 1 min. At time 0 the intraliposomal radioactivity was 24,200 cpm and did
not varied in the absence of external substrate for 30 min.
Ki values for substrates competing with
[14C]oxoglutarate uptake
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FOOTNOTES |
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* This work was supported by the Consiglio Nazionale delle Ricerche Target Project "Biotechnology," by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST), by the Piano Biomedicina, Progetto No. 1 Cluster C04 L.488/92, by MURST 5%-Consiglio Nazionale delle Ricerche program Biomolecole per la Salute Umsna L. 95/95, and by the European Social Fund.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) AJ278148 and AJ289714 (for the human and rat ODC cDNAs, respectively).
** To whom correspondence should be addressed. E-mail: walker@mrc-dunn.cam.ac.uk.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M009607200
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ABBREVIATIONS |
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The abbreviations used are: ODC, oxodicarboxylate carrier protein; bp, base pair(s); EST, expressed sequence tag; FISH, fluorescence in situ hybridization; kb, kilobase(s); nt, nucleotide; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription polymerase chain reaction.
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27. | Bisaccia, F., Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369[Medline] [Order article via Infotrieve] |
28. | Tobes, M. C., and Mason, M. (1977) J. Biol. Chem. 252, 4591-4599[Abstract] |
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