The Mechanism of Adenosine Formation in Cells
CLONING OF CYTOSOLIC 5'-NUCLEOTIDASE-I*
Graciela B.
Sala-Newby
,
Andrzej C.
Skladanowski
§, and
Andrew
C.
Newby
¶
From the
University of Bristol, Bristol Heart
Institute, Bristol BS2 8HW, United Kingdom and § Department
of Biochemistry, Medical University of Gdansk, ul. Debinki
1 80-211, Gdansk, Poland
 |
ABSTRACT |
Adenosine increases blood flow and decreases
excitatory nerve firing. In the heart, it reduces rate and force of
contraction and preconditions the heart against injury by prolonged
ischemia. Based on indirect kinetic arguments, an AMP-selective
cytosolic 5'-nucleotidase designated cN-I has been implicated in
adenosine formation during ATP breakdown. The molecular identity of
cN-I is unknown, although an IMP/GMP-selective cytosolic
5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have
been cloned. We utilized the high abundance of cN-I in pigeon heart to
purify a 40-kDa subunit for partial protein sequencing and subsequent cDNA cloning. We obtained a full-length clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in
heart, brain, and breast muscle. Immunolocalization in heart showed a
striated cytoplasmic location, suggesting association with contractile
elements. Transient expression in COS-7 cells, generated a
5'-nucleotidase that catalyzed adenosine formation from AMP, which was
increased during ATP catabolism. In conclusion, the cloning and
expression of cN-I provides definitive evidence of its ability to
produce adenosine during ATP breakdown.
 |
INTRODUCTION |
The formation of adenosine during net catabolism of cytosolic ATP
has been proposed to function as a signal of metabolic stress that
brings about appropriate retaliatory actions against the conditions
leading to stress (1, 2). The actions of adenosine include
A1 receptor-mediated opening of K+ channels
leading to decreased stimulatory neurone firing and hyperpolarization
of postsynaptic cells and antagonism of the effects of catecholamines
on adenylate cyclase (3, 4). A2A receptor-mediated effects
include vasodilatation leading to the increased delivery of substrates
and oxygen to underperfused tissues (4, 5). In the heart, these
mechanisms lead to a spectrum of responses including decreased heart
rate, slower atrioventricular conduction, decreased force of
contraction, and increased blood flow, all of which tend to reverse an
imbalance between ATP formation and utilization (3, 6). Adenosine,
acting through A1 or A3 receptors, also appears
to play a role in ischemic preconditioning, a process by which brief
ischemia can protect the heart against a subsequent longer period of
infarction (4, 7, 8).
Cloning of e-N1 (EC 3.1.3.5)
(9) confirmed a wealth of biochemical
findings that this enzyme is involved exclusively in the hydrolysis of
extracellular AMP, produced for example during cholinergic nerve
transmission (10) and at the endothelial surface from degranulating
platelets (11). Despite their importance, the mechanisms responsible
for cytosolic AMP breakdown to adenosine have been based until now
solely on indirect kinetic arguments. These have led to the conclusion
that an IMP-preferring cytosolic 5'-nucleotidase, cN-II, abundant in
the liver, probably plays a minor role in adenosine formation,
especially in tissues such as heart that have an active AMP deaminase
(12). An AMP preferring enzyme, cN-I, has been purified from pigeon,
rabbit, rat, dog, and human hearts (13-17). The kinetic properties of
cN-I, particularly its potent activation by ADP but not ATP (14, 15)
and regulation by pH (18), are more consistent with a role in adenosine
formation. However, the high apparent Km for the
enzyme of 5 mM (obtained at the optimum pH and with ADP
concentrations above 50 µM) has cast doubt on its ability
to generate adenosine under the conditions of the cell. Indeed, the
capacity of any cytosolic 5'-nucleotidase to generate adenosine in
intact cells has not previously been tested directly. The primary
purpose of the present experiments was therefore to clone cN-I.
Furthermore, we sought to obtain the first definitive evidence that a
cytosolic 5'-nucleotidase was capable of catabolizing cytosolic
AMP to adenosine under the conditions present within cells,
particularly after metabolic poisoning to induce ATP breakdown.
 |
EXPERIMENTAL PROCEDURES |
Protein Purification and Sequencing--
The cN-I enzyme was
purified from pigeon (Columba livia) heart ventricles as
described (14). A 40-kDa peptide was excised from 10%
SDS-polyacrylamide gels, and peptides were generated by in
situ tryptic proteolysis, separated by small bore reverse phase
hplc, and N-terminally sequenced by Edman degradation (19).
Cloning of the cDNA of cN-I--
A pigeon ventricle
directional cDNA library was constructed in the vector
Zap II
(Stratagene, Cambridge, UK). RNA was extracted from one pigeon heart
apex using ULTRASPEC (AMS, Witney, UK), poly(A)+ RNA was
isolated using Oligotex resin (Qiagen, Crawley, UK). Degenerate
antisense oligonucleotide primers were designed from the microsequenced
peptide doubly underlined in Fig. 1. This was used paired
with an oligonucleotide primer corresponding to the T3 promoter
sequence present at the 5' end of the
phage cloning site. Touchdown
PCR (20) was performed on 1 µl of the amplified pigeon library
(corresponding to 3 × 107 plaque forming units or 20 copies of each clone). The PCR product was subcloned into pGEM-T
(Promega, Chilworth, UK), and a clone coding for the remainder of the
peptide sequence was obtained. This fragment (corresponding to
nucleotides 107-285; Fig. 1) was excised, 32P-labeled by
random priming, and used to screen 5 × 105 plaques
from the library using the Rapid-hyb system (Amersham Pharmacia
Biotech, Little Chalfont, UK). Pure
phage from positive clones
was in vivo excised to generate clones in the pBluescript SK
plasmid as described by the manufacturer. Automated
sequencing was conducted in both directions using the dideoxy chain
termination method and an ABI (Perkin-Elmer, Applied Biosystems
Division, Warrington, UK) automated sequencer.
Expression of the cN-I cDNA in COS-7 Cells--
The
full-length cDNA clone was subcloned into the
BamHI-KpnI site of pTargeT (Promega). COS-7 cells
were routinely cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM glutamine,
100 µg/ml streptomycin, 100 units/ml penicillin. COS-7 cell were
transfected in 6-well plates using per well 10 µl of LipofectAMINE
(Life Technologies, Inc., Paisley, UK) with 0.8 µg of cN-I coding
plasmid plus 0.2 µg of pSV-
-galactosidase (Promega) control
plasmid or 1 µg of pSV-
-galactosidase control plasmid and used
48 h after transfection. Cells were transferred to KRH (containing
120 mM NaCl, 48 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgSO4,
1.3 mM CaCl2, 25 mM HEPES, pH 7.3)
and preincubated for 15 min with 10 µM
erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of
adenosine deaminase (EC 3.5.4.4) (21). Cells were then incubated for 5 min in fresh medium containing
erythro-9-(2-hydroxy-3-nonyl)adenine and 50 µM
5'-amino-5'-deoxyadenosine to inhibit adenosine kinase (EC 2.7.1.20)
(21). Glycolysis was inhibited by addition of 10 mM
2-deoxy-D-glucose, and mitochondrial ATP synthesis was
inhibited with 1 µM carbonyl cyanide
p-(trifluoromethoxy) phenyl-hydrazone. Cell incubations were
terminated and metabolite and protein concentrations were determined as
described below. The cell viability was assessed by measurement of LDH
in cell supernatants at the end of the incubations (cytotoxicity test,
Roche Molecular Biochemicals, Lewis, UK) and expressed as a percentage
of the total LDH present in parallel wells extracted in KRH containing
0.1% Triton X-100.
Characterization of Recombinant cN-I in COS-7 Cell
Extracts--
Untransfected and cN-I-transfected COS-7 cells were
extracted with a buffer containing 40 mM
N-dimethylglutarate, 40 mM
-glycerophosphate, 200 mM KCl, 0.2 mM dithiothreitol, 12.6 mM MgCl2, 0.2% Triton X-100, pH 6.9. Extracts
were subjected to polytronic homogenization for 15 s and then
centrifuged at 10,000 × g for 10 min at 4 °C. Aliquots of extract were incubated for between 0 and 10 min at 37 °C
with 10 µM
erythro-9-(2-hydroxy-3-nonyl)adenine, 10 µM
5'-deoxy-5'aminoadenosine, 50 µM
,
-methylene-ADP
(final concentrations) and with 4.8 mM ATP, 2.8 mM ADP, 0.66 mM AMP, or (for the
Km determinations) a range of AMP concentrations as
described previously (22). Adenosine and protein concentrations were
measured as described below.
Measurement of Nucleoside, Nucleotide, and Protein
Concentrations--
Addition of 0.75 volumes of 1.6 M
HClO4 was used to terminate reactions followed by
neutralization of supernatants with 0.125 volumes of 3 M
K3PO4. Nucleosides and nucleotides were
measured by reverse phase hplc as described (23). To resolve AMP from 5'-deoxy-5'-aminoadenosine, an additional run was performed at pH 6.7, and the following gradient (gradient B: 15% acetonitrile in buffer A
containing 150 mM KCl and 150 mM
KH2PO4) changed linearly as follows: 0 min, 0%
B; 0.1 min, 3% B; 3 min, 9% B; 7.5 min, 100% B; 8.5 min, 0% B for
3.5 min. Protein concentrations were measured in the HClO4
pellets or in separate Triton X-100 extracts by micro BCA assay
(Pierce, Chester, UK).
Northern Blot Analysis--
Total RNA dissolved in RNA sample
buffer (Sigma, Poole, UK) was size fractionated on a 1.2% agarose gel
(24) and transferred to Hybond N+ nylon membrane and
hybridized using Rapid-hyb (Amersham Pharmacia Biotech) following the
manufacturer's instructions.
Immunocytochemistry and Western Blots--
A rabbit polyclonal
antibody was generated by immunization with a multiple antigenic
peptide (25) of amino acids 222-233. The specificity of the antibody
was confirmed by Western blot analysis. Equal quantities of protein (35 µg/lane) extracted from pigeon ventricle, untransfected COS-7 cells,
and COS-7 cells expressing cN-I were fractionated by SDS-polyacrylamide
gel electrophoresis (10%) and transferred to nitrocellulose. Antibody
binding was detected by ECL (Amersham Pharmacia Biotech).
Immunocytochemistry was conducted using the peroxidase antiperoxidase
method (26) on 4-µm frozen sections of pigeon ventricle that had been
fixed with cold acetone.
Statistical Methods--
Values are expressed throughout as the
means ± S.E. Comparisons are made by the Student's t
test using paired data.
 |
RESULTS |
We took advantage of the high abundance of cN-I in pigeon heart to
purify the enzyme using a published method (14). The product yielded a
major 40-kDa subunit on SDS-polyacrylamide gel electrophoresis (results
not shown). The band was excised and digested with trypsin, peptides
were separated by hplc, and four partial N-terminal sequences were
obtained (19) (underlined in Fig.
1). One sequence (doubly
underlined in Fig. 1) was used to design degenerate
oligonucleotide antisense primers. Touchdown PCR (20) was then
performed on a pigeon heart cDNA library in
Zap using the T3
promoter sequence as the sense primer. The resulting PCR product (178 base pairs) was used to screen the library and 10 clones were obtained.
The clone with the longest insert (1710 base pairs) was sequenced in
both directions to yield the sequence shown in Fig. 1. The clone
contains an open reading frame that codes for a peptide of 358 amino
acids with a predicted molecular mass of 39472 Da. The sequence
contains not only that used to generate oligonucleotides for PCR but
also each of the three other peptide sequences; all four were preceded,
as expected, by a lysine residue. The protein sequence displays no
homology (>20% overall) to any other in the Swissprot, PIR,
translated EMBL, and GenBankTM data bases. A potential
nuclear localization signal was recognized using the program PSORT
(Fig. 1). Interestingly, there is no homology to the previously
published human and bovine sequences of cN-II (27, 28) or with that for
e-N (9, 29). We therefore reached the surprising conclusion that cN-I
is a novel protein, unrelated to cN-II or e-N. The clone did, however,
contain a sequence (amino acids 253-336) with 79% identity to a
translated human testis expressed sequence tag (accession number
AA446194), implying this EST might code for a human cN-I.

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Fig. 1.
Nucleotide and deduced amino acid sequence of
cN-I cDNA. The asterisks indicate stop codons. The
amino acid sequences determined are underlined. The
doubly underlined sequence was used to design degenerate
oligonucleotide primers for PCR. The predicted peptide sequence used to
generate a polyclonal antibody is in bold.
|
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The cDNA subcloned into pTargeT for expression in COS-7 cells
directed expression of 5'-nucleotidase activity measured under conditions previously shown to be optimal for the detection of pigeon
heart cN-I (22). Activities were 0.23 ± 0.02 µmol·min
1·mg protein
1, which compared
with activities of 0.004 ± 0.001 in untransfected cells. Parallel
experiments using a plasmid encoding active
-galactosidase indicated
that more than 30% of COS-7 cells were transfected under these
conditions. The activity in transfected COS-7 cells was approximately
14-fold that previously reported in pigeon heart (14). The
5'-nucleotidase activity had properties similar to published values
from pigeon heart (Table I). In
particular, the cloned enzyme had a Km (measured at
pH 7.0 and with greater than 50 µM ADP) similar to that
of the pigeon heart cN-I and had a similar absolute requirement for
adenine nucleotides as an activator. The enzyme had a preference for
AMP over IMP and was only weakly inhibited by isobutylthioadenosine,
which distinguishes it from cN-II. Like purified cN-I, it was inhibited by p-nitrophenyl and also by 2',3' dideoxycytidine,
dideoxythymidine, and dideoxyuridine, which have recently been shown to
be potent and selective inhibitors of cN-I but not cN-II from rabbit
heart (30).
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Table I
Comparison of recombinant and native cN-I
Experimental values are either from two or three separate experiments.
Values for the native enzyme are taken from Ref. 22. ddT,
dideoxythymidine; ddU, dideoxyuridine; ddC, dideoxycytidine.
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Incubation of cells with inhibitors of adenosine deaminase and
adenosine kinase was used to measure rates of adenosine formation from
intact COS-7 cells (Fig. 2). Incubation
with 2-deoxy-D-glucose and carbonyl cyanide
p-(trifluoromethoxy) phenyl-hydrazone was used to accelerate
ATP catabolism. The effects of cN-I and
-galactosidase transfection
(as a control for the effects of transfection per se) were compared
with untransfected cells. In untransfected cells, the sum of ATP + ADP + AMP + adenosine was 107 ± 9 nmol/mg protein (n = 6). Purine metabolite concentrations remained unchanged during a
control incubation for 5 min in the presence of inhibitors of adenosine
metabolism (Fig. 2). Metabolic poisoning led to a 91 ± 1% fall
in ATP concentration in 5 min, 85 ± 3% of which was accounted
for by increased AMP concentration and 4 ± 1% of which was
accounted for by increased adenosine concentration. IMP inosine and
hypoxanthine concentrations remained undetectable (data not shown).
These results indicate that flux through endogenous 5'-nucleotidase and
AMP deaminase was low under these conditions. Cells transfected with
the control
-galactosidase encoding plasmid had a similar initial
total adenine metabolite (ATP + ADP + AMP + adenosine) concentration
(103 ± 20 nmol/mg, n = 4). However, in contrast to untransfected cells, they showed a significant fall in ATP concentration (p < 0.05) and rise in AMP concentration
(p < 0.01) over 5 min of control incubation (Fig. 2).
This adverse effect of transfection did not result in any increase in
adenosine concentration (Fig. 2). After incubation with metabolic
poisons ATP concentration fell to levels similar to those in
untransfected cells. Of the fall in ATP concentration, 99 ± 2%
was accounted for by increased AMP concentration and 4 ± 1% was
accounted for by increased adenosine concentration (Fig. 2), similar to
values in untransfected cells. Cells transfected with the cN-I plasmid
had a significantly lower initial total adenine metabolite
concentration (64 ± 7 nmol/mg, n = 6) compared
with untransfected cells (p < 0.05). However, when
values were normalized (Fig. 2), they showed the same percentage fall
in ATP concentrations during incubation without metabolic poisons as
the cells transfected with
-galactosidase. In contrast to the
-galactosidase-transfected cells, there was no increase in AMP
concentrations, but instead adenosine concentrations rose significantly
(p < 0.02). These data show that there was basal flux
through cN-I under these conditions. After metabolic poisoning of
cN-I-transfected cells, ATP concentrations fell to a similar extent as
in untransfected or
-galactosidase-transfected cells. However, only
60 ± 6% of the fall in ATP concentration was accounted for by
increased AMP concentration, whereas 39 ± 3% was accounted for
by increased adenosine concentration (both p < 0.001 versus either untransfected or
-galactosidase-expressing
control cells). From these data it is clear that AMP was
stoichiometrically converted to adenosine in cN-I-transfected cells.
Metabolic poisons clearly increased adenosine formation by 2.6-fold
(Fig. 2).

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Fig. 2.
Nucleotide metabolism in untransfected,
-galactosidase-expressing, and cN-I-expressing
COS-7 cells. The levels of adenine metabolites were measured in
cells that had been preincubated for 15 min with an inhibitor of
adenosine deaminase (time 0). Batches of cells were then incubated for
5 min in fresh medium containing inhibitors of both adenosine deaminase
and adenosine kinase. Separate batches of cells were incubated in the
presence (+) or absence ( ) of 2-deoxy-D-glucose
(2-DOG) and carbonyl cyanide p-(trifluoromethoxy)
phenyl-hydrazone (FCCP) to inhibit energy metabolism. ATP
(open bars), ADP (shaded bars), AMP
(hatched bars), and adenosine (filled bars)
concentrations were measured by hplc, and the values are expressed as a
percentage of the total concentration of ATP + ADP + AMP + adenosine,
the values of which are reported in the text. The values are the means
for four separate experiments, each performed in duplicate.
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To control for possible effects on cell viability, we measured release
of LDH under the various experimental conditions. Untransfected cells
released 0.3 ± 0.2 and 1.3 ± 0.7% of LDH during incubation without and with metabolic poisons (n = 4). Cells
transfected with
-galactosidase or cN-I released similarly increased
levels of LDH (1.5 ± 1.3 and 2.6 ± 1.6%, respectively,
without and 2.5 ± 1.7 and 4.6 ± 1.6%, respectively, with
metabolic poisons). These increases in LDH release are consistent with
the evidence from measurements of ATP and AMP concentrations that
transfection per se causes metabolic stress. However, the effects on
LDH release were small compared with the rises in adenosine in
cN-I-expressing cells, which implies that most adenosine formation
occurred inside intact cells. Further data were obtained against the
possibility that released AMP metabolized by e-N accounted for the
rises in adenosine formation. The selective e-N inhibitor,
,
-methylene-ADP (50 µM), did not decrease the
percentage of ATP converted to adenosine in two separate batches of
metabolically poisoned, cN-I-expressing cells (28 or 56% without
,
-methylene-ADP versus 41 or 61% with
,
-methylene-ADP).
The distribution of cN-I was investigated by reverse transcriptase-PCR
(not shown) and Northern blotting (Fig.
3). Reverse transcriptase-PCR products of
the correct size were obtained from all the pigeon tissues tested,
albeit at trace levels in aorta, liver, and kidney. Products were most
abundant in heart, brain, and skeletal (breast) muscle. These results
were corroborated by Northern analysis (Fig. 3), in which a single
2.3-kilobase transcript was prominent in heart, brain, and breast
muscle.

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Fig. 3.
Distribution of cN-I mRNA in various
pigeon tissues by Northern blot analysis. Total RNA (10 µg) was
electrophoresed, transferred to nylon, and then hybridized with
32P random primed DNA fragments. A PCR fragment of cN-I
cDNA (nucleotides 87-850) (panel a) or a pigeon heart
glyceraldehyde 3-phosphate dehydrogenase (GAPDH, AF036934)
(panel b) reverse transcriptase-PCR fragment (nucleotides
116-465). Ao, aorta; Br, brain; Ki,
kidney; Li, liver; Sk, breast muscle;
Ht, ventricle. kb, kilobase.
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The distribution of cN-I in pigeon ventricle was further investigated
by immunocytochemistry using a polyclonal antibody raised against the
peptide indicated in Fig. 1. The antibody recognized a predominant
40-kDa band in extracts of pigeon heart and in cN-I-transfected but not
untransfected COS-7 cells (Fig.
4a). Staining of frozen sections showed diffuse cytoplasmic staining of myocytes (Fig. 4b), which gave a striated pattern in cells sectioned
longitudinally (Fig. 4, b and c).

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Fig. 4.
Location of cN-I in pigeon heart by
immunocytochemistry. a, the specificity of the rabbit
polyclonal antibody anti cN-I was confirmed by Western blot analysis.
Equal quantities of protein (35 µg/lane) extracted from pigeon
ventricle, untransfected COS-7 cells, and COS-7 cells expressing cN-I
were fractionated by SDS-polyacrylamide gel electrophoresis (10%).
b-d, immunoperoxidase staining (26) of 4-µm frozen
sections of pigeon heart fixed with acetone using a 1:400 dilution of
antiserum against cN-I (b and c) or preimmune
rabbit serum (d). The counterstain is hematoxylin.
Scale bars correspond to 25 µm.
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 |
DISCUSSION |
An IMP-selective cytosolic 5'-nucleotidase (cN-II) (27, 28) and an
ecto-5'-nucleotidase (e-N) (9) have been previously cloned. One of the
most surprising conclusions of our study is that the sequence of cN-I
bears no similarity to cN-II. Interestingly e-N is related neither to
cN-I nor to cN-II. Presumably the potent, selective activation of cN-I,
the less potent and less selective activation of cN-II, and inhibition
of e-N by ADP represent cases of convergent evolution. The only
recognizable structural motif in cN-I was a putative nuclear
localization signal, and this proved to be inactive, as indicated by
immunocytochemistry. Indeed, cN-I was confirmed to be cytoplasmic, in
agreement with a previous study (31). However, our results imply
association with the contractile apparatus, as has been suggested
previously from subcellular fractionation studies. These experiments
(22) showed that the addition of KCl to buffers used for homogenization
led to a redistribution of cN-I from the particulate to the soluble
fraction, implying a loose interaction with contractile elements. This
association may provide a mechanism for sensing local AMP
concentrations in the region of the contractile proteins. Our results
showed that cN-I is abundant in oxidative muscles (heart and breast
muscle) and also in brain.
Adenosine production from endogenous pathways amounted to 4% of
ATP breakdown in untransfected cells. We therefore had to obtain
expression of recombinant cN-I in a substantial proportion of cells to
exceed this background. We achieved this by liposome-mediated transfection and used transfection with the irrelevant gene,
-galactosidase, as a control. Our results demonstrate that
transfection per se caused metabolic stress, as evidenced by increased
AMP concentrations under basal conditions. Given this, it was not
surprising that when we inhibited adenosine metabolism, we demonstrated
substantial adenosine formation in cN-I-transfected cells, even in the
absence of metabolic poisons. However, flux through the enzyme was
clearly elevated during ATP catabolism, consistent with the role
proposed for cN-I in catalyzing the cytoprotective actions of
adenosine. Interestingly, overexpression of cN-I also caused a long
term decline in total adenine nucleotide concentrations in the absence of inhibitors of adenosine metabolism. It will be interesting to
investigate in future experiments whether decreasing cN-I activity causes a corresponding increase in the total adenine nucleotide pool.
In conclusion, the aims of this study were to clone cN-I and provide
definite evidence for its role in ATP catabolism and adenosine
formation. Our results clearly demonstrate that we have achieved these
two aims.
 |
ACKNOWLEDGEMENTS |
We thank Kate Keen and Jason Johnson for the
expert technical assistance. Protein sequence analysis was performed by
Dr. M. Wilkinson (University of Liverpool, Liverpool, UK).
 |
FOOTNOTES |
*
This work was supported by grants from the British Heart
Foundation and the British Council.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) AJ131243.
¶
To whom correspondence should be addressed. Tel.:
44-1179283582; Fax: 44-117928 358; E-mail: A.Newby{at}bris.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
e-N, ecto-5'-nucleotidase;
cN, cytosolic 5'-nucleotidase;
hplc, high
pressure liquid chromatography;
PCR, polymerase chain reaction;
LDH, lactate dehydrogenase.
 |
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