From the State Key Laboratory of Genetic Engineering,
Institute of Genetics, School of Life Science, Fudan University,
Shanghai 200433, People's Republic of China, § United Gene
Holdings, Ltd., Shanghai 200092, People's Republic of China, and the
Cell Regulation and Signalling Group, School of Biological
Sciences, University of Liverpool, Liverpool L69 7ZB, United
Kingdom
Received for publication, December 11, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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ITP and dITP exist in all cells. dITP is
potentially mutagenic, and the levels of these nucleotides are
controlled by inosine triphosphate pyrophosphatase (EC
3.6.1.19). Here we report the cloning, expression, and characterization
of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an
enzyme whose activity has been reported in many animal tissues and
studied in populations but whose protein sequence has not been
determined before. At the optimal pH of 10.0, recombinant hITPase
hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their
respective monophosphates whereas activity with other nucleoside
triphosphates was low. Km values for ITP, dITP, and
xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mM,
respectively, and kcat values were 580, 360, and 640 s ITP has been found in many animal tissues. It is generated by
pyrophosphorylation or stepwise phosphorylation of IMP, an essential metabolite of purine biosynthesis and a precursor of both AMP and GMP
(1-3). The deoxyribonucleotide dITP may be generated from dATP by
slow, non-enzymatic hydrolysis or by reduction of ITP (3). Normally,
the cellular ITP/dITP concentration is very low. The inability to
demonstrate the synthesis of ITP/dITP in cellular preparations has been
attributed to the presence in the cytoplasm of an inosine
triphosphatase (ITPase),1 an
enzyme that does not permit accumulation of these nucleotides (2).
ITPase hydrolyzes ITP/dITP to IMP/dIMP and PPi. XTP is also a
substrate, but activity toward other purine nucleoside triphosphates is
low, whereas no activity is found with IDP or IMP (2-6). Different studies have measured different Km values for ITP
(2, 6-12), but in all cases an alkaline pH optimum and an absolute requirement for a divalent ion such as Mg2+ or
Mn2+ has been found. The range of ITPase activity in
erythrocytes from Caucasian populations has been measured as 120-320
µmol of IMP produced/h/g Hb. In one population study, evidence
was presented that a deficiency of ITPase was responsible for the high
level of ITP found in 7 of >6,000 samples from mainly unrelated
individuals. The frequency of heterozygosity for ITPase deficiency in
Caucasian populations is estimated to be ~5% (6, 13-15). The gene
coding for ITPase, ITPA, is located on the short arm of
chromosome 20 (16).
Although mammalian ITPase activities have been identified in human
erythrocytes (9), rabbit liver (10), and several rat tissues (17), no
gene has been cloned and characterized. Recently a novel bacterial
nucleoside triphosphate pyrophosphatase Mj0226, from
Methanococcus jannaschii, was revealed by structure-based identification and subsequent biochemical analysis (18). In the
presence of Mg2+ or Mn2+ ions Mj0226 can
efficiently hydrolyze non-standard nucleotides such as XTP to
xanthosine 5'-monophosphate and ITP to IMP but not the canonical
standard nucleotides. A yeast protein, Ham1p, was reported to be the
product of a gene controlling sensitivity of yeast strains to
6-N-hydroxylaminopurine (HAP) (19). This yeast protein is
homologous to the Mj0226 protein, with about 30% sequence identity.
Experiments with yeast HAM1 Here we report the cloning, expression, and characterization of a human
ITPase, hITPase, that converts ITP and dITP to their respective
monophosphates and PPi. The RNA transcription profiles of hITPase were
determined by Northern blot and cDNA microarray analysis. The
chromosomal localization of the hITPase gene, ITPA, is also described.
Materials--
dITP was from Roche Molecular Biochemicals, and
other dNTPs were from Promeaga. ITP, XTP, IDP, IMP, and other NTPs were
from Sigma. Advantage cDNA polymerase mix was from
CLONTECH, and all other enzymes were from New
England Biolabs.
Identification and Cloning of hITPase cDNA--
A high
quality cDNA library was constructed using human fetal brain
poly(A)+ RNA and a SMART PCR cDNA library
construction kit (CLONTECH). cDNA synthesis was
performed from 100-200 ng of mRNA, followed by 20-24 cycles of
PCR amplification. The cDNAs were inserted into the pBlueScript SK
plasmid vector and transformed into E. coli DH5 Expression in E. coli and Purification of Human
ITPase--
The hITPase cDNA was cloned between the
BamHI and HindIII sites of the bacterial
expression vector pQE31 (Qiagen) after PCR amplification. Transformants
of E. coli M15/pREP4 with the resulting pQE31- hITPase
construct were grown at 30 °C in 100 ml of LB medium with 100 µg/ml ampicillin and 25 µg/ml kanamycin. The plasmid pREP4
constitutively expresses the Lac repressor protein encoded by the
lacI gene to reduce the basal level of expression (Qiagen). When the culture had grown to an A600 of
0.6, isopropyl Enzyme Assay--
Substrates were screened, and kinetic
parameters for substrate hydrolysis were determined by measuring the
Pi released in a coupled assay by coincubation of substrate
with hITPase and inorganic pyrophosphatase (22). The standard assay
(800 µl) for triphosphate nucleotide substrates involved incubation
of 1.25 mM substrate with 15 ng of hITPase and 1 unit of
inorganic pyrophosphatase for 10 min at 37 °C in 50 mM
Tris-HCl, pH 8.5, 50 mM MgCl2, 1 mM
dithiothreitol. The Pi released was measured colorimetrically. Pi released from triphosphate nucleotide
substrates in control assays without hITPase was subtracted.
Reaction products were identified by high performance liquid
chromatography. Reaction mixtures described above but without inorganic
pyrophosphatase were incubated at 37 °C for 10 min in a volume of
800 µl. The reaction was stopped by adding 200 µl of 20%
trichloroacetic acid. Aliquots (10 µl) were injected onto a 4.6 × 250 mm Zorbax 300SB-C18 5-µm column (Hewlett Packard) at 30 °C.
Chromatography conditions were as described previously (23).
Northern Blot Analysis--
Multiple tissue Northern
(MTNTM) blots containing 2 µg of poly(A+) mRNA
isolated from a variety of human tissues were purchased from
CLONTECH. Blots were probed with a full-length
human ITPase cDNA that had been radioactively labeled with
[ Chromosome Mapping of the ITPA Gene--
To determine the
chromosomal localization of the human ITPA gene, the
Stanford G3 radiation hybrid panel (Research Genetics) consisting of 83 hybrid cell lines was screened by PCR. The PCR primer pair used for
amplification was 5'-CTCTGAGAAACTCTGGCAAGTGGACG Identification and Cloning of the Human ITPA Gene--
The
GenBankTM non-redundant and EST data bases were searched
with individual sequences from our collection of full-length assembled cDNAs using the NCBI BLAST server to identify sequences of interest to our laboratories. One such sequence, represented in our collection by three clones, was found to encode a putative protein similar to the
M. jannaschii Mj0226 and Saccharomyces cerevisiae
Ham1p proteins (amino acid identities of 33 and 42% and
E-values of 5 × 10
The longest hITPase cDNA clone we obtained had 1085 base pairs
containing an open reading frame of 585 nucleotides from the first
translation initiation codon ATG to the termination codon TAA
(nucleotides 105-689; GenBankTM accession number
AF219116). The 3'-end of the sequence contains a poly(A) stretch,
preceded by a putative polyadenylation signal AATAAA (nucleotides
1062-1067). The open reading frame encodes a 194-amino acid protein
with a predicted molecular mass of 21,501 Da and pI of 5.50. There is
an in-frame stop codon upstream of the open reading frame whereas the
proposed initiating ATG is in the highly favorable sequence context
ACCATGG, indicating that the predicted sequence probably
represents the full-length protein.
Expression and Purification of hITPase--
After E. coli M15/pQE31-ITPase was induced with isopropyl
Properties of the Enzyme--
Activity of the hITPase toward
inosine nucleotides (ITP, IDP, IMP, and dITP), XTP, and the eight
canonical ribo- and deoxyribonucleoside triphosphates was determined.
At a fixed concentration of 1.25 mM, ITP, dITP, and XTP
were the best substrates for the recombinant hITPase followed by GTP
and dGTP (Fig. 2). In the absence of
inorganic pyrophosphatase, no Pi production was detected by
the colorimetric assay. The hITPase showed very low activity toward
other nucleoside triphosphates; UTP, (d)CTP, TTP, and (d)ATP were
hydrolyzed 10-100-fold less than dITP (Fig. 2). IDP and IMP were not
hydrolyzed at all, showing that hITPase is a nucleoside triphosphate
pyrophosphohydrolase. High pressure liquid chromatography analysis
confirmed the production of IMP and dIMP as primary products; no IDP or
dIDP was observed (data not shown).
Similar to the purified human erythrocyte ITPase, recombinant hITPase
had an absolute requirement for Mg2+ ions and a high
Mg2+ optimum (6, 12). Activity increased sharply up to
about 10 mM MgCl2 and reached a plateau between
about 30 and 100 mM (data not shown). The recombinant
hITPase also had a markedly high pH optimum of ~10.0 in glycine
buffer, similar to the value of 9.6 from previous reports (12).
Activity in the absence of 1 mM dithiothreitol was about
half that in its presence indicating a requirement (though not
absolute) for reducing conditions (data not shown). When analyzed by
gel filtration, the recombinant 23-kDa tagged hITPase behaved as a
homogeneous 45-kDa dimer (Fig. 3).
With ITP, dITP, and XTP as substrates, the recombinant hITPase followed
Michaelis-Menten kinetics but with clear evidence of inhibition at
higher substrate concentrations, most easily seen in reciprocal
Lineweaver-Burk plots of the data (Fig.
4). Both substrate inhibition and
inhibition by contaminating (deoxy)nucleoside diphosphates have been
documented before for this enzyme (6, 12). For this reason, kinetic
parameters were calculated by non-linear regression using data points
only at concentrations that did not appear to result in
inhibition according to visual analysis of the reciprocal plots
(Table I). Within the errors of
the kcat/Km values calculated
in this way, all three substrates appear to be used with similar
efficiencies.
Tissue-specific Expression of hITPase--
To determine the size
and distribution of hITPase mRNA transcripts, Northern blots
containing 2 µg of poly(A)+ RNA from different adult
human tissues were probed with [32P]dCTP-labeled hITPase
cDNA. This revealed a transcript of about 1.4 kb in all 24 adult
human tissues examined, with the most abundant expression in heart,
liver, sex glands, thyroid, and adrenal gland (Fig.
5). These results were largely confirmed
by cDNA microarray analysis, which showed heart, liver, thyroid,
and thymus tissues to have the highest normalized expression ratios of
those examined compared with housekeeping genes (all >2.6; data not
shown).
Sequence Comparisons--
There are more than 40 protein and
putative protein sequences in GenBankTM that have moderate
similarity to human ITPase. All of these proteins are about 200 amino
acids long and have similar secondary structures as predicted by the
PHDsec program (24). Multiple sequence alignment of hITPase and related
sequences revealed a new conserved protein family representing inosine
triphosphatases from bacteria to mammals. In the alignment (Fig.
6), the amino acid residues corresponding to positions comprising the nucleotide binding site predicted by
three-dimensional structural analysis of Mj0226 are shown (18). Not all
are conserved; however, in some cases the side chain may be of lesser
importance than the backbone amide in binding the substrate (18).
Fig. 7 shows the dendrogram of the
alignment results generated by the TREE-PUZZLE 5.0 program (25). For
each internal branch, the bootstrap proportion was estimated by the
Dayhoff method with 1000 replications (26). It is clear that these
homologues fall into three divisions representing the three
superkingdoms of eukaryota, archaea, and eubacteria and that the ITPase
family, therefore, has an ancient origin and function.
Chromosomal Localization--
The chromosomal location of the
human ITPA gene was determined with the Stanford G3
radiation hybrid panel with a 3'-untranslated region-specific primer
pair for human ITPA gene. The expected 133-base pair PCR
product was amplified from 12 of 83 DNAs from the hybrid cell lines.
Data were submitted to the Stanford Human Genome Center for statistical
evaluation. The human ITPA gene was located on chromosome
20, close to markers SHGC-14786, SHGC-2800, and SHGC-21112 from the
Stanford G3 chromosome 20 radiation hybrid map data and between
microsatellite marker AFM240zf4 (D20S181) and microsatellite
anchor marker AFM036ya3 (D20S97). Our cDNA clone corresponds to
NCBI Unigene Hs.6817, which was located on the same interval
D20S113-D20S97 on GeneMap '99. We found two genes, PTPRA
(protein tyrosine
phosphatase, receptor type, alpha polypeptide) and CENPB (centromere protein
B), upstream and downstream, respectively, of Unigene
Hs.6817 on the same region. These genes also flank the mouse
Itpa gene. This result confirmed our conclusion that the
cDNA clone encodes the human ITPA gene product, hITPase (Table II).
In this report, we have described the isolation and
characterization of a cDNA clone encoding a human ITP/dITP/XTP
pyrophosphatase, hITPase, from a human fetal brain cDNA library.
Initially, the library was searched for potential homologues of the
M. jannaschii nucleoside triphosphate pyrophosphatase
Mj0026. The cDNA thus obtained encodes a 195-amino acid protein
with a predicted molecular mass of 21,501 Da and 33% amino acid
identity to Mj0026. The chromosomal localization of the cDNA clone
matches that of the previously described ITPA locus on the
short arm of chromosome 20 between the PTPRA and CENPB genes. These
results confirm our view that this clone encodes the human
ITPA gene product. The presence of hITPase mRNA in all
human tissues examined and its high expression in endocrine glands was
shown by Northern blot analysis and by cDNA microarray
hybridization. The distribution of ITPA transcripts in human
tissues agrees with the distribution of ITPase enzyme activity in
different rat tissues (17). These and other studies also suggested that
the enzyme was located in the cytoplasm (6, 17). We have confirmed this
location by transfection of COS-7 cells with a construct in which
hITPase was fused to the C terminus of green fluorescent protein. A
diffuse but clearly non-nuclear distribution of fluorescence was
observed.2
Enzymological analysis of the recombinant human protein expressed from
this cDNA clone showed a high specificity for the hydrolysis of
ITP, dITP, and XTP to their respective monophosphates. Characteristics of the recombinant enzyme were similar to those from earlier reports of
ITPase isolated from human tissues, except the Km value for ITP, which at 0.51 mM was higher than those
previously reported, 0.13 (12) and 0.07 mM (6). Possible
explanations for this discrepancy are (i) different levels of
inhibitory IDP in the substrate preparations used, (ii) lack of a
post-translational modification in the recombinant enzyme that is
present in the enzyme isolated from tissues, or (iii) reduced affinity
for ITP and other substrates caused by the N-terminal tag.
Three different human populations have been reported with respect to
their ITPase activity. The first has high activity, the second has a
mean activity exactly 25% that of the first, whereas the third
population has very low activity. A theoretical explanation of this
study is that normal (active) and mutant (inactive) alleles exist in
the population and that the ITPase is only active as a dimer with two
normal subunits (14, 15). In heterozygotes, only one of the four
hypothetical dimers would be composed of two normal monomers, leading
to 25% activity. Consistent with this hypothesis is our finding that
recombinant hITPase behaves as a homogeneous dimer under physiological
conditions. This agrees with results generated by structural analysis
of Mj0226 (18). In ITPase-deficient populations, there is an obvious
elevation of ITP concentration in erythrocytes and other cells, showing that hITPase may be the major enzyme responsible for regulating the ITP
concentration in human cells. Although no clinical abnormalities have
been reported to be associated with complete hITPase deficiency, one
study has reported a significant reduction in tissue ITPase activity in
paranoid schizophrenics (56.3 ± 5.5 µmol/h/g of Hb) compared
with normal controls (94.1 ± 5.3 µmol/h/g of Hb)
(p < 0.0002), and it has been suggested that the
resulting elevated ITP may inhibit the activity of glutamate
decarboxylase, the enzyme responsible for generating the
neurotransmitter ITP and dITP can be incorporated into RNA and DNA, respectively, by
polymerases (29-32). As an unusual nucleoside in RNA, inosine arising
from incorporation from ITP could lead to the same effects on RNA
sequence-specific interactions as the inosine arising from RNA editing
by ADARs, adenosine deaminases that
act on RNA (33, 34). Therefore, effects on
structure, translatability, and degradation rate are all possible.
Further, because adenosine deamination can alter RNA structure,
sequence-independent processes also could be affected.
The deoxyribonucleotide dITP behaves as a dGTP analogue and is
incorporated opposite cytosine with about 50% efficiency. Both isolated nuclei and purified DNA polymerases rapidly incorporated dITP
into DNA. In the presence of ATP, dITP is stabilized in extracts of
nuclei (35) and E. coli (36), allowing the possibility that
a small amount of dIMP will also be incorporated into DNA in
vivo. Although hypoxanthine DNA glycosylase can remove the base
from DNA (37), evidence has been presented that this enzyme only
efficiently removes a hypoxanthine base from an I-T base pair,
whereas removal from an I-C base pair is 15-20 times slower (38).
Because of the relative stability of an I-C base pair, inosine can
remain until the next round of DNA replication, increasing the risk of
direct mutagenesis. In vitro polymerase studies have shown
that the presence of dITP in reaction mixtures may induce a high
frequency of mutation (39, 40). Other reports shows that ITP and IDP
added to cell cultures can cause elevated rates of chromosomal
structural aberrations (41, 42).
In human erythrocytes ITP is continuously synthesized and broken down
at a relatively high rate, forming a futile cycle that has been
proposed to regulate the concentration of ATP. Additionally, ITP
appears to be a substrate for the cartilage pyrophosphorylase associated with articular calcium crystal deposition (43) and a
substrate for receptor/G proteins to activate effector systems (44,
45).
Other naturally occurring unusual purine nucleoside triphosphates could
be considered as possible substrates of hITPase, such as the
triphosphates of HAP (46) and
2-amino-N6-hydroxyladenine (47). The
HAM1 protein, the hITPase homologue in yeast, can control
the sensitivity of yeast strains to HAP added in medium. Overexpression
of HAM1 can protect E. coli from both the toxic
and mutagenic effects of HAP (21). Therefore, Ham1p must be acting as a
HAP triphosphate pyrophosphatase to prevent the incorporation of HAP
into DNA (20). Given the possibility that HAP could be generated inside
living cells (48) and induce mutations and chromosomal aberrations
(49), a role for the ITPA family in the prevention of HAP-induced
mutagenesis must be considered.
A BLAST search of protein data bases revealed hITPase/Mj0026/Ham1p
homologues in most of the bacterial and eukaryotic organisms that have
been fully or partially sequenced. Sequence alignment shows that the
previously identified nucleotide binding sites are conserved in these
proteins. The results of a phylogenetic analysis showed that these
homologues fall into three divisions, in accordance with the three
superkingdoms of eukaryota, archaea, and eubacteria. The widespread
presence of hITPase homologues suggests an ancient origin for ITPases,
perhaps arising after the appearance of de novo purine synthesis.
An interesting comparison may be drawn between hITPase and its
homologues and the nudix hydrolase gene family. This family is composed
of mostly small, soluble nucleotide phosphohydrolases that possess a
sequence signature motif called the nudix box, formerly called the MutT
motif, named after the antimutagenic E. coli mutT gene
product (50-52). The MutT protein can hydrolyze 8-oxo-dGTP, a highly
mutagenic oxidized nucleotide, to 8-oxo-dGMP and PPi (50, 51). Because
of gene duplication, any one genome may have several nudix family
members. There are 13 paralogs in E. coli, 5 in S. cerevisiae, and at least 15 in man, several of which have been
characterized as nucleotide hydrolases with widely varying substrate
specificity. Almost all of the substrates of nudix hydrolases possess a
nucleoside diphosphate linked to another moiety, "x" (51). These
enzymes are believed to eliminate toxic nucleotide derivatives from the
cell and regulate the levels of important signaling nucleotides and
their metabolites (51, 52). Unlike nudix family members, the ITPA
family has only one member per genome without paralogs. This may
indicate a simpler but still important role in cleansing minor
nucleotides from the cell. Another example of a non-nudix nucleoside
pyrophosphohydrolase that restricts the accumulation of a minor NTP is
deoxyuridine 5'-triphosphatase, which both prevents the
incorporation of uracil into DNA and generates dUMP for dTMP synthesis
(53, 54).
Although the biochemical properties of ITPases have been well
characterized, no definite biological function has yet been defined.
One direction for future research is to study the toxicity and
mutagenicity of its substrates ITP and dITP, as well as the other
purine derivatives that may be hydrolyzed by ITPase in vivo. Confirmation of its cellular and disease-related function must await
the result of gene knock-out studies and further population investigations.
1, respectively. A divalent cation was
absolutely required for activity. The gene encoding the hITPase
cDNA sequence was localized by radiation hybrid mapping to
chromosome 20p in the interval D20S113-D20S97, the same interval
in which the ITPA inosine triphosphatase gene was
previously localized. A BLAST search revealed the existence of many
similar sequences in organisms ranging from bacteria to mammals. The
function of this ubiquitous protein family is proposed to be the
elimination of minor potentially mutagenic or clastogenic purine
nucleoside triphosphates from the cell.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants have shown
that adenine-requiring haploid strains are unable to grow on HAP as the
sole adenine source (19, 20), whereas Escherichia
coli transformed with the HAMI gene are resistant to
HAP mutagenesis (21). This phenomenon presumably occurs because the
conversion of HAP to HAP triphosphate and subsequent incorporation into
DNA is lethal (20). Based on biochemical information and sequence
similarity, these two proteins may be microbial ITP hydrolases that can
convert mutagenic, non-canonical purine nucleoside triphosphates to
monophosphates and thus protect DNA from the incorporation of modified
purine bases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by
electroporation (Gene Pulser; Bio-Rad). A 96-well R.E.A.L. plasmid kit
(Qiagen) was used to prepare double-stranded plasmid DNA. Sequencing
reactions were performed with Big-Dye primer cycle sequencing and
Big-Dye terminator cycle sequencing Kits (PerkinElmer Life
Sciences) with the
21M13 or M13Rev primers to obtain the 5' or 3'
sequences. The complete sequences were determined and confirmed by
primer walking using the Big-Dye terminator cycle sequencing kit.
Sequencing was performed on a PE-ABI 377 sequencer. The Acembly program
was used to assemble the full-length cDNA sequences. Vector
sequences were removed, and data base searches were performed with the
BLAST using the NCBI web site.
-D-thiogalactopyranoside was added to a
final concentration of 1 mM. After inducing the expression
of the hITPase protein for 4 h at 30 °C, cells were harvested,
washed, and resuspended in 20 ml of 20 mM sodium phosphate buffer, pH 7.4, containing 300 mM NaCl and 10 mM imidazole. The cell suspension was sonicated, and
the lysate was cleared by centrifugation at 20,000 × g
and 4 °C for 20 min. The supernatant was then poured into a
Ni2+-nitrilotriacetic acid-agarose column, washed with
phosphate buffer containing 60 mM imidazole, and eluted
with buffer containing 500 mM imidazole. The purity of the
eluted protein was checked by SDS-PAGE. Fractions containing the
recombinant hITPase were dialyzed overnight against two changes of 1 liter of 50 mM Tris-HCl, pH 7.5, 1 mM
dithiothreitol, 50% glycerol. Dialyzed samples were stored at
20 °C.
32P]dCTP by random priming using a random primer
labeling kit (Amersham Pharmacia Biotech). Northern
hybridization was performed according to the manufacturer's
recommendations. The blot was hybridized at 68 °C overnight and
washed in solution 1 (2× SSC and 1% SDS) three times at 65 °C and
twice in solution 2 (0.1× SSC and 0.5% SDS) at 50 °C. The blot was
stripped by incubation for 10 min in 0.5% SDS at 95 °C and reprobed
with radiolabeled
actin cDNA as an indicator of mRNA loading.
3' (forward primer)
and 5'
CACGCCCTCACTCCCACCAAGTAC-3' (reverse primer), derived from the
3'
untranslated region of the human ITPA gene. PCR products, 133 base
pairs in length, were identified by electrophoresis on a high
resolution agarose gel. The results were submitted to the Stanford
Human Genome Center for statistical evaluation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21 and 2 × 10
29, respectively). We then searched the UniGene data
base and found a cluster of similar expressed sequence tags located at
chromosome 20p. A search of the OMIM data base with the terms
nucleotide and 20p revealed that the previously described, but
unsequenced, inosine triphosphatase ITPA gene mapped to 20p by
population analysis. This suggested that our protein may be the ITPA
gene product. Based on the confirmatory evidence outlined below, we
have named this putative protein human inosine triphosphatase, or hITPase.
-D-thiogalactopyranoside, the 6× His-tagged recombinant
hITPase yielded a major band with an apparent molecular mass of 25 kDa on an SDS-PAGE gel corresponding to about 15-20% of the total applied
protein (Fig. 1). Purification by binding
to Ni2+-nitrilotriacetic acid-agarose resin and elution
with buffer containing 500 mM imidazole resulted in a near
homogeneous preparation (Fig. 1). To confirm that this was the expected
expression product, an accurate molecular mass of 23,072 Da was
determined by mass spectrometry (data not shown). This result is very
close to the predicted mass of 23,069 Da for the recombinant hITPase,
which includes the N-terminal vector-derived tag sequence
MRGSHHHHHHTDP. The larger apparent molecular mass determined by
SDS-PAGE may be because of incomplete heat denaturation and SDS
binding.
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Fig. 1.
SDS-PAGE of E. coli extracts
expressing hITPase and of the purified enzyme. E. coli
M15 cells transformed with pQE31-hITPase were induced with 1 mM isopropyl -D-thiogalactopyranoside for up
to 4 h. Lanes 1-5, aliquots were taken at hourly
intervals from 0 to 4 h, analyzed on a 12% SDS gel, and stained
with Coomassie Blue. Lane 6, hITPase protein after
purification on a Ni2+-nitrilotriacetic acid-agarose column
as described under "Experimental Procedures." The protein standards
were rabbit phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43 kDa), bovine carbonic anhydrase (31 kDa),
trypsin inhibitor (20 kDa), and hen egg white lysozyme (14.4 kDa).
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Fig. 2.
Substrate specificity of hITPase.
Purified hITPase was incubated with 1.25 mM substrate at
37 °C for 10 min in 50 mM Tris-HCl, pH 8.5, 1 mM DTT, 50 mM MgCl2. The hydrolysis
of the substrates was assayed using the colorimetric procedure
described under "Experimental Procedures" and depicted relative to
the activity with XTP. Each result is the mean ± S.D. from three
experiments.
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Fig. 3.
Molecular weight determination of native
recombinant hITPase by gel filtration chromatography. A sample of
homogeneous hITPase protein was applied to a 10 600-mm Superdex 75 column (Amersham Pharmacia Biotech) in 0.05 M sodium
phosphate buffer, pH 7.3, 0.1 M
Na2SO4 and eluted at 0.5 ml/min in the same
buffer. Fractions were assayed for enzyme activity as described under
"Experimental Procedures." The column was calibrated with the
following standards under the same conditions: A, bovine
serum albumin, 68 kDa; B, hen ovalbumin, 45 kDa;
C, bovine chymotrypsinogen A, 25 kDa; D,
cytochrome c, 12.5 kDa. The elution position of the hITPase
and the V0 are marked with
arrows.
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Fig. 4.
Lineweaver-Burk plots for the hydrolysis of
ITP, dITP, and XTP by hITPase. Enzyme assays were performed with
15 ng of enzyme over the substrate range 0.056-3.35 mM,
and the products were quantified by the colorimetric procedure
described under "Experimental Procedures." Values are the means of
four separate determinations. Error bars are omitted for
clarity.
Kinetic parameters of recombinant human ITPase
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Fig. 5.
Expression of hITPase mRNA in human
tissues. Northern blots of poly(A)+ RNA from 23 human
tissues (2 µg per lane) were hybridized with
32P-labeled full-length hITPase cDNA probe. The
membranes were stripped and reprobed with a -actin cDNA probe.
Lane 1, heart; lane 2, brain; lane 3,
placenta; lane 4, lung; lane 5, liver; lane
6, skeletal muscle; lane 7, kidney; lane 8,
pancreas; lane 9, spleen; lane 10, thymus;
lane 11, prostate; lane 12, testis; lane
13, ovary; lane14, small intestine; lane 15,
colon; lane 16, peripheral blood leukocyte; lane
17, stomach; lane 18, thyroid; lane 19,
spinal cord; lane 20, lymph node; lane 21,
trachea; lane 22, adrenal gland; lane 23, bone
marrow.
View larger version (116K):
[in a new window]
Fig. 6.
Sequence alignment of hITPase with homologous
proteins or putative proteins. Full-length hITPase and homologous
protein or putative protein sequences were aligned using the Clustal W
program and displayed using Genedoc software. Sequences are from (with
GenBankTM accession numbers in brackets) Homo
sapiens hITPase (AF219116), Mus musculus (W54379),
Drosophila melanogaster (AE003608), Caenorhabditis
elegans (U13642), Arabidopsis thaliana (T05241),
S. cerevisiae Ham1p (S57088), M. jannaschii
Mj0226 (C64328), Archaeoglobus fulgidus (E69529),
Pyrococcus horikosii (C71206), E. coli (A65081),
Bacillus subtilis (C69986), Hemophilus influenzae
(D64146), and Deinococcus radiodurans (G75550). Black
shading indicates 100% conservation of amino acid similarity,
white on gray is 80-100%, black on gray is
60-80%, and no shading is <60% conservation. Amino acid
residues comprising the predicted nucleotide binding site are marked
with an asterisk.
View larger version (23K):
[in a new window]
Fig. 7.
Phylogenetic tree of hITPase and homologous
sequences from other organisms. The TREE-PUZZLE program with the
Dayhoff model was applied. The length of each branch is proportional to
the estimated number of amino acid substitutions. The
numbers at the internodes represent the bootstrap values of
each branch.
Human ITPA and mouse Itp chromosome linkage maps
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid (27, 28).
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ACKNOWLEDGEMENT |
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We thank Yueqiong Chao from United Gene Holdings, Ltd. for providing facilities for DNA sequencing and Junxia Zhu and Xiongying Fang for technical assistance. Thanks are also because of Prof. Yang Zhong for help with calculating and constructing the dendrogram alignment and to Prof. Wanxiang Xu for helpful discussions.
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FOOTNOTES |
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* This work was supported by funds from United Gene Holdings, Ltd.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) AF219116.
¶ Contributed equally to this paper.
** To whom correspondence may be addressed: Inst. of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. Tel.: 86-21-65643573; Fax: 86-21-65642502; E-mail: yxie@fudan.edu.cn.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M011084200
2 S. Lin and J. Shi, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ITPase, inosine triphosphate pyrophosphatase; HAP, 6-N-hydroxylaminopurine; XTP, xanthosine 5'-triphosphate; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
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