From the Departments of Pharmacology & Cancer Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Discovery of a structurally conserved
metal-dependent lithium-inhibited phosphomonoesterase
protein family has identified several potential cellular targets of
lithium as used to treat manic depression. Here we describe
identification of a novel family member using a "computer cloning"
strategy. Human and murine cDNA clones encoded proteins sharing
92% identity and were highly expressed in kidney. Native and
recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3'-phosphate from 3'-5' bisphosphate nucleosides and
3'-phosphoadenosine 5'-phosphosulfate with Km and
Vmax values of 0.5 µM and 40 µmol/min/mg. Lithium uncompetitively inhibited activity with a
Ki of 157 µM. Interestingly, BPntase
was competitively inhibited by inositol 1,4-bisphosphate with a
Ki of 15 µM. Expression of mammalian
BPntase complemented defects in hal2/met22 mutant yeast.
These data suggest that BPntase's physiologic role in nucleotide
metabolism may be regulated by inositol signaling pathways. The
presence of high levels of BPntase in the kidney are provocative in
light of the roles of bisphosphorylated nucleotides in regulating salt
tolerance, sulfur assimilation, detoxification, and lithium toxicity.
We propose that inhibition of human BPntase may account for
lithium-induced nephrotoxicity, which may be overcome by
supplementation of current therapeutic regimes with inhibitors of
nucleotide biosynthesis, such as methionine.
Lithium is a major drug used to treat manic depression, yet its
molecular mechanism of action has not been conclusively elucidated. Insight into lithium's pharmacological activity has come with the
identification of a magnesium-dependent phosphomonoesterase family whose members are inhibited by lithium at subtherapeutic concentrations (1). Despite relatively low overall sequence similarity,
in some cases undetectable, the family shows a conserved three-dimensional core structure. Functional and structural studies of
three members, fructose 1,6-bisphosphatase
(fbptase)1, inositol
monophosphatase (impase), and inositol polyphosphate 1-phosphatase
(1ptase), indicate that residues involved in metal binding and
catalysis are conserved within a sequence motif
D-Xn-EE-Xn-DP(i/l)D(s/g/a)T-Xn-WD-X11-GG (2-7). Of interest, several studies indicate that lithium interacts at
one or more of the metal binding sites (6, 8,
9).2 Together, these data
support the idea that the signature motif confers the common enzymatic
characteristics of the family, including lithium sensitivity.
The lithium-sensitive family includes proteins with multiple cellular
roles. The roles of fbptase in gluconeogenesis, and impase and 1ptase
in inositol signaling, are well established. Additionally, this family
includes HAL2(MET22), SAL1, and cysQ, gene
products implicated in sulfur assimilation and salt tolerance in yeast,
plants, and bacteria (10-14). Met22p and isoallelic Hal2p were
identified as yeast proteins involved in methionine biosynthesis (10)
and sodium tolerance (11, 12). SAL1 was identified in a screen for
plant genes that produced salt tolerance in yeast (13). The bacterial
gene cysQ was found to be involved in the biosynthesis of
cysteine (14). Biochemical analysis of these proteins demonstrated a
Mg2+-dependent, Li+-sensitive
phosphomonoesterase activity on 3' phosphoadenosine 5' phosphate
(PAP) and 3' phosphoadenosine 5' phosphosulfate (PAPS) (11-13).
Additionally, SAL1 has been reported (13) to possess 1ptase activity
which removes the 1-position phosphate from either inositol
1,4-bisphosphate (Ins(1,4)P2) or Ins(1,3,4)P3
(15, 16). As SAL1 overexpression confers salt tolerance on yeast and
complements the methionine auxotrophy of hal2 mutants (13), roles were ascribed for both hydrolytic activities in the functioning of SAL1.
Direct evidence links physiological effects of lithium to members of
this phosphomonoesterase family. Treatment of cells with lithium
results in accumulation of inositol mono- and polyphosphates, suggesting that impase and 1ptase are inhibited in vivo.
Dichtl et al. (17) recently showed that the mechanism of
lithium toxicity in yeast involves inhibition of the cytosolic RNA
processing enzyme Xrn1p due to the inhibition of Hal2p and subsequent
accumulation of PAP. Concurrent PAP-independent lithium-mediated
inhibition of RNase MRP leads to an accumulation of immature ribosomal
RNA molecules (17). In addition, the data of Acharya et al.
indicate that 1ptase is a target of lithium in Drosophila
melanogaster (18). Defects in neuronal function observed in
Drosophila 1ptase mutants were phenocopied precisely by the
administration of lithium to wild-type flies (18).
The conservation of the sequence motif of this family is important for
identifying novel family members from human genome sequencing efforts.
To this end, we have used this motif to clone a novel mammalian
metal-dependent lithium-inhibited bisphosphate 3'-nucleotidase (BPntase) from expressed sequence tag (EST) data bases.
Characterization and tissue distribution of a mammalian BPntase suggest
that it is involved in nucleotide metabolism, sodium homeostasis, and
the physiological effects of lithium.
Protein concentrations were determined by the method of Bradford
(19), using bovine serum albumin (BSA) as a standard or by measuring
A280 (assuming an extinction coefficient of 1 cm/ml/mg). SDS-PAGE was run using the Laemmli method (20). Gels were
silver-stained as described within the Silver Stain Plus system
(Bio-Rad). DNA-manipulating enzymes were from Life Technologies, Inc.
or Roche Molecular Biochemicals. All other materials were reagent
grade, typically purchased from Sigma. Polyclonal antibodies were
generated by injecting PAP-agarose-purified enzyme as described below
into rabbits following a standard protocol (Alpha Diagnostic, San
Antonio, TX). The Western blot procedure followed a standard protocol
and was developed with a phosphorescent reagent activated by
horseradish peroxidase conjugated to donkey-anti-rabbit IgG (Amersham
Pharmacia Biotech).
Clone Identification and Sequencing--
Mouse and human EST
data bases were searched with BLASTP, gapped BLASTP, or TBLASTN (21)
with appropriate query sequences. Candidate cDNAs were obtained
from Research Genetics, Inc. (Huntsville, AL) and completely sequenced
on both strands by dideoxynucleotide chain termination (22) with
Sequenase version 2.0 (Amersham Pharmacia Biotech) and/or by
fluorescent terminator sequencing (Duke University Medical Center
Genome Facility, Durham, NC).
Plasmid Construction--
Full-length mouse and human
cDNA's 439033 and 645079 were obtained from Research Genetics,
Inc. (Huntsville, AL) as inserts in pBluescriptSK(
The HAL2 coding sequence was produced by polymerase chain
reaction (PCR) amplification of genomic Saccharomyces
cerevisiae DNA with primers 5'-GAGCTCCCGGGTGATCATATGGCATTGGAAAGAG
and 5'-GGTACCTGATCAGCGGGCCGCAAGCTTAGGCGTTTCTTGACTG as described
(24). Bacterial cysQ was cloned using oligonucleotide primers 5'-CATATGTTAGATCAAGTATGCCAGC and
5'-AAGCTTCTGTTTCTGCCATCTGAATTTAG. PCR was performed in 100-µl
reactions using a GeneAmp PCR System 2400 temperature cycler
(Perkin-Elmer). Reactions contained 100 ng of yeast genomic DNA or
approximately 104 Escherichia coli DH5
Recombinant virus was made by co-transfecting Spodoptera
frugiperda (Sf9) cells (2 × 106) with 2 µg of sterile pVL439033 and 1 µg of BaculoGold baculovirus DNA
(PharMingen) as described in the BaculoGold user manual. Viable, recombinant viruses were harvested 4 days after infection and amplified
to a 300-ml quaternary stock in Sf9 cells. Virus stocks had an
approximate titer of 1 × 108 and were stored at
4 °C.
Northern Blot Analysis--
Approximately 30 ng of the 430-bp
StyI fragment of the human clone 251113 was radiolabeled
with [ Preparation of 5' [32P]PAP
Substrate--
Nucleotide 3'-monophosphates (3'-AMP, -dAMP, -CMP,
-GMP, -TMP, and -UMP) (Sigma) were 5'-labeled with 32P
isotope by incubating an excess of the unlabeled nucleotide with
[ Preparation of PAPS--
PAPS (Sigma) is supplied as a lithium
salt. Lithium was removed by desalting over a gravity-flow 5 × 280-mm G10-Sepharose gel filtration column equilibrated in 1 mM Tris-Cl, pH 8.7, 50 mM KCl as described
(25).
Expression and Purification--
Bacterial expressions were
performed by growing recombinant bacteria in 1-liter cultures of LB
broth (10 g/liter Bacto-tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl) containing 100 µg/ml ampicillin. At mid-log phase
(OD600 = 0.6-1.0), cultures were induced with 0.2-0.4
mM isopropyl-1-thio-
Sf9 cells were maintained in spinner flasks at 28 °C in
Grace's medium (Life Technologies, Inc.) at pH 6.1 supplemented with 3.3 g/liter yeastolate, 130 mg/liter lactalbumin hydrolysate, 50 µg/ml gentamycin, and 10% heat-inactivated fetal bovine serum (FBS)
until cell densities reached 1 × 106 cells/ml (log
phase). Viral infections were performed by incubating cells (1 × 109) with approximately 15 ml of virus solution for 4 h at 28 °C with periodic gentle agitation. The cells were then
diluted to 1 liter with Grace's medium complete with 10% FBS. The
infected cell suspension was incubated with stirring for 60 h.
Cells were harvested by centrifugation at 6000 × g for
15 min, followed by washing with phosphate-buffered saline. Cells were
either frozen immediately at
Purification of the GST fusion protein was performed using glutathione
agarose as described in the manual provided by Amersham Pharmacia
Biotech. Purification of the non-fusion recombinant mouse BPntase
initially involved phenyl-Sepharose high performance liquid
chromatography (HPLC). Cell pellets were resuspended in lysis buffer
(25 ml/5 g wet cells) containing 50 mM HEPES, pH 7.5, 5 mM MgCl2, 3 mM NaCl, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride
(PMSF). Cells were lysed in a French pressure cell, and cell debris was
removed by centrifuging at 50,000 × g for 20 min. The
supernatant was filtered through a 0.22-µm cellulose acetate filter
and applied to Bio-Rad Q and S ion exchange cartridges connected in
series and equilibrated in 50 mM HEPES, pH 7.5, 3 mM MgCl2 (buffer A). The flow-through and one
column volume wash were collected and combined. Ammonium sulfate was
added gradually to 30%, and the 50,000 × g
supernatant was applied to a 2-ml phenyl-Sepharose HPLC (Bio-Rad)
column equilibrated with 30% ammonium sulfate in buffer A (buffer B).
The column was washed with 10 column volumes of buffer B, and the
protein was eluted with a 15-column volume reverse linear gradient from
30% to 0% ammonium sulfate saturation in buffer A. The protein eluted
in 18-22% ammonium sulfate saturation.
In order to improve the yield and reliability of the purification, an
affinity purification step was developed based on the enzyme's
activity on PAP. PAP-agarose resin (Sigma) was swelled in PAP-agarose
buffer (50 mM HEPES, pH 7.5, 10 mM
CaCl2, and 50 mM KCl) and poured into a 10-ml
disposable polypropylene chromatography column (Bio-Rad). A 1-ml bed
volume was sufficient to bind up to 6 mg of enzyme. Calcium, a potent
inhibitor of homologous phosphomonoesterases, was added to satisfy the
metal requirement for substrate binding while preventing the hydrolysis
of immobilized PAP. Cell pellets were resuspended in PAP-agarose buffer
containing 1 mM PMSF, and the cells were disrupted in a
French pressure cell and centrifuged as above. The supernatant was
applied to the column by gravity flow, and the column was washed with
15 column volumes of PAP-agarose buffer plus 0.5 M NaCl and
then reequilibrated with PAP-agarose buffer containing no additional
salt. The enzyme was eluted with PAP-agarose buffer containing 300 µM 2'/3',5'-PAP (Sigma). The protein was concentrated and
the PAP removed by dialysis in Microcon centrifugal concentrators,
30,000 nominal molecular weight limit (Millipore; Bedford, MA). The
protein was purified further from small molecules by size exclusion
chromatography on Sephadex G50 (fine) QuickSpin columns (Roche
Molecular Biochemicals). The protein was stored in 50 mM
NaHEPES, pH 7.5, 100 mM KCl, 3 mM
MgCl2, and 0.02% NaN3. Aliquots were frozen at
Native protein was also isolated with PAP-agarose affinity
purification. Kidneys, lungs, liver, and heart from a freshly dissected mouse were homogenized separately with a Dounce homogenizer in 1 ml of
ice-cold PAP-agarose buffer containing 1 mM PMSF. The homogenates were spun at 22,800 × g in a 4 °C
microcentrifuge. The soluble fractions were applied to 50-µl
PAP-agarose columns, which were washed and eluted as above. The eluants
were analyzed by SDS-PAGE and visualized with silver staining and
Western blotting with the anti-BPntase antibody. Eluted enzyme from the
kidney preparations was pooled, and BSA was added to a final
concentration of 0.1 mg/ml. The protein was dialyzed approximately
50,000-fold against a reaction mixture containing 50 mM
NaHEPES, pH 7.5, 100 mM KCl, 1 mM EGTA, and 3 mM MgCl2 (HEKM) with Microcon centrifugal concentrators (Millipore) prior to performing kinetic analyses.
Determination of Substrate Specificity--
Purified GST-439033
fusion protein was utilized to investigate reactions catalyzed by the
protein. Sequence homology with the consensus Li+-sensitive
phosphomonoesterase family active site motif led us to predict that the
enzyme hydrolyzed small phosphorylated molecules, possibly including
inositols and nucleotides, in a Mg2+-dependent
manner. Trace molar amounts of [3H] inositol standards
(American Radiolabel Corp., St. Louis, MO) were incubated with protein
in HEKM. Following incubation at 37 °C, the reaction was stopped
with 1 volume of 20 mM ammonium phosphate (AP) and was
loaded onto a 4.6 × 250-mm PartiSil 10 SAX-HPLC strong anion
exchange column (Whatman, Clifton, NJ). Pre-reaction inositol standards
and post-reaction potential inositol products typically were separated
with linear gradients from 10 mM to 1.7 M AP
over 50 min. Radioactivity of the eluant was measured continuously using a Beta-RAM in-line detector (INUS Systems, Tampa, FL).
Alternatively, GST-439033 was used to treat PAP and PAPS as potential
substrates. Enzyme was incubated with both 10 µM
unlabeled PAPS (Sigma) and approximately 105 cpm
5'-[32P]PAP. Following incubation at 37 °C in HEKM,
the reaction was stopped as above and loaded onto the PartiSil 10 SAX
column. In this case, the gradient used was 10 mM to 1.02 M AP over 60 min. One-milliliter fractions were collected
and monitored for absorbance at Assays of Enzyme Kinetics--
Phosphatase activity was analyzed
in the following manner. Unless otherwise noted, reactions were carried
out in HEKM buffer containing 0.4 mg/ml BSA. For inhibitor studies, the
reaction mixtures contained appropriate concentrations of LiCl,
unlabeled PAPS, or unlabeled Ins(1,4)P2. Enzyme was added
to reaction mixtures containing appropriate concentrations of
trace-labeled 3H-inositols or
5'-32P-bisphosphorylated nucleotides. Reactions were
performed at 37 °C for specific times and were stopped with 20 volumes of appropriate formate buffer (see below). The stopped reaction
mixture was applied to a 200-µl bed volume of Dowex equilibrated in
formate buffer. Reaction products were eluted by washing with 20 column
volumes of formate buffer whereas higher phosphorylated product
remained bound to the column. Quantitation of hydrolysis was performed by liquid scintillation counting of the eluent. Formate buffer for
Ins(1,4)P2 assays contained 0.05 M
NH4COO, 0.1 M COOH. For Ins(1,3,4)P3 experiments, formate buffer consisted of 0.35 M NH4COO, 0.01 M COOH. PAP formate
buffer consisted of 0.137 M NH4COO, 0.273 M COOH.
HAL2 Disruption and Complementation--
Yeast were propagated
in standard rich (YPD) medium, or in complete minimal (CM) medium
lacking appropriate nutrients to maintain plasmids. The entire
HAL2 open reading frame was disrupted from S. cerevisiae W303 and replaced with LEU2. LEU2 was
amplified from pRS305 (23) by PCR with the sense primer
5'-GGCATTGGAAAGAGAATTATTGGTTGCAACTCAAGCTGTACGAAACAGATTGTACTGAGAGTGC and the antisense primer
5'-GGCGTTTCTTGACTGAATGACATCGCATGATGTAGACACCACCAACCTTACGCATCTGTGCGG (where underlined bases correspond to the HAL2 coding
sequence and the remainder is common to the pRS series of plasmids).
The resulting hal2::LEU2 PCR product
was transformed into diploid strain W303
(MATa/MAT
Complementation analysis of the methionine auxotrophy of
hal2::LEU2 was performed by transforming
appropriate plasmids into haploid yeast containing the disruption using
a standard lithium acetate protocol (24). Cells were grown initially in
CM-ura containing the non-repressing sugar raffinose (2% w/v) and
induced at mid-log phase by the addition of 2% galactose. Cells were
counted with a hematocytometer and inoculated into CM-ura lacking
methionine at 104 cells/ml. Cells were grown with shaking
at 30 °C, and growth was determined by measuring
OD600.
Cloning and Tissue Distribution of a Putative Lithium-sensitive
Phosphomonoesterase Family Member--
The wealth of sequence
information obtained through genome and EST data bases has been
invaluable in identifying and extending protein families. In order to
"computer clone" novel members of the structurally conserved
lithium-sensitive phosphomonoesterase protein family, basic local
sequence alignment tool (BLAST) (21) comparisons were initiated. A
query sequence of human 1ptase (accession number L08488) was used to
search human and mouse EST data bases specifically looking for
conservation of the motif
"D-Xn-EE-Xn-DP(i,l)D(g,s)T-Xn-WD-X11-GG," which contains the signature residues important for metal/lithium binding and catalysis. A partial sequence of the murine EST clone 439033, accession number AA008240, was found to have weak similarity to
1ptase and contained part of the above motif,
"D-Xn-EE-Xn-DPlDgT." The entire 439033 cDNA clone was sequenced and found to be 1.4 kb
and had a predicted open reading frame of 308 amino acids (data not
shown; deposited into GenBank accession number AF125043). Importantly,
the coding sequence was found to also contain the "WD-X11-GG" motif, indicating that it is a
bona fide member of this family. In addition, several
truncated human clones were initially found, and subsequent searches
have identified a full-length human cDNA, clone 645079, that has a
length of 2.1 kb and a predicted coding sequence of 309 amino acids
(deposited into Genbank accession number AF125042). Polyadenylation
signals and An tails were found indicating the
3' end of both clones had been identified. The reason for the different
size 3' untranslated sequences between the mouse and human cDNA's,
300 versus 1000 nucleotides, is uncertain. Alignment of the
human and mouse primary structures showed 92% identity and 96%
similarity (data not shown).
In order to define the evolutionary relationship of clone 645079 to the
other family members, the primary structure was compared with the
non-redundant GenBank data base using a gapped BLASTP search (version
2.0.6; BLOSUM62 matrix). Probability scores (in brackets) were used to
rank a number of similar proteins including: U42833,
Caenorhabditis elegans CEESN37F [2e-54]; AL032655, C. elegans cDNA EST yk255e11.5 [1e-20]; L08488, human
1ptase [3e-14]; U14003, E. coli cysQ (amtA) [1e-07];
P29218 -human impase [3e-06]. As clone 645079 was most related to
1ptase and PAP phosphatase, a multiple sequence alignment was performed
among 645079 (hBPntase), 1ptase (h1pt), CEESN37F (U42833), and yeast and plant PAP phosphatase (HAL2 and SAL1) using the program MATCHBOX (27) as shown in Fig. 1 (panel
A). Despite relatively low overall sequence identities,
eleven regions conserved throughout all five proteins were found as
indicated by lowercase lettering (determined without human
intervention), and the gray boxes (for enhanced visualization).
The core structure of the lithium-sensitive family encompasses 160 residues and is composed of 5
In order to determine the expression pattern and size of the 645079 transcript, a multitissue Northern blot analysis was performed. A
radiolabeled 0.4-kb region of the human cDNA was used to probe 2 µg of human messenger RNA from a variety of tissue sources, and the
results are shown in Fig. 2. A single
2.5-kb message was visible at various levels in all tissues examined,
indicating that the clone 645079 was approximately full-length. As a
control for amount of mRNA loaded, the same blot was reprobed with
Substrate Specificity Determination--
Computer cloning
represents a unique "reverse" biochemistry problem, i.e.
determining the substrate specificity for a clone of unknown function.
Sequence homology with proteins of the lithium-sensitive phosphomonoesterase family led us to predict that the enzyme would be
metal-dependent and hydrolyze small phosphorylated
molecules, possibly including phosphorylated inositols and nucleotides.
To investigate this possibility, the coding sequence from the murine clone 439033 was inserted into a pGEX vector and expressed in bacteria
generating a GST-439033 fusion protein. Recombinant GST-439033 was
readily purified using glutathione affinity resin and incubated in
Mg2+ buffer containing radiolabeled Ins(1,4)P2,
Ins(1,3,4)P3, and Ins(1,4,5)P3. Reactants from
GST control, GST-439033, or pure 1ptase were separated by strong anion
HPLC, and representative radiograms of eluted compounds are shown (Fig.
3A, lower,
middle, and upper traces,
respectively). GST and untreated (not shown) samples show three peaks,
B, D, and E, corresponding to
Ins(1,4,)P2, Ins(1,3,4)P3, and
Ins(1,4,5)P3, respectively. Treatment with 1ptase provides
additional standards Ins(4)P (peak A) and
Ins(3,4)P2 (peak C), consistent with
removal of the 1-position phosphate. Treatment with GST-439033 shows
qualitatively similar results in that peaks A and
C have appeared at the expense of disappearance of
peaks B and D. In both cases,
Ins(1,4,5)P3 is not hydrolyzed, as the size of
peak E is unchanged. As GST-439033 does not
metabolize the Ins(4)P product to inositol nor does it hydrolyze
Ins(1)P (data not shown), we conclude it does not function as an
impase. Additionally, Ins(3,4)P2 is not converted,
indicating that GST-439033 is not acting as a 3- or 4-phosphatase.
Furthermore, other inositol polyphosphates that were tested included
Ins(1,3,4,5)P4, Ins(1,3,4,5,6)P5, and
InsP6, none of which was dephosphorylated. Together, these data demonstrate that GST-439033 functioned qualitatively as an inositol polyphosphate 1-phosphatase.
Given the sequence similarity to HAL2, cysQ, and
to a lesser extent SAL1, we tested GST-439033 using PAP and PAPS
substrates. GST-439033 or GST protein was incubated in the presence of
Mg2+, 5'-[32P]PAP, and unlabeled PAPS, the
reactants were separated by HPLC, and fractions were collected and
monitored by absorbance at 260 nm and scintillation counting (Fig.
3B). Upon reaction, the PAP and PAPS peaks disappear,
whereas quantitative peaks at the elution times of 5'-AMP and 5'-APS
appear. Thus, the enzyme has phosphatase activity on PAP and PAPS.
Because the substrate is labeled at the 5' position and no free
32Pi appears, we conclude that the 3'-position
phosphate is hydrolyzed.
To qualitatively address whether or not the nucleotide played a role in
determining substrate selectivity, several additional bisphosphorylated
nucleotides were tested as potential substrates. As described under
"Materials and Methods," 3'-monophosphates of 2'-dAMP, CMP, GMP,
TMP, and UMP were 5'-labeled with polynucleotide kinase and
[ Purification of Non-fusion Protein--
In order to determine
detailed biochemical parameters of mammalian nucleotide/inositol
phosphatase, purification of non-fusion protein was performed. The open
reading frame of the mouse clone was inserted into a baculovirus
cloning vector. Recombinant virus was amplified and used to infect
Sf9 cells in medium containing 10% FBS. Typical infections
resulted in production of approximately 10 mg of soluble enzyme/liter
of cells, representing around 4% of the total soluble protein.
Initial investigations indicated that the protein flowed through both
S-Sepharose cation exchange columns and Q-Sepharose anion exchange
columns at pH 7.5. Therefore, a negative purification step involving
both ion exchange columns in series was employed. Subsequently,
ammonium sulfate was added to 30%, and soluble proteins were applied
to an HPLC phenyl-Sepharose column. The protein eluted in 18-22%
ammonium sulfate in a reverse linear gradient from 30% to 0% ammonium
sulfate. Following concentration and removal of ammonium sulfate,
typical yields were on the order of 20% with purification of
approximately 25-fold.
In order to increase yield, an improved affinity purification method
based on previously reported PAP-agarose chromatography (28) was
developed. Calcium exhibits micromolar inhibition of phosphomonoesterase activity of other members of the lithium-sensitive family. Cell lysates therefore were applied to PAP-agarose equilibrated in 10 mM CaCl2 as described under "Materials
and Methods" in order to prevent hydrolysis of the immobilized
substrate. Typically, a 1-ml resin bed volume was sufficient to bind
approximately 6 mg of enzyme. The resin was washed with buffer
containing up to 500 mM NaCl to elute nonspecifically bound
proteins, and the enzyme was eluted in approximately 300 µM free PAP. The single-step purification resulted in a
high yield of homogeneous protein (Fig. 3). This protein was
concentrated and dialyzed to remove calcium and free PAP prior to
enzymatic assays. Aliquots were stored frozen at
In order to confirm the in vivo expression, we isolated it
from freshly harvested mouse tissues. Following the success of using a
PAP-agarose affinity matrix to obtain purified baculovirus-expressed 439033 protein in a single step, we employed this technique to isolate
the native enzyme. The soluble fraction from centrifuged mouse kidney
homogenate was applied directly to a PAP-agarose column equilibrated in
10 mM CaCl2. As shown by the silver stain in
Fig. 4B, a single band
co-migrating with recombinant BPntase appears in the column elution
fraction. In addition to similar PAP-agarose binding and SDS-PAGE
migration, polyclonal antibodies to recombinant 439033 protein react
with the 37-kDa band (Fig. 4C), giving further evidence that
the native enzyme has been isolated from fresh mouse tissues. Several
mouse tissues, including kidney, lung, heart, and liver, were analyzed
(Fig. 4C). Western blot data corroborated Northern blot
analysis (Fig. 2), indicating highest levels of expression in the
kidney and little expression in lung.
Kinetics of Phosphomonoesterase Activity--
Previously reported
kinetic parameters for bisphosphate nucleotidase activity were
determined using non-radioactive PAP and/or PAPS substrate. Due to
relative insensitivity of this assay, Michaelis constants for all
nucleotides were approximated. As a means to accurately determine the
affinity constant and catalytic efficiency of mammalian protein, a
radiolabeled nucleotide assay was developed. [32P]PAP
labeled in the 5' position was generated simply by incubating 3'-AMP
with [
Phosphatase activity on PAP, Ins(1,4)P2, and
Ins(1,3,4)P3 is linearly dependent on amount of enzyme and
time within a range of 10-50% hydrolysis (data not shown). Therefore,
dose and time were adjusted within experiments to keep fraction of
substrate hydrolyzed around 30%. Plots of reaction velocity
versus substrate concentration for recombinant 439033 protein are displayed in Fig. 5
(A-C). The Michaelis-Menten constants were determined from Lineweaver-Burk analyses (Fig. 5, insets) and are displayed
in Table I, part A. PAP is hydrolyzed to
5' AMP with a Km of 520 nM and a
Vmax of 42 µmol/min/mg of enzyme.
Ins(1,4)P2 is hydrolyzed to Ins(4)P with a
Km of 113 µM and a
Vmax of 11 µmol/min/mg.
Ins(1,3,4)P3 is hydrolyzed to Ins(3,4) P3 with a Km of 100 µM and a
Vmax of 8 µmol/min/mg. According to these
parameters, PAP is clearly the favored substrate under these
conditions, and the protein is thus named bisphosphate 3'-nucleotidase, or BPntase. The kcat/Km for the
hydrolysis of PAP, 5.0 × 107 s
Purified native protein was assayed for hydrolysis of PAP and
Ins(1,4)P2. Native enzyme hydrolyzes PAP
(Km = 475 nM,
Vmax = 60 µmol/min/mg) and
Ins(1,4)P2 (Km = 130 µM, Vmax = 8 µmol/min/mg) in a
Li+-sensitive manner and with kinetics similar to those
determined for the recombinant protein (data not shown).
PAP hydrolysis is dependent on the presence of Mg2+.
Optimum activity was obtained within a Mg2+ concentration
range of 2.5-3.5 mM. Similar to other enzymes in the
lithium-sensitive family, activity is inhibited at high concentrations of Mg2+ (Fig. 6A)
(2). A Hill plot of data within a range of Mg2+
concentrations of 0.01-0.2 mM gives a Hill coefficient of
1.60, indicating positive cooperativity but an undetermined number of metal binding sites (Fig. 6B). The pH optimum for PAP
hydrolysis is approximately 7.5 (Fig. 6C). Similar results
were obtained for the hydrolysis of Ins(1,4)P2 and
Ins(1,3,4)P3 (data not shown).
Inhibition of Phosphatase Activity--
Based on its limited
sequence similarities to other Mg2+-dependent
phosphomonoesterases in the 1ptase family, we predicted that BPntase
would also be uncompetitively inhibited by Li+. The
velocity of PAP phosphatase activity versus lithium
concentration at various substrate concentrations was determined and
displayed as a Dixon plot in Fig.
7A. Parallel
lines in the Dixon plot indicate that lithium inhibition is
uncompetitive with respect to PAP. The Ki for this
inhibition is 157 ± 17 µM LiCl (Table I, part B).
Similarly, Li+ inhibition is uncompetitive with respect to
Ins(1,4)P2, and the calculated Ki is 540 µM (data not shown).
As BPntase markedly favors nucleotides, we sought to determine if the
inositol substrates compete for identical sites. The data are
summarized in Table II. The cellular
ranges of inositol polyphosphates are estimated to be near or below
micromolar levels in unstimulated cells and 10-50 µM in
stimulated cells. Thus we analyzed inhibition at 50 and 100 µM concentrations. Remarkably, 50 µM
Ins(1,4)P2 inhibited 84.4% of PAP hydrolytic activity,
whereas other inositol polyphosphates even at 100 µM
concentrations had less potent inhibitory activity. This was carefully
analyzed in Fig. 7B. Nucleotide hydrolysis in the presence
of Ins(1,4)P2 displays competitive inhibition with a
Ki of 15 µM (Fig. 7B; Table I, part B). This provides a basis that Ins(1,4)P2 may play
a regulatory role via inhibition of phosphatase activity as opposed to
hydrolysis in the action of BPntase in vivo.
Functional Complementation of Yeast HAL2--
The similar
substrate selectivity of BPntase, SAL1, and Hal2p prompted experiments
to determine if BPntase could functionally complement hal2
yeast mutants. The HAL2 gene therefore was disrupted in
diploid W303 S. cerevisiae by single gene replacement with the LEU2 marker gene. The diploid yeast were sporulated,
tetrads were dissected, and spores were found to segregate 4:0 on rich media consistent with a nonessential function. Previous studies show
that the hal2 disruption confers methionine auxotrophy (10); therefore, spores with the disruption are expected to be methionine auxotrophic and leucine prototrophic. Replica plating tetrads on
Leu
Complementation of the hal2 disruption was examined by
overexpressing mBPntase, Hal2p, cysQ protein, and human 1ptase. The genes were expressed from a high copy plasmid (pRS426) containing a
galactose-inducible promoter. Following growth in media containing methionine and 2% galactose, cultures were inoculated at
104 cells/ml into synthetic media lacking methionine and
containing 2% galactose. Fig. 8 displays
the functional complementation of the hal2 knockout. Exogenous Hal2p,
mBPntase, and cysQ complement at similar levels. As expected, the
vector control displays methionine auxotrophy. In addition, 1ptase,
which does not hydrolyze nucleotides, does not complement the defect.
This is consistent with the idea that hydrolysis of bisphosphorylated
nucleotides and not inositol polyphosphates is required for growth in
the absence of methionine.
Enzyme overexpression was monitored by changes in specific activities.
At 1 µM PAP, the cultures had the following specific activities in nanomoles of PAP hydrolyzed/min/mg of total protein: HAL2, 23.7; mBPntase, 16.7; cysQ, 3.5; 1Ptase, 0; vector control, 0. At
8 µM Ins(1,4)P2, the cultures had the
following specific activities (pmol/min/mg): HAL2, 130.4; mBPntase,
415.7; cysQ, 66.1; 1Ptase, 746.2; vector control, 41.7. These data
suggest the following: 1) that the cultures were expressing the
recombinant proteins near expected levels; 2) that overexpression of
HAL2, as well as cysQ, results in a significant increase in specific activity against Ins(1,4)P2, suggesting that these proteins
have hydrolytic activity against bisphosphorylated inositols. This result contradicts an earlier report that suggested that HAL2 does not
hydrolyze Ins(1,4)P2 (12). To address this, we analyzed the
kinetic properties of recombinant Hal2p and cysQ. Hal2p hydrolyzes PAP
(Km = 720 nM) and Ins(1,4)P2
(Km = 430 µM). Similarly, cysQ
hydrolyzes PAP (Km = 1.1 µM) and
Ins(1,4)P2 (Km = 1.2 mM).
Catalytic efficiencies are displayed in Table I, part A.
The Lithium-sensitive Phosphomonoesterase Family--
Lithium's
role in biology is of intense interest, yet the mechanisms by which it
exerts its effects in human disease and development remain to be firmly
established. The identification of a family of structurally conserved
lithium-sensitive phosphomonoesterases has provided insight into this
mechanism in several ways. First, biochemical and biological
characterization of these proteins demonstrated that potent inhibition
of enzymatic activity occurs at submillimolar concentrations,
indicating that they are relevant targets. In vivo studies
have validated this point. Perhaps most compelling are genetic studies
of Drosophila 1ptase mutants demonstrating that deletion of
the ipp phenocopies treatment of normal flies with lithium
(18). Additionally, lithium induces changes in the levels of substrates
and products of several family members in vivo. Second, the
initial connection of lithium to inositol signaling pathways was made
through impase leading to an "inositol depletion" hypothesis (30).
Discovery of other lithium-sensitive signaling and metabolic proteins,
such as 1ptase, fbptase, Hal2p, SAL1, and now BPntase, has greatly
expanded our view of pathways involved. The notion that manic
depression is a polygenic disorder is consistent with the involvement
of multiple signaling pathways. Third, the conservation of the
structures of this family has provided a key demonstration that
they have evolved from a common ancestor. The ability to identify
members such as fbptase required knowledge of the three-dimensional
structure, as the overall sequence similarity is undetectable by
current local alignment strategies. Importantly, the comparison of the
structures has resulted in the identification of the signature motif of
this family:
D-Xn-EE-Xn-DP(i/l)D(s/g/a)T-Xn-WD-X11-GG.
The hypothesis that proteins having this motif will also function as
metal-dependent lithium inhibited phosphomonoesterases has
been validated by this study. This is of significant importance especially in the upcoming "post-genomic" era of modern biology. Repeating the strategy defined in this study on emerging or completed EST and genome data bases will facilitate rapid identification of novel
human lithium targets. The demonstration that BPntase was
uncompetitively inhibited by Li+ with the lowest inhibition
constant described to date is significant. Additionally, structural
information was obtained from multiple sequence alignments with 1ptase
and mapping regions of conservation to the core elements. Of note,
although the signature motif is able to definitively identify a
protein's membership in the family and lithium sensitivity, it appears
unable to predict a protein's substrate selectivity. This is evident
from characterization of the mammalian BPntase that appeared more
related to 1ptase than to nucleotidases, yet the substrate preference
favored bisphosphate nucleotides.
The Role of Nucleotidase Versus Inositol Phosphatase Activities of
BPntase--
The ability of BPntase to hydrolyze both nucleotide and
inositol polyphosphates raises the question which activity is important biologically? Previous reports characterizing nucleotidase biochemical parameters were limited in interpretation due to the relative insensitivity of measuring phosphate release from unlabeled substrate and/or spectroscopic measurements. This was overcome by developing a
simple radiolabeling procedure for making 5'-labeled PAP, and our data
show that the Michaelis affinity constant for nucleotides was in the
nanomolar range, an order of magnitude below previously estimated
values (13, 28, 29, 31). Previous biochemical studies of Hal2p
nucleotidase showed that it did not possess 1-phosphatase activity
(12). In contrast, our data clearly demonstrate that Hal2p does have
1-phosphatase activity, with similar selectivity and catalytic
efficiency to mammalian and plant counterparts (13). We demonstrate
that the catalytic efficiency of BPntase, Hal2p, and CysQ toward
nucleotide substrates is highly favored. Furthermore, 1ptase, which
does not possess nucleotidase activity, is unable to complement
hal2 mutant yeast providing further biological support favoring the nucleotidase function. Thus, we conclude that the biologically relevant activity is the 3'-nucleotidase.
An unexpected finding of our studies was that inositol 1,4-bisphosphate
potently inhibits nucleotidase activity, having an inhibitory constant
in the physiological range. This provides a novel connection that,
rather than functioning to hydrolyze inositol polyphosphates, BPntase
is potently regulated by these potential messengers. Kidney tissue has
been shown to possess ample levels of 1ptase, which has a catalytic
activity 100 times higher than the 1-phosphatase activity of BPntase
(16). Of interest, a previous report of Quintero et al. (13)
suggested that the 1-phosphatase activity of SAL1 was important for
mediating the sodium/lithium efflux response. This interpretation was
based on the observation that both the inhibition of inositol-specific phospholipase C using the inhibitor 48/80, and the overexpression of
SALI promote lithium efflux in cells. The authors concluded from these
experiments that phospholipase-mediated inositol signaling regulates
cation efflux through ENA pumps and that SALI participates in this process by promoting turnover of messenger molecules. Another
interpretation, consistent with our model, is that blocking phospholipase C activity reduces levels of inositol 1,4-bisphosphate, relieving inhibition of the nucleotidase activity, thereby mimicking the effects of overexpression. Further studies are needed to clarify which model is correct.
The ability of this family of nucleotidases to utilize either
bisphosphate nucleosides or PAPS has further confounded determination of the physiologically relevant substrate. Mutations in either HAL2/MET22 or cysQ result in defective sulfur
assimilation (10, 14), providing evidence the PAPS nucleotidase
activity is biologically relevant. Neuwald et al. suggested
that PAPS or a derivative may be cytotoxic when allowed to accumulate
(14). As support for this idea, they cite the fact that poor growth due
to mutations in the PAPS-utilization pathway can be rescued by
inhibiting the formation of PAPS with additional mutations in
cysC, a 5'-APS 3'-kinase (14). Furthermore, Peng and Verma
have shown that supplementation of media with methionine but not
sulfite supports growth of hal2 mutants, indicating that the
PAPS nucleotidase activity is most relevant (31).
Alternatively, due to their similar structures, PAP and PAPS may play
cooperative roles in the sulfur assimilation pathway. For example,
Ozeran et al. showed that a PAPS translocase transports PAPS
across mitochondria membranes via an antiport mechanism with PAP as the
returning ligand (32, 33). BPntase may function to deplete the pool of
PAP, thereby facilitating the transport of PAPS across membranes and
into the vicinity of the sulfotransferase machinery. The PAPS
translocase itself was found to be competitively inhibited by PAP with
respect to PAPS (32, 33). Therefore, accumulation of PAP via a decrease
in BPntase activity could have drastic effects on sulfur assimilation
pathways. In addition to a possible effect on sulfur assimilation,
recent evidence points to a role of PAP nucleotidase activity in
regulating RNA processing. Dichtl et al. (17) reported
deletion of hal2 results in defects in Xrn1p-mediated RNA
processing due to direct inhibition by PAP. This enzyme is not
essential, but the redundant function is accomplished by RNase MRP, an
enzyme that may itself be inhibited directly or indirectly by lithium.
Therefore, Dichtl et al. propose that lithium toxicity, at
least in yeast, is mediated by inhibition of RNase MRP and by
concurrent inhibition of the cytosolic enzyme Xrn1p via inhibition of
HAL2 and subsequent PAP accumulation (17). Under growth
conditions containing high Na+ or Li+
concentrations, overexpression of the PAP-metabolizing enzymes HAL2 and SAL1 would rescue growth by an increase
in enzyme activity, thus reducing accumulated PAP pools. Methionine
supplementation would also rescue growth by down-regulating the
production of PAP from PAPS (34). As it appears that BPntase is a true
functional homologue of HAL2 and SAL1, our results augment
the findings of Dichtl et al. BPntase is active on
bisphosphorylated nucleotides other than PAP such as PGP and PCP. Thus,
other bisphosphorylated nucleosides that may accumulate in the absence
of nucleotide phosphatase activity may also inhibit Xrn1p.
The Role of BPntase in Kidney Function--
It is particularly
interesting that the levels of nucleotidase are highest in kidney,
consistent with the notion increases in nucleotidase activity are
associated with resistance to salt. Both SAL1 and HAL2 were
identified on screens for proteins that, when overexpressed, conferred
tolerance to cations such as sodium and lithium. Furthermore, the
up-regulation of SAL1 expression in response to increased tonicity (13)
suggests a physiological role for nucleotidase activity in osmoregulation.
In addition, one of the major side effects of lithium treatment is
nephrogenic diabetes insipidus, characterized by polyuria and
polydipsia. The root of this side effect may lie in the inappropriate inhibition of BPntase phosphatase activity by Li+.
Disruption of sodium extrusion machinery on either the lumenal or basal
side of kidney cells could upset sodium balances. Such a disruption in
sodium homeostasis could be manifested as the movement of large amounts
of fluid across kidney cells, resulting in the voiding of copious
amounts of liquid. Of particular clinical significance is the ability
of methionine supplementation to suppress lithium toxicity in yeast,
through a mechanism that down-regulates production of PAP and
ultimately effects RNA processing, sulfur assimilation, and cation
efflux pathways. We suggest that a similar strategy in which lithium is
co-administered with methionine in patients treated for bipolar
disorder may prove clinically useful in abrogating the harmful effects
of nucleotidase inhibition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
). The mouse coding
sequence was inserted into pGEX3X (Amersham Pharmacia Biotech) to
generate an in-frame glutathione S-transferase (GST)-439033
fusion expression construct. The pBluSK439033 plasmid was digested with
DraIII and BclI, and a fragment representing residues 5-308 was purified. Residues 1-5 were rebuilt with the complementary linker oligonucleotides
5'-AATTCGGGATCCCCATCATGGCTCCAGCCACACC and
3'-GCCCTAGGGGTAGTACCGAAGGTCGGTGTGG. Triple body ligation of the
1-kb fragment, linker adapter, and pGEX3X/BamHI cut was
performed to create pGEX439033. The non-fusion 439033 protein was made
by generating recombinant 439033 baculovirus DNA. The baculovirus transfer vector pVL1393 (PharMingen, San Diego, CA) was
digested with BamHI, and the
BamHI/BclI fragment of pGEX439033 was inserted to
create pVL439033. For yeast complementation studies, the
BamHI-NotI fragment of pVL439033 was ligated into
BamHI/NotI-linearized pRS426GAL (23) (obtained
from Susan Wente, Washington University, St. Louis, MO) to create
pRS439033, a yeast high copy plasmid with expression under control of a
galactose-inducible promoter.
cells,
100 ng of each primer, 0.2 µl of Expand High-Fidelity Taq
polymerase (Roche Molecular Biochemicals), and 1× Expand PCR
buffer. PCR proceeded for 30 cycles of 30 s of denaturation
(94 °C), 30 s of annealing (52 °C), and 1 min of extension
(72 °C). The amplified products were gel-purified and cloned into
pCR2.1 using the TA cloning kit (Invitrogen, San Diego, CA) to create
pCRHAL2 and pCRcysQ. The BclI-NotI fragment of
pCRHAL2 and the BamHI-NotI fragment of pCRcysQ
were cloned into BamHI/NotI-linearized pRS426GAL
to create pRSHAL2 and pRScysQ. The NdeI-BclI
fragment of pCRHAL2 and the NheI-BamHI fragment of pCRcysQ were isolated and ligated into
NdeI/BamHI-linearized pET11c or
NheI/BamHI-linearized pET11c bacterial expression
vector (Novagen, Madison, WI) to create pETHAL2 and pETcysQ. These
plasmids were transformed into the E. coli strain BL21(DE3)
for overexpression.
-32P]ATP to a specific activity of approximately
1 × 109 cpm/µg as described with the Random Hexamer
primer labeling kit (Roche Molecular Biochemicals). A human multiple
tissue Northern blot, prepared with 2 µg of poly(A)-selected mRNA
isolated from various tissues, was purchased from
CLONTECH. The membrane was prehybridized and
hybridized at 42 °C in 10 ml of 50% formamide, 5× SSPE (0.9 M NaCl, 50 mM phosphate, pH 7.4, 5 mM EDTA), 10× Denhardt's solution, 0.1 mg/ml sheared
salmon sperm DNA, 2% SDS. The membrane was washed twice with 2×
standard saline citrate (SSC), 0.05% SDS at room temperature and twice
with 0.1× SSC, 0.1% SDS at 50 °C and then exposed to film. The
membrane was stripped and reprobed with radiolabeled human actin DNA
supplied with the blot.
-32P]ATP (NEN Life Science Products) and T4
polynucleotide kinase (Roche Molecular Biochemicals) under conditions
recommended by the manufacturer. The reaction was stopped by the
addition of formate to a concentration of 0.01 M COOH, 0.5 M NH4COO. The mixture was applied to a 25-µl
Dowex (AG 1-X8 Resin, 200-400-mesh, formate form; Bio-Rad) column
equilibrated in formate. Following extensive washing, the product was
eluted with 0.03 M COOH, 1.05 M
NH4COO. Ammonium formate was removed by repeated lyophilizing.
-D-galactopyranoside and
grown at 37 °C for an additional 4-6 h.
80 °C for later use or were lysed and
purified at once.
80 °C and were stable for at least 3 months with no detectable
loss of activity.
= 260 nm and then for
32P radioactivity by scintillation counting. The
experimental chromatogram was compared with a standard chromatogram
containing commercially obtained 5'-adenosine phosphosulfate (APS),
5'-AMP, PAP, and PAPS (Sigma).
), and stable
transformants were selected on defined media plates lacking leucine as
described (26). Gene disruption was confirmed by PCR using a sense
primer complementary to the chromosomal region 5' of the
HAL2 locus (5'-GACCAGCAATATCACCTGTTG) and an antisense
primer corresponding to a sequence in LEU2
(5'-CCATGCCACGGTTCTGCTC). Heterozygous diploids were sporulated and
dissected as described (26). The
hal2::LEU2 spores segregated 2:2 (data
not shown) and were identified by growth on -Leu as well
as PCR analysis as above.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Multiple sequence alignment and
structural relationship of 645079 to selected members of the
lithium-sensitive phosphomonoesterase family. A, the
sequences of human clone 645079 predicted coding region, human 1ptase
(h1pt), yeast and plant 3'-nucleotidases (HAL2 and SalI),
and Caenorhabditis elegans clone CEESN37F (U42833) were
aligned with MATCHBOX (27). Conserved regions among these sequences
were identified by the program are displayed in lowercase
letters and visually enhanced with gray
boxes. Secondary structure elements, determined from
the high resolution structure of bovine 1ptase (1), are mapped onto the
alignment as arrows (strands) and coils
(helices). B, the -carbon trace of the core of 1ptase is
displayed as a stereo image with conserved structural elements, 5
-helices, and 11
-strands, labeled.
-helices, 11
-strands, and at 2 two
metal binding sites. A stereo image of the 1ptase
-carbon core is
shown in Fig. 1B. To characterize the sequence/structure relationship, the secondary structure elements were mapped onto the
alignment as shown by the gray arrows (strand)
and coils (helix) in Fig. 1A. Remarkably, the
MATCHBOX alignment identified 11 out of 16 core structure regions,
providing further evidence that clone 645079 is structurally and
functionally related to the lithium-sensitive protein family.
-actin probe, as shown in the lower panel.
Relative expression was determined by comparing the ratio of 645079 and
actin radioactivity (as quantified by phosphorimage analysis). The
lowest level of expression was observed in lung and was assigned a
value of 1.0 arbitrary units to which the other tissues were
normalized. The highest level of expression was observed in kidney,
which has 90.5-fold increased level relative to lung. Additionally,
Northern analysis of murine kidney mRNA using a radiolabeled
fragment of clone 439033 demonstrated a single band of 1.4 kb
consistent with size observed from sequencing of the clone (data not
shown). Of interest, the relative amount of expression in brain was
low.
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Fig. 2.
Distribution of clone 645079 mRNA in
human tissues. A human multitissue Northern blot was probed for
the expression of the 645079 message. The blot was probed with a
radiolabeled fragment of the human gene and exposed to film for 32 h at 80 °C with two intensifying screens (top). The
same filter was then stripped and re-probed with a radiolabeled actin
cDNA fragment and exposed to film for 2 h at room temperature
(bottom). Radioactivity for each band was quantified using a
PhosphorImager, and expression levels of 645079 are provided relative
to actin and normalized to levels observed in lung (assigned a value of
1). RNA molecular weight standards (kb) are displayed.
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Fig. 3.
Analysis of reactions catalyzed by
439033. A, various inositols were treated with:
439033-GST (1), 1ptase (2), and GST control
(3). Reaction products were separated on PartiSil 10 SAX-HPLC using a linear gradient from 10 mM to 1.7 M AP over 50 min. Radioactivity was measured continually
with an in-line liquid scintillation counter. Elution times of
commercially prepared Ins(1,4)P2 (B),
Ins(1,3,4)P3 (D), and Ins(1,4,5)P3
(E) are indicated by the letters and
corresponding arrows. Products of the 1ptase reaction
correspond to peaks A (Ins(4)P) and C
(Ins(3,4)P2). B, 5'-[32P]PAP and
unlabeled PAPS were treated for 1 h with GST-439033 (1)
and GST (2). Reaction products were separated by HPLC using
a linear gradient from 10 mM to 1.02 M AP over
60 min. One-milliliter fractions were collected and monitored for
substrate via absorbance at = 260 nm (dashed
lines) and 32P liquid scintillation counting.
Letters and arrows indicate elution times of
commercially prepared 5'-AMP (A), 5'-APS (B), PAP
(C), and PAPS (D).
-32P]ATP. The purity of labeled nucleotides was
analyzed by PartiSphere SAX HPLC to ensure that no contaminating PAP
was present. Although no standards are commercially available, purity
of the nucleotides was inferred because, under conditions of HPLC,
their elution times differed individually and from that of PAP. Only
the purity of deoxy-PAP remained ambiguous due to its co-elution with
the 2'-oxy form. The labeled nucleotides were incubated with purified GST-439033 or GST alone and subjected to Dowex anion exchange chromatography as described under "Materials and Methods" using PAP
reaction conditions. In each case, product was detected upon reaction
with GST-439033, indicating that each of the nucleotides tested is a
substrate for the enzyme (data not shown).
80 °C for up to 3 months with no apparent loss of activity.
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Fig. 4.
Overexpression of recombinant BPntase and
purification of recombinant and native proteins. Proteins were
separated on 9% SDS-PAGE. Standards (kDa) migrated as indicated.
A, Coomassie Brilliant Blue staining of recombinant mouse
BPntase produced in a baculovirus/Sf9 system. Lane 1,
SF9 crude supernatant, 60 h after infection (5 µg of total
protein); lane 2, PAP-agarose flow-through and 7 column
volume wash (5 µg); lane 3, final wash; lanes
4-9, profile of elution with 300 µM free PAP.
B, silver staining of native BPntase isolated from mouse
kidney. Lane 1, crude mouse kidney supernatant (1 µg);
lane 2, mouse liver PAP-agarose flow-through and 7 column
volume wash (1 µg); lane 3, final wash; lanes
4-6, free PAP elutions. C, Western blot analysis using
antibodies against recombinant BPntase (rabbit host). Lane
1, pure recombinant mBPntase (50 ng); lanes 2-5, crude
mouse tissues (30 µg of total protein/lane) (lane
2, kidney; lane 3, lung;
lane 4, heart; lane 5,
liver); lanes 6-9, third elution from PAP-agarose
purifications (lane 6, kidney; lane
7, lung; lane 8, heart;
lane 9, liver).
-32P]ATP and T4 polynucleotide kinase. In this
way, nucleotide concentrations in the nanomolar range were easily
assayed. Similarly, radioassays for inositol phosphatase activity were
used as described previously (15).
1
M
1, is approximately 1000-fold higher than
the kcat/Km for the hydrolysis of
Ins(1,4)P2 and Ins(1,3,4)P3, which are 6.0 × 104 s
1 M
1 and
8.1 × 104 s
1
M
1, respectively. Lithium-free PAPS was used
to inhibit PAP phosphatase activity in order to determine an
approximate association constant for the sulfated nucleotide. As
expected, inhibition was competitive (data not shown), with a
Ki of 700 nM (Table I, part B).
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Fig. 5.
Kinetic parameters of BPntase
phosphomonoesterase activity. Reactions were run as described
under "Materials and Methods." In each case, reaction times were
varied to keep the fraction hydrolyzed within the linear range of
10-50%. Inset of each velocity versus substrate
concentration plot shows a Lineweaver-Burk plot of the data as
1/velocity (µmol/min/mg) 1 versus 1/substrate
concentration (µM)
1. A, PAP
hydrolysis of recombinant mBPntase. B,
Ins(1,4)P2 hydrolysis of recombinant mBPntase.
C, Ins(1,3,4)P3 hydrolysis of recombinant
mBPntase. D, PAP hydrolysis of native mBPntase.
E, Ins(1,4)P2 hydrolysis of native
mBPntase.
Biochemical parameters of nucleotide bisphosphatases
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Fig. 6.
Mg2+ and pH dependence of BPntase
PAP-phosphatase activity. PAP phosphatase reactions were run as
described under "Materials and Methods." Each reaction contained 2 µM PAP, 50 mM Na-HEPES, and the appropriate
concentration of MgCl2. Mg2+ dependence
reactions were performed at pH 7.5, and pH dependence reactions were
run at 3 mM MgCl2. Reactions were performed in
triplicate, and error bars represent the
mean ± standard deviation. A, Mg2+
dependence over a concentration range of 0.5-10 mM
MgCl2. B, Mg2+ dependence over a
concentration range of 0-0.2 mM MgCl2.
Inset shows a Hill plot of the data as ln(velocity)
versus ln(Mg2+). The slope of the plot (Hill
coefficient) is 1.60. C, pH dependence.
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Fig. 7.
Inhibition of BPntase PAP-phosphatase
activity. PAP phosphatase reactions were run as described under
"Materials and Methods." A, each reaction contained
labeled PAP and an appropriate concentration of LiCl.
Numbers next to each line represent the concentration of PAP
(µM) in the corresponding set of reactions. The data are
displayed as a Dixon plot, in which parallel lines signify
uncompetitive inhibition with respect to substrate. The calculated
inhibition constant is 157 ± 12 µM LiCl.
B, each reaction contained labeled PAP and an appropriate
concentration of unlabeled Ins(1,4)P2. Numbers
next to each line represent the concentration of Ins(1,4)P2
(µM) in the corresponding set of reactions. Data are
displayed as a Lineweaver-Burk plot. Lines intersecting at the
y axis signify competitive inhibition with respect to
substrate. The calculated inhibition constant is 15.0 ± 0.3 µM Ins(1,4)P2.
Inhibition of PAP hydrolysis by inositol phosphates
and Met
plates showed a 2:2 segregation
of both leucine and methionine auxotrophy (data not shown).
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Fig. 8.
Complementation of
hal2 methionine auxotrophy. The native
HAL2 gene in W303 was disrupted by replacement with a LEU2
cassette. Complementation analysis on haploid cells was performed with
indicated gene products on a 2µ plasmid (pRS426), selected by uracil
prototrophy, and under control of a GAL promoter. Transformed yeast
were inoculated at 104 cells/ml in synthetic media lacking
uracil and methionine and containing 2% galactose.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Leslie Stolz for help in generating the yeast mutant strain.
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FOOTNOTES |
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* This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, a Whitehead Scholar award, and National Institutes of Health Grant R01-HL 55672.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) AF125042 and AF125043.
To whom correspondence should be addressed. Tel.: 919-681-6414;
Fax: 919-684-8922; E-mail: yorkj{at}acpub.duke.edu.
2 J. D. York and J. P. Xiong, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: fbptase, fructose 1,6 bisphosphatase; impase, inositol monophosphatase; 1ptase, inositol polyphosphate 1-phosphatase; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; Ins(1)P, inositol 1-monophosphate; Ins(4)P, inositol 4-monophosphate; Ins(1, 4)P2, inositol 1,4-bisphosphate; Ins(3, 4)P2, inositol 3,4-bisphosphate; Ins(1, 3,4)P3, inositol 1,3,4-trisphosphate; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; Ins(1, 3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; Ins(1, 3,4,5,6)P2, inositol 1,3,4,5,6-pentakisphosphate; InsP6, inositol hexakisphosphate; BSA, bovine serum albumin; GST, glutathione S-transferase; PCR, polymerase chain reaction; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; AP, ammonium phosphate; PMSF, phenylmethylsulfonyl fluoride; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); bp, base pair(s); APS, 5' adenosine phosphosulfate.
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REFERENCES |
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