Báo cáo khoa học: Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays pdf

Purification of a plant nucleotide pyrophosphatase as a protein that
interferes with nitrate reductase and glutamine synthetase assays
Greg B. G. Moorhead*, Sarah E. M. Meek, Pauline Douglas*, Dave Bridges*, Catherine S. Smith*,
Nick Morrice and Carol MacKintosh
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, UK
An activity that inhibited both glutamine synthetase (GS)
and nitrate reductase (NR) was highly purified from cauli-
flower (Brassica oleracea var. botrytis) extracts. The final
preparationcontainedanacyl-CoAoxidaseandasecond
protein of the plant nucleotide pyrophosphatase family. This
preparationhydrolysedNADH,ATPandFADtogenerate
AMP and was inhibited by fluoride, Cu
2+
,Zn
2+
and Ni
2+
.
The purified fraction had no effect on the activity of NR
when reduced methylviologen was used as electron donor
insteadofNADH;andinhibitedtheoxidationofNADHby
both spinach NR and an Escherichia coli extract in a time-
dependent manner. The apparent inhibition of GS and NR
and the ability of ATP and AMP to relieve the inhibition of
NR can therefore be explained by hydrolysis of nucleotide
substrates by the nucleotide pyrophosphatase. We have no
evidence that the nucleotide pyrophosphatase is a specific
physiological regulator of NR and GS, but suggest that
nucleotide pyrophosphatase activity may underlie some
confusion in the literature about the effects of nucleotides
andproteinfactorsonNRandGSin vitro.

Keywords: nucleotide pyrophosphatase (nucleotide pyro-
phosphohydrolase); nitrate reductase; glutamine synthetase;
AMP; nudix hydrolase.
In response to water stress or when photosynthesis is
blocked, the cytosolic enzyme nitrate reductase (NR) is
inhibited by a two-step mechanism; firstly, a serine residue
(Ser543 in the spinach enzyme) is phosphorylated [1,2]. The
phosphorylation alone has no effect on enzyme activity. The
addition of a phosphate to this serine, within this amino acid
context, generates a phosphopeptide motif (Arg-Ser-X-
phosphoSer- X-Pro) that is recognized by and binds to NIP
(nitrate reductase inhibitor protein) [3,4], which comprises
isoforms of 14-3-3 proteins [5,6]. Binding of 14-3-3 proteins
inhibits the phosphorylated NR. The inhibition of phos-
phorylated NR by 14-3-3 proteins also requires millimolar
Mg
2+
or Ca
2+
[7]. The inhibited, 14-3-3-bound NR can be
activated by dephosphorylation [4,5], dissociation of 14-3-3
proteins by a competitor 14-3-3-binding phosphopeptide [6],
or chelation of metal ions [7].
During purification of 14-3-3 proteins, a second protein
factor was found to ÔinterfereÕ with the inactivation of NR by
phosphorylation and 14-3-3 proteins [6]. The inhibition of
NR by the ÔinterferingÕ protein was blocked by ATP. This
means that theinhibition ofNR by Mg-ATP, NR kinase,and
14-3-3 proteins can be counteracted by what seems like
Mg-ATP-dependent activation of NR in fractions containing

the ÔinterferingÕ protein (see Results). The effect of Mg-ATP
on the ÔinterferingÕ protein was reminiscent of a reportedly
ÔNR-specific inhibitorÕ from spinach leaves, termed NRI
[8–11]. This ÔfactorÕ has been discussed in the literature for
over a decade and is still described in reviews today [12].
Recently, forms of glutamine synthetase (GS) from
cauliflower [13] and Chlamydomonas reinhartii [14] were
purified by 14-3-3-affinity chromatography. Moreover,
cytosolic GS extracted from leaves of Brassica napus L.
and plastid GS from tobacco have been found to be
activated and/or stabilized by interaction with 14-3-3
proteins [15,16].
Here, we aimed to purify GS for further characterization
of its regulation by 14-3-3 proteins. However, during the
first purification step, a poly(ethylene glycol) fractionation,
we repeatedly noticed an apparent threefold to fourfold
increase in total GS activity compared with the crude
extract. A similar observation was made [17] while purifying
soybean hypocotyl glutamine synthetase. We report that
this apparent activation of GS is due to separation of GS
from a protein that has been identified as a nucleotide
pyrophosphatase. We demonstrate that the nucleotide
pyrophosphatase has identical properties to the NR Ôinter-
feringÕ protein [6], and shares some of the reported
properties of the NRI nucleotide pyrophosphatase [8,11].
However, in contrast to Sasaki et al. [8] and Sonoda et al.
[11], we have no evidence that the nucleotide pyrophospha-
tase is a physiologically relevant, specific inhibitor of NR
that promotes oligomerization of NR. All the apparent
inhibitory effects on NR and GS can be explained simply by

the enzymatic properties of the nucleotide pyrophosphatase.
Correspondence to C. MacKintosh, MRC Protein Phosphorylation
Unit, Department of Biochemistry, University of Dundee,
MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK.
Fax: + 44 1382 223778,
E-mail:
Abbreviations: GS, glutamine synthetase; NR, nitrate reductase;
Con-A, concanavalin A.
*Present address: Department of Biological Sciences,
University of Calgary, 2500 University Drive NW, Calgary,
Alberta, Canada T2N 1N4.
(Received 15 November 2002, revised 8 January 2003,
accepted 10 February 2003)
Eur. J. Biochem. 270, 1356–1362 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03509.x
Materials and methods
Purification of a nitrate reductase/glutamine
synthetase inhibitor protein
All steps were performed at 4 °C. The outer curd
( 1000g)oftwocauliflower’s(Brassica oleracea var.
botrytis; Tesco Supermarket, Dundee) was homogenized in
a Waring Blender in 1 vol ice-cold buffer (50 m
M
Hepes/
OH (pH 7.5), 1 m
M
dithiothreitol, 0.2 m
M
phenyl-
methanesulfonyl fluoride, 1 m
M

benzamidine and 1%
(w/v) insoluble polyvinylpolypyrollidone) and clarified by
centrifugation at 12 000 g,4°C,for30min.Afterfiltra-
tion through glass wool and two layers of miracloth, the
inhibitor was precipitated with 0–9% poly(ethylene glycol)
added from a 50% (w/v) solution of poly(ethylene glycol)
8000 dissolved in 25 m
M
Tris pH 7.5, 1 m
M
dithiothreitol,
1m
M
MgCl
2
(buffer A). After stirring for 15 min and
centrifugation at 12 000 g,4°C, for 20 min, the pellet was
resuspended in 25 m
M
Mes-OH pH 6, 1 m
M
dithiothreitol,
1m
M
MgCl
2
(buffer B) and clarified by centrifugation at
100 000 g,4°C, for 45 min. The sample was then filtered
through a 0.4-lm syringe filter and loaded at 3 mLÆmin
)1

onto a (6 · 1.6 cm) Hiload S-Sepharose column equili-
brated in buffer B and eluted with a 0–0.4
M
NaCl gradient
in buffer B over 200 mL with 5-mL fractions. Peak
fractions were pooled and dialysed into 25 m
M
Tris
pH 8.5, 1 m
M
dithiothreitol and 1 m
M
MgCl
2
(buffer C).
The sample was filtered through a 0.2-lm syringe filter and
loaded onto a HR (5/5) Mono-Q anion-exchange column
equilibrated in buffer C and fractionated over 20 mL with
a0–0.5
M
NaCl gradient in buffer C with 1-mL fractions.
Peak fractions were pooled and dialysed into buffer A,
concentrated to less than 200 lL, and chromatographed
on a Superose 12 gel filtration column equilibrated in
buffer A plus 100 m
M
NaCl at 0.4 mLÆmin
)1
. Fractions of
0.2 mL were collected. Peak fractions were pooled,

dialysed into buffer B and fractionated over 2 mL with
0.1-mL fractions and a 0–0.3
M
NaCl gradient in buffer B
on a Mono-S (PC 1.6/5) cation-exchange column using a
Pharmacia Smart chromatography system. The Superose
12 column was calibrated with the following standards:
thyroglobulin (670 kDa), c-globulin (158 kDa), bovine
serum albumin (66 kDa), ovalbumin (44 kDa), myoglobin
(17 kDa).
When using concanavalin A (Con-A) Sepharose, the
pooled fractions from the Mono-Q step were made 0.25
M
NaCl and loaded at 1 mLÆmin
)1
onto a 1-mL Con-A-
Sepharose column equilibrated in buffer A plus 0.25
M
NaCl. The column was washed with buffer A plus 0.25
M
NaCl and protein eluted with 0.25
M
methyl a-
D
-gluco-
pyranoside in buffer A plus 0.25
M
NaCl.
Enzyme assays
GS activity was measured by the formation of c-glutamyl

hydroxamate using the transferase assay [18]. Reaction
mixtures contained, in a final volume of 100 lL: 50 m
M
Hepes/OH pH 7.7, 50 m
M
monosodium glutamate,
7.5 m
M
ATP, 75 m
M
MgCl
2
,0.5m
M
EDTA, 1 m
M
dithiothreitol and clarified source of GS. A 9–20%
poly(ethylene glycol) fraction from cauliflower curd was
the source of GS for routine assays of the GS inhibitor,
and each inhibitor assay contained 4 milliunits (mU) of
GS activity. One mU of GS activity produced 1 nmol
c-glutamyl hydroxamate per min at 30 °C.
Reactions were started by the addition of hydroxylamine
(pH 7.2) to a final concentration of 2.5 m
M
. After incuba-
tion for 10 min at 30 °C, assays were stopped by the
addition of 25 lL of a 1 : 1 : 1 mixture containing 10%
(w/v) FeCl
3

ÆH
2
Oin0.2
M
HCl, 50% (v/v) HCl and 24%
(w/v) trichloroacetic acid. The A
504
was measured and the
c-glutamyl hydroxamate produced was quantified using
commercial c-glutamyl hydroxamate as standard. Control
assays were performed in the absence of ATP to ensure that
the reaction was dependent on ATP.
Nitrate reductase (NR) was assayed in a total volume of
100 lL in buffer D (50 m
M
Hepes pH 7.5, 10 m
M
MgCl
2
,
10 l
M
FAD, 1 m
M
dithiothreitol). Assays were incubated
for 5 min at 30 °C and the reaction initiated by the addition
of 50 lL buffer E (buffer D containing 2 m
M
KNO
3

plus
0.5 m
M
NADH). After 5 min, reactions were stopped with
10 lLof0.5
M
zinc acetate and NADH removed by adding
10 lL155l
M
phenazine methosulfate and incubating for
20 min at room temperature in darkness. Sulfanilamide
(50 lLof1%(w/v)in3
M
(HCl) and 50 lLof0.02%
(w/v) N-(1-naphthyl)ethylenediamine dihydrochloride were
added. After 5 min, the mixtures were clarified by centri-
fugation at 16 000 g,4°C, for 2 min and the amount of
nitrite was determined by measuring A
540
and comparing
with a standard curve. For routine assays of NR inhibitor,
NR was partially purified by ammonium sulfate fraction-
ation (0–30%) of a spinach leaf crude extract prepared as
described in [4]. Each inhibitor assay contained 0.5 mU of
NR, where one mU of NR activity is defined as 1 nmol
nitrite produced per min at 30 °C.
Assays of NADH hydrolysis by purified NR were
followed continuously as the decrease in A
340
in the presence

of NADH (0.4 m
M
or as stated in results) and 2 m
M
KNO
3
.
Nucleotide pyrophosphatase assays were performed as
described in Frick and Bessman [19] using the coupled
enzyme assay. In stage I of the assay, 5 lL of purified NR/
GS inhibitor was incubated with the indicated amounts of
NADH, FAD, or ATP in 100 m
M
Tris pH 7.5, 0.1 m
M
MgCl
2
in a total volume of 100 lLfor10minat30 °Cafter
which the reaction tube was heated at 95 °Cfor5min.The
AMP generated was determined as follows in stage II of the
assay. Fifty microlitres of the AMP-containing sample was
added to 950 lL of reaction mixture (62 m
M
Tris pH 7.5,
20 m
M
KCl, 6 m
M
ATP, 10 m
M

MgCl
2
,4m
M
phos-
phoenolpyruvate, 0.4 m
M
NADH, 10 UÆmL
)1
lactate
dehydrogenase (Worthington) and 10 UÆmL
)1
pyruvate
kinase. The reaction was started with the addition of 5 U of
adenylate kinase and monitored by A
340
and converted to
nmol using a molar extinction coefficient of 6.22
M
)1
Æcm
)1
.
The change in A
340
was subtracted from a control with
no enzyme, which was run parallel to each assay. The
assays which contained ATP in stage I were diluted
100-fold for stage II. All assays were performed at least in
duplicate.

NADH oxidase in a desalted, cell-free extract of Escheri-
chia coli DH5a was assayed continuously as the decrease in
A
340
in the presence of NADH (0.4 m
M
)in50m
M
Mops/
NaOH, pH 7.3, 5 m
M
MgCl
2
.
Ó FEBS 2003 Apparent inhibition of NR and GS (Eur. J. Biochem. 270) 1357
Amino acid sequencing
Proteins ( 5 lg) were excised from SDS/PAGE gels that
had been lightly stained with Coomassie blue. The gel slices
were washed in Milli-Q water (5 · 1 mL) for 1 h, brought
to near dryness by rotary evaporation, suspended in 250 lL
buffer F (50 m
M
Tris/HCl pH 8.0, 0.01% alkylated Triton
X-100) containing 1 lg of alkylated trypsin (Roche) and
incubated with shaking for 20 h at 30 °C. The supernatant
was removed and a further 250 lL buffer F without trypsin
was added for 4 h. The combined supernatants were dried
to 50 lL and applied to capillary C18 column
(0.5 · 150 mm) from Applied Biosystems (Warrington,
UK) equilibrated in 0.1% (v/v) trifluoroacetic acid attached

to a Applied Biosystems ABI 173A Microblotter Capillary
HPLC system. The column was developed with a linear
acetonitrile gradient in 0.09% (v/v) trifluoroacetic acid with
an increase in acetonitrile concentration of 0.5% per min.
A
214
was recorded with an on-line monitor. The flow
rate was 7 lLÆmin
)1
. Selected peptides were sequenced on
an Applied Biosystems 476A protein sequencer. For
N-terminal sequencing, proteins were run on SDS/PAGE,
transferred to Problott (Applied Biosystems), stained on
the membrane as described by the manufacturer, and the
relevant bands were excised and sequenced from the
membrane.
Results
Properties of a nitrate reductase inhibitor
On reporting the purification and identification of 14-3-3
proteins as the NR inhibitor protein, NIP, we reported
‘‘another protein that interfered with the NIP-14-3-3 assay,
and that was eluted in the 0.2
M
NaCl wash’’ during anion-
exchange chromatography of extracts of spinach leaves [6],
or cauliflower (Fig. 1). In contrast to 14-3-3 proteins, the
ÔinterferingÕ protein inhibited both phosphorylated and
dephosphorylated NR (Fig. 1 and data not shown). A
protein inhibitor of NR, NRI, that bound to Con-A had
been reported previously [9,10]. Similar to NRI, we found

that our interfering protein bound to Con-A (Fig. 2). The
inhibition of NR by the interfering protein could be
prevented by addition of either EDTA, ATP, or AMP
directly to NR activity assays (Fig. 1 and data not shown).
A similar Con A-binding NR inhibitor was also present in
extracts of Chlamydomonas (M. Pozuelo Rubio, MRC Unit,
University of Dundee, UK, personal communication).
Identification and purification of a GS inhibitor
with identical properties to the NR inhibitor
We partially purified GS from cauliflower curd in prepar-
ation for studies aimed at discovering whether GS activity is
regulated by its interaction with 14-3-3 proteins. During the
first purification step, a 0–9% poly(ethylene) glycol frac-
tionation, we noticed an apparent threefold to fourfold
increase in total GS activity compared with the activity in
the crude extract (GS activity remains in the 0–9%
poly(ethylene glycol) supernatant). The apparent activation
of GS did not occur if a 0–20% poly(ethylene glycol)
fraction was made. Addition of a 0–9% poly(ethylene
glycol) fraction inhibited the GS activity in a 9–20%
poly(ethylene glycol) cut (Table 1).
The protein responsible for the apparent inhibition of GS
was purified further (Fig. 2 and Table 1). Similar to the
interfering protein, the GS inhibitor was eluted from
Q-Sepharose by 0.2
M
NaCl (Table 1 and data not shown).
Consistent with the possibility that the NR inhibitor and GS
inhibitor were identical proteins, the EDTA-sensitive NR
inhibitor and GS inhibitor cochromatographed throughout

the purification (Fig. 2 and data not shown). The final
fractions contained three protein bands with apparent
molecular masses of 70, 47 and 45 kDa on SDS/PAGE that
were most abundant in the fractions containing highest NR/
GS inhibitory activity (Fig. 2). The NR/GS inhibitor
behaved on Superose 12 gel filtration as a 55-kDa protein
(not shown). The intact proteins and peptides produced
from tryptic digests of the protein bands were sequenced.
BLAST
searches of sequence databases revealed that the band
of 70 kDa belonged to the acyl-CoA oxidase protein family,
while all of the peptides derived from the 47 and 45 kDa
bands matched most closely with an Arabidopsis thaliana
nucleotide pyrophosphatase-like protein (Table 2).
Sequenced peptides covered 22% of the nucleotide pyro-
phosphatase with 71% identity and 79% similarity. The
N-terminal sequence obtained began at residue 49 and the
predicted mass of the protein from this residue onwards
(45.9 kDa) closely matches the observed mass of the
sequenced bands on the gel (Fig. 2) indicating the protein
was proteolytically cleaved.
All of the glutamine synthetase and nitrate reductase
inhibitory activity bound to Con-A and was eluted with
Fig. 1. Separation of two NR inhibiting activities by anion exchange
chromatography. A desalted 4–70% (NH
4
)
2
SO
4

fraction ( 250 mg)
prepared from 100 g of cauliflower harvested in the light was chro-
matographed on Q-Sepharose. The column was washed in buffer A
until the A
280
hadreturnedtobaseline,andwasdevelopedwithalinear
gradient (broken line) of 0–500 m
M
NaCl in buffer A over 70 min at
3mLÆmin
)1
. Fractions (3 mL) were desalted by microdialysis (BRL)
and aliquots (15 lL) assayed for inhibition/inactivation of NR in a
0–30% (NH
4
)
2
SO
4
fraction that contained NR and NR kinase (pre-
pared as in MacKintosh et al., 1995). Assays were performed in the
presence of 12 m
M
EDTA (d), 2 m
M
ATP (j) or the absence of ATP
(h). Similar profiles were seen for extracts of spinach leaves and cul-
tured Arabidopsis cells, though the relative inhibitory activities of peaks
1 and 2 varied among preparations.
1358 G. B. G. Moorhead et al. (Eur. J. Biochem. 270) Ó FEBS 2003

0.25
M
methyl a-
D
-glucopyranoside, with a recovery of
between 20 and 40% activity in different preparations.
However, the Con-A step would not have improved the
overall purification because both the acyl-CoA oxidase and
the nucleotide pyrophosphatase bound to, and were eluted
from, the Con-A column, as determined by amino acid
sequencing of the  70 and  47 kDa proteins seen in
Fig. 2C. Similarly, both proteins bound to AMP-Sepharose
(not shown).
The fractions from the final Mono-S column containing
NR/GS inhibitory activity were found to catalyse the
production of AMP from NADH, ATP and FAD
(Table 3). Ninety percent of the amount of NADH used
in our standard NR assay, and 60% of the amount of ATP
inaGSassay,wasconvertedintoAMPwithin10minat
30 °C by an amount of a Mono Q fraction that appeared to
inhibit NR by 55% (Table 3). These findings suggest that
the NR/GS inhibitor functions during both the assay
preparation and the assays by converting the cofactors
necessary for the NR or GS reactions into AMP. Consistent
with this notion, the hydrolysis of NADH to NAD by either
purified NR or an extract of E. coli were clearly inhibited by
the Mono-S fractions in a time-dependent manner, and
transiently restored by adding extra NADH (data not
shown). The inhibitor preparation had no effect on the
activity of NR when reduced methylviologen was used as

electron donor instead of NADH (not shown). In addition,
using NADH as substrate, the enzyme displayed a K
m
of
70 l
M
and a V
max
of 20 lmol AMP produced per min per
mg protein. The enyzme activity was inhibited by >95%
using 1 m
M
Cu
2+
,Zn
2+
and Ni
2+
(all as chloride salts) in
the assay, in common with other nucleotide pyrophospha-
tases [20]. The nucleotide pyrophosphatase was inhibited
54% by 10 l
M
NaF, but was unaffected by 10 l
M
NaCl,
Fig. 2. Co-purification of glutamine synthetase and nitrate reductase inhibitor proteins. (A) Activity of glutamine synthetase (h) and nitrate reductase
(j) in the presence of fractions from the final Mono-S chromatography step of the purification of inhibitor from cauliflower curd. (B) Fractions
from the same Mono-S chromatography run shown in (A) were run on a 12% SDS-gel and stained with Coomassie blue. (C) The fractions
containing peak activity from an earlier step in the purification (Mono-Q) were pooled and loaded onto a Con-A-Sepharose column and eluted after

extensive washing (see Materials and methods) and run on SDS/PAGE and stained with Coomassie blue. Molecular mass standards (in kDa) are
phosphorylase (97), bovine serum albumin (66), ovalbumin (43), carbonic anhydrase (30) and soybean trypsin inhibitor (21.5).
Ó FEBS 2003 Apparent inhibition of NR and GS (Eur. J. Biochem. 270) 1359
again when employing NADH as substrate. Similarly, the
apparent inhibition of NR was blocked 48% by 10 l
M
NaF, using an amount of Mono S fraction that gave 20%
inhibition of NR in the standard assay.
Discussion
An activity that inhibited both GS and NR was highly
purified from cauliflower (B. oleracea var. botrytis) extracts.
The final preparation contained an acyl-CoA oxidase and a
second protein of the plant nucleotide pyrophosphatase
family. Nucleotide pyrophosphatases belong to a family of
widely distributed hydrolases that are active on a variety of
derivatives of nucleoside diphosphates (hence the name
nudix hydrolases), and/or non-nucleotide diphosphoinositol
polyphosphates, and characterized by the mutT motif
(GX
5
EX
7
REUXE
3
GU; where U represents one of the
bulky hydrophobic amino acids, usually I, L or V) [21–25].
These enzymes are often extracellular and their physiologi-
cal substrates may include signalling metabolites, including
toxic derivatives.
The purified protein catalysed the hydrolysis of NADH,

ATP and FAD (Table 3). Moreover, the apparent inhibi-
tion of NR is consistent with the hydrolysis of NADH by
the nucleotide pyrophosphatase, and the ability of ATP to
relieve the inhibition of NR (Fig. 1) is because ATP protects
NADH from hydrolysis, most likely by providing an
alternative substrate for the nucleotide pyrophosphatase
(Table 3). The apparent inhibition of GS can be explained
by hydrolysis of the Mg-ATP substrate by the nucleotide
pyrophosphatase, and generation of 5¢-AMP, a GS inhi-
bitor [26]. While we were unable to separate the active
Table 1. Purification of GS inhibitor from cauliflower curd. One unit of activity is the amount of inhibitor that will decrease the activity of 4 mU of
GS by 50% during the assay.
Step Volume (mL) Protein (mg) Activity (U) Specific activity (UÆmg
)1
) Purification (fold) Yield (%)
Extract 1300 4030 – – – –
0–9% Poly(ethylene glycol) 140 644 9333 14.5 1 100
S-Sepharose 35 10 2333 233 16 25
Mono-Q 2 1.27 296 233 16 3.2
Superose 12 0.6 0.22 200 909 63 2.1
Smart Mono-S 0.3 0.09 100 1111 77 1.1
Table 2. Sequences, identities, and accession numbers of proteins that were copurified with inhibitory activity towards glutamine synthetase and nitrate
reductase. *, N-terminal sequence.
Band (kDa) Identity Sequence obtained/sequence matched Accession no.
70 Acyl-CoA oxidase
LFEEAXKDPLXDXV
LFEEALKDPLNDSV
Genpept 3044214
70 Acyl-CoA oxidase
XVATDPVFXXVNXR

LVASDPVFEKSNRA
Genpept 3044214
70 Acyl-CoA oxidase
XLSLAR
WLSLAN
Genpept 3044214
47 Nucleotide pyrophosphatase
KLNKPVVLMISSDGFDFGYQN*
KLNKPVVLMISCDGFRFGYQF Genpept 13430714
47 Nucleotide pyrophosphatase
IPPIIGMVGEGLVVR
IPPIIGIVGEGLMVR
Genpept 13430714
45 Nucleotide pyrophosphatase
KLNKPVVLM*
KLNKPVVLM Genpept 13430714
45 Nucleotide pyrophosphatase
XLCPHFSLSVPFEECSR
GYCPHFNLSVPLEERVD
Genpept 13430714
45 Nucleotide pyrophosphatase
ALAYFXPGREVXR
AVTYFWPSSEVLK
Genpept 13430714
45 Nucleotide pyrophosphatase
VDLILNQFDLPPR
PDLLMLYFDEPDQ
Genpept 13430714
45 Nucleotide pyrophosphatase
XXLGEPLVVMXLEE

WWLGEPLWVTAVNQ
Genpept 13430714
Table 3. Generation of AMP from NADH, ATP and FAD by purified
NR/GS inhibitor in a 100-lL incubation at 30 °C for 10 min, using the
amounts and concentrations of ATP and NADH used in standard GS
and NR assays, respectively. Data are presented as mean ± SEM.
Cofactor
AMP generated
(nmol)
% cofactor hydrolysed
to AMP
None 0.4 –
ATP (750 nmol) 662 ± 17 88
NADH (50 nmol) 33 ± 1.4 66
FAD (5 nmol) 1.64 ± 0.20 32
1360 G. B. G. Moorhead et al. (Eur. J. Biochem. 270) Ó FEBS 2003
nucleotide pyrophosphatase from the acyl-CoA oxidase by
a number of procedures that maintained the activity of the
nucleotide pyrophosphatase (Fig. 1, Table 2 and data not
shown) there is no obvious mechanistic reason to implicate
the acyl-CoA oxidase in the apparent inhibition of NR and
GS.
Sonoda et al. [11,12] reported purification of an irrever-
sible inhibitor of NR, termed NRI, and its identification as a
spinach nucleotide pyrophosphatase. In contrast to our
findings, the enzyme purified by Sonoda et al. [11,12] did
not affect the NADH-dependent activities of glutamate
dehydrogenase or lactate dehydrogenase [8], and was
speculated to be NR specific with a possible physiological
role in NR inactivation during leaf senescence [11,12].

Moreover, NRI was reported to promote the assembly of
NR into oligomeric forms that had a retarded electro-
phoretic mobility, and oligomerization was suggested to be
mediated via action of the nucleotide pyrophosphatase on
the FAD cofactor bound to NR [8–12]. In contrast, we have
no evidence that the inhibitory activity we have purified here
causes NR polymerization. Thus, while the cauliflower
nucleotide pyrophosphatase has very similar chromato-
graphic properties, size on SDS/PAGE and inhibition by
EDTA to the enzyme purified by Sonoda et al. [11,12], we
have no clear evidence to suggest that the nucleotide
pyrophosphatase has regulatory effects on NR or GS
in planta. The binding to Con-A indicates that the protein is
glycosylated and may therefore be extracellular, as are many
nucleotide pyrophosphatases [25].
The nonspecific hydrolysis by nucleotide pyrophospha-
tases has previously caused confusion in regulatory systems
that use ATP and adenine dinucleotides [27]. We suggest
that the nucleotide pyrophosphatase may have caused much
confusion in studies on NR regulation. For example, we and
others have found that the nucleotide pyrophosphatase
activity in plant extracts can often be so high that when
Mg-ATPisaddedtoanextracttheactivityofNRappears
to go up because Mg-ATP prevents the hydrolysis of
NADH, instead of down due to the effect of phosphory-
lation and binding to NIP-14-3-3 proteins (Fig. 1). In
addition, NR inactivating factors found in rice and
Neurospora extracts were dependent on NADH and
blocked by EDTA [28–30] and a protein inhibitor was
reported[31]thathadsimilareffectsonGStothenucleotide

pyrophosphate that we have found here.
5¢-AMP has been widely reported to activate NR and GS
in cell-free extracts [7,14,26,32,33]. The mechanism of
5¢-AMP activation of NR may, in part, involve binding to
14-3-3 [32]. However, the apparent inhibition of NR by the
nucleotide pyrophosphatase was largely relieved by milli-
molar concentrations of 5¢-AMP, presumably acting as a
product inhibitor, and it seems likely that this mechanism
contributes to the reported 5¢-AMP activation of NR and
GS.
Both the NADH pyrophosphatase activity and the
inhibition of NR were inhibited  50% by 10 l
M
NaF,
which is consistent with the proposal that the apparent
inhibition of NR is due to the pyrophosphatase. Other
nucleotide pyrophosphatases have been reported to be
inhibited by micromolar concentrations of NaF [34]. The
inhibitory effect of NaF on the nucleotide pyrophosphatase
is useful: NaF is commonly used to inhibit protein serine/
threonine phosphatases, including the PP2A that dephos-
phorylates NR, and we know that at concentrations up to
15 m
M
, fluoride has no obvious effect on NR kinases [35]
anduptoatleast2.5m
M
hasnoeffectonNRactivityor
14-3-3 binding [36]. We therefore suggest that analysis of the
regulation by phosphorylation/14-3-3 proteins of NR or GS

in crude fractions be performed in the presence of >1 m
M
NaF, or after passing through a Con-A column to remove
the ÔinterferenceÕ from extracts containing high nucleotide
pyrophosphatase activity.
Acknowledgements
This work was supported by funds from the UK Biotechnology and
Biological Sciences Research Council (to C. M.).
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