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.. Smith*, Nick Morrice and Carol MacKintosh

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

preparation contained an acyl-CoA oxidase and a second

protein of the plant nucleotide pyrophosphatase family.This

preparation hydrolysed NADH, ATP and FAD to generate

AMP and was inhibited by fluoride, Cu2+, Zn2+and Ni2+

The purified fraction had no effect on the activity of NR

when reduced methylviologen was used as electron donor

instead of NADH; and inhibited the oxidation of NADH by

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 and protein factors on NR and GS in 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

Mg2+or Ca2+[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
interfere with the inactivation of NR by

phosphorylation and 14-3-3 proteins [6].The inhibition of

NR by the
interfering protein was blocked by ATP.This

means that the inhibition of NR 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
interfering protein (see Results).The effect of Mg-ATP

on the
interfering protein was reminiscent of a reportedly

NR-specific inhibitor from spinach leaves, termed NRI [8–11].This
factor 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
inter-fering 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: c.mackintosh@dundee.ac.uk

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)

Materials and methods

Purification of a nitrate reductase/glutamine

synthetase inhibitor protein

All steps were performed at 4C.The outer curd

( 1000 g) of two cauliflower’s (Brassica oleracea var

botrytis; Tesco Supermarket, Dundee) was homogenized in

a Waring Blender in 1 vol ice-cold buffer (50 mM Hepes/

OH (pH 7.5), 1 mM dithiothreitol, 0.2 mM

phenyl-methanesulfonyl fluoride, 1 mM benzamidine and 1%

(w/v) insoluble polyvinylpolypyrollidone) and clarified by

centrifugation at 12 000 g, 4C, for 30 min After

filtra-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 mMTris pH 7.5, 1 mMdithiothreitol,

1 mM MgCl2 (buffer A).After stirring for 15 min and

centrifugation at 12 000 g, 4C, for 20 min, the pellet was

resuspended in 25 mMMes-OH pH 6, 1 mMdithiothreitol,

1 mM MgCl2(buffer B) and clarified by centrifugation at

100 000 g, 4C, 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 0–0.4MNaCl gradient

in buffer B over 200 mL with 5-mL fractions.Peak

fractions were pooled and dialysed into 25 mM Tris

pH 8.5, 1 mMdithiothreitol and 1 mM MgCl2 (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

a 0–0 5MNaCl 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 mMNaCl 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 0–0.3MNaCl 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 0.25M

NaCl and loaded at 1 mLÆmin)1 onto a 1-mL

Con-A-Sepharose column equilibrated in buffer A plus 0.25M

NaCl.The column was washed with buffer A plus 0.25M

NaCl and protein eluted with 0.25M methyl a-D

-gluco-pyranoside in buffer A plus 0.25MNaCl

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 mM

Hepes/OH pH 7.7, 50 mM monosodium glutamate,

7.5 mM ATP, 75 mM MgCl2, 0 5 mM EDTA, 1 mM

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 30C

Reactions were started by the addition of hydroxylamine (pH 7.2) to a final concentration of 2.5 mM.After incuba-tion for 10 min at 30C, assays were stopped by the addition of 25 lL of a 1 : 1 : 1 mixture containing 10% (w/v) FeCl3ÆH2O in 0 2MHCl, 50% (v/v) HCl and 24% (w/v) trichloroacetic acid.The A504was 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 mMHepes pH 7.5, 10 mMMgCl2,

10 lMFAD, 1 mMdithiothreitol).Assays were incubated for 5 min at 30C and the reaction initiated by the addition

of 50 lL buffer E (buffer D containing 2 mMKNO3plus 0.5 mMNADH).After 5 min, reactions were stopped with

10 lL of 0 5Mzinc acetate and NADH removed by adding

10 lL 155 lMphenazine methosulfate and incubating for

20 min at room temperature in darkness.Sulfanilamide (50 lL of 1% (w/v) in 3 M (HCl) and 50 lL of 0 02% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride were added.After 5 min, the mixtures were clarified by centri-fugation at 16 000 g, 4C, for 2 min and the amount of nitrite was determined by measuring A540 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 30C

Assays of NADH hydrolysis by purified NR were followed continuously as the decrease in A340in the presence

of NADH (0.4 mMor as stated in results) and 2 mMKNO3 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 mM Tris pH 7.5, 0.1 mM MgCl2in a total volume of 100 lL for 10 min at 30C after which the reaction tube was heated at 95C for 5 min 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 mMTris pH 7.5,

20 mM KCl, 6 mM ATP, 10 mM MgCl2, 4 mM phos-phoenolpyruvate, 0.4 mM 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 A340and converted to nmol using a molar extinction coefficient of 6.22M )1Æcm)1 The change in A340 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 coliDH5a was assayed continuously as the decrease in

A340in the presence of NADH (0.4 mM) in 50 mMMops/ NaOH, pH 7.3, 5 mMMgCl

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 mMTris/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 30C.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

A214 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 0.2MNaCl wash’’ during

anion-exchange chromatography of extracts of spinach leaves [6],

or cauliflower (Fig.1).In contrast to 14-3-3 proteins, the

interfering 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 0.2MNaCl (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 BLASTsearches 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 had returned to baseline, and was developed with a linear gradient (broken line) of 0–500 m M NaCl in buffer A over 70 min at

3 mLÆmin)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.

0.25M 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

in a GS assay, was converted into AMP within 10 min at

30C 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 Kmof

70 lMand a Vmaxof 20 lmol AMP produced per min per

mg protein.The enyzme activity was inhibited by >95% using 1 mMCu2+, Zn2+and Ni2+(all as chloride salts) in the assay, in common with other nucleotide pyrophospha-tases [20].The nucleotide pyrophosphatase was inhibited 54% by 10 l NaF, but was unaffected by 10 l 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).

again when employing NADH as substrate.Similarly, the

apparent inhibition of NR was blocked 48% by 10 lM

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 (GX5EX7REUXE3GU; 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 (%)

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.

LFEEALKDPLNDSV Genpept 3044214

LVASDPVFEKSNRA 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

ATP (750 nmol) 662 ± 17 88

NADH (50 nmol) 33 ± 1.4 66

FAD (5 nmol) 1.64 ± 0.20 32

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-ATP is added to an extract the activity of NR appears

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] that had similar effects on GS to the nucleotide

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 lM 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 mM, fluoride has no obvious effect on NR kinases [35] and up to at least 2 5 mMhas no effect on NR activity or 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 mM NaF, or after passing through a Con-A column to remove the
interference 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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