Báo cáo Y học: Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures ppt

Plaxton1,2 Departments of1Biology and2Biochemistry, Queen’s University, Kingston, Ontario, Canada;3Department of Horticulture and Landscape Architecture, Purdue University, Indiana, USA

Purification and characterization of two secreted purple acid

phosphatase isozymes from phosphate-starved tomato

Gale G Bozzo1, Kashchandra G Raghothama3and William C Plaxton1,2

Departments of1Biology and2Biochemistry, Queen’s University, Kingston, Ontario, Canada;3Department of Horticulture and Landscape Architecture, Purdue University, Indiana, USA

Two secreted acid phosphatases (SAP1 and SAP2) were

markedly up-regulated during Pi-starvation of tomato

suspension cells.SAP1 and SAP2 were resolved during

cat-ion-exchange FPLC of culture media proteins from

8-day-old Pi-starved cells, and purified to homogeneity and final

p-nitrophenylphosphate hydrolyzing specific activities of 246

and 940 lmol PiproducedÆmin)1mgÆprotein)1,

respect-ively.SDS/PAGE, periodic acid-Schiff staining and

analyt-ical gel filtration demonstrated that SAP1 and SAP2,

respectively, exist as 84 and 57 kDa glycosylated monomers

SAP1 and SAP2 are purple acid phosphatases (PAPs) as

they displayed an absorption maximum at 518 and 538 nm,

respectively, and were not inhibited by L-tartrate.The

respective sequence of a SAP1 and SAP2 tryptic peptide was

very similar to a portion of the deduced sequence of several

putative Arabidopsis thaliana PAPs.CNBr peptide mapping

indicated that SAP1 and SAP2 are structurally distinct.Both

isozymes displayed a pH optimum of approximately pH 5.3

and were heat stable.Although they exhibited wide substrate specificities, the Vmaxof SAP2 with various phosphate-esters was significantly greater than that of SAP1.SAP1 and SAP2 were activated by up to 80% by 5 mMMg2+, and demon-strated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively.Inter-estingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive

to Mg2+or molybdate.This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species dur-ing the oxidative burst associated with the defense response

of plants to pathogen infection

Keywords: phosphate starvation (plants); acid phosphatase (purple); peroxidase; Lycopersicon esculentum

Acid phosphatases (APs; orthophosphoric-monoester

phos-phohydrolase) catalyze the hydrolysis of a broad and

overlapping range of P-monoesters with a pH optimum

below pH 7.0 [1] APs are ubiquitous and abundant in

plants, animals, fungi, and bacteria.They are believed to

function in the production, transport and recycling of Pi,

which is crucial for cellular metabolism and energy

trans-duction processes.Eukaryotic APs exist as tissue- and/or

cellular compartment-specific isozymes that display

vari-ation in subunit Mr, substrate specificity, localization and

sensitivity to inhibition by various divalent cations and

metabolites [1,2]

Control of plant AP expression is mediated by a variety

of environmental and developmental factors [2].APs are

induced under various stresses, such as water deficiency, salinity stress, and nutritional Pi-deficiency [2,3].Plant AP activity is also abundant in storage tubers, ripening fruit, and germinating seeds [2,4,5].APs can be distinguished based on relative substrate specificities, and are categorized

as one of the following: (a) nonspecific AP, having no clear substrate specificity; and (b) APs having defined but nonabsolute substrate specificity [2].The latter type may play a specific metabolic role.For example, phytase is an AP induced during seed germination that preferentially utilizes phytic acid, a major seed phosphorus storage compound Another specialized plant AP is phosphoenolpyruvate phosphatase, hypothesized to function as an inducible glycolytic bypass reaction to the ADP-limited pyruvate kinase during Pi-stress [6,7]

The induction of AP is a universal response of vascular plants to Pistarvation [2].Piis a critical macronutrient that limits plant growth in many natural ecosystems [7,8].Soil Pi

is often chelated to inorganic mineral cations, or is bound organically, and therefore is not directly available for plant uptake.A correlation exists between intracellular and/or extracellular AP activity and cellular Pi status [2,7].An increase in secreted AP and Pi-uptake activity is believed to assist in the acquisition of limiting Pifrom the environment

by Pi-deficient (–Pi) plants [7,8].Secreted APs are likely involved in Pi scavenging from extracellular organic P-monoesters [7–9].Pi starvation inducible secreted APs have been documented in tomato suspension cell cultures

Correspondence to W.C.Plaxton, Department of Biology,

Queen’s University, Kingston, Ontario, Canada K7L 3 N6,

Fax: + 1 613 533 6617, Tel.: + 1 613 533 6150,

E-mail: plaxton@biology.queensu.ca

Abbreviations: AP, acid phosphatase; pNPP, p-nitrophenylphosphate;

PAP, purple acid phosphatase; +P i and –P i , P i -sufficient and

P i -deficient, respectively; ROS, reactive oxygen species;

SAP, secreted acid phosphatase.

Enzymes: acid phosphatase (EC 3.1.3.2); pyruvate kinase

(EC 2.7.1.40); peroxidase (EC 1.11.1.7).

(Received 6 June 2002, revised 5 September 2002,

accepted 4 November 2002)

and roots [10,11], white-lupin proteoid roots [12], and

Arabidopsis thalianaseedlings [13].Cell wall localized APs

are associated with Pi-starvation of white clover [14],

Brassica nigra suspension cells [15], and duckweed [16]

The cell wall-localized AP from –PiB nigrasuspension cells

appeared to be identical to an AP secreted into the cell

culture media [15], whereas the

glycosylphosphatidyl-anchored AP from duckweed was demonstrated to be a

purple AP (PAP) [17]

PAPs represent a distinct class of nonspecific AP

containing binuclear transition metal centers [1,18–21]

PAPs are characterized by their purple color in solution

and insensitivity to inhibition byL-tartrate [1,18,21].Plant

PAPs purified to date exist as 110 kDa homodimers [19,22–

24], whereas mammalian PAPs are generally 35 kDa

monomers [19].Moreover, plant PAPs contain a Fe(III)–

Zn(II) or Fe(III)–Mn(II) binuclear transition metal center,

but mammalian PAPs typically contain a Fe(III)–Fe(II)

unit in their active site [20].A number of plant genes

putatively encoding low MrPAPs have been identified [25]

Recent studies indicate that PAPs may also display

peroxi-dase activity, which has been hypothesized to function in the

production of reactive oxygen species (ROS) during the

hypersensitive defense response of animals and plants

[24,26]

Several reports have documented the identification and

partial characterization of APs from –Pitomato plants or

suspension cell cultures [10,11,27].Tadano and Sakai [28]

reported the secretion of significant AP activity from the

roots of –Pitomato and lupin, which was greater than most

crop species tested under similar conditions.A 130-kDa

homodimeric secreted AP was purified from –PiL

esculen-tum roots, and was found to have comparable kinetic

properties to a homodimeric AP secreted from –Pi lupin

roots [11].Recently, Baldwin and coworkers [27] isolated a

cDNA sequence encoding a putative 30 kDa AP (LePS2)

from –Pi tomato roots.The subcellular localization and

physiological role of LePS2 have yet to be elucidated.Bosse

and Ko¨ck [29] reported similar Pi-starvation inducible acid

hydrolases for tomato seedlings and suspension cell cultures

A 57-kDa AP was partially purified from the cell culture

medium of 3-day-old –Pitomato suspension cells [10], but a

thorough kinetic and structural characterization was not

performed.In the present study, we report the purification

and detailed comparison of two distinct PAP isozymes from

the culture media of –Pitomato suspension cells

M A T E R I A L S A N D M E T H O D S

Chemicals and plant material

Fractogel EMD-SO3650 (S) cation exchange resin, and KCl

were from BDH Chemicals.A Superose 12 HR10/30 column

and gel filtration Mr standards were from Amersham

Biosciences.Acrylamide, bisacrylamide, and dithiothreitol

were from ICN Pharmaceuticals.Horseradish peroxidase

was from Roche Applied Sciences.All other chemicals were

obtained from Sigma Chemical Co.All solutions were

prepared using Milli-Q processed water.Heterotrophic

tomato (Lycopersicon esculentum, cv Moneymaker) cell

suspensions were kindly provided by E.Blumwald

(Univer-sity of California at Davis, USA), and cultured in

Murashige-Skoog media containing 2.5 mMP as described previously

[30].P deficiency treatments were initiated 7 days after subculturing the cells into fresh Pi-sufficient (+Pi) media

A 50-mL portion of +Picell suspension was centrifuged axenically at 4000 g for 12 min at 25C.The cells were washed with –Pimedia, and used to inoculate 500 mL of fresh –Pimedia.After 8 days the –Picells were harvested by filtration through Whatman 541 filter paper on a Buchner funnel, and the filtrate concentrated as described below

AP assays All assays were linear with respect to time and concentration

of enzyme assayed.One unit (U) of activity is defined as the amount of AP resulting in the hydrolysis of 1 lmol of substrate per min at 25C

Phosphatase assay A For routine measurements of AP activity, the hydrolysis of phosphoenolpyruvate to pyruvate was coupled to the lactate dehydrogenase reaction and assayed at 25C by monitoring the oxidation of NADH at 340 nm using a Gilford 260 recording spectrophotometer.Standard AP assay condi-tions were 50 mM Na-acetate (pH 5.3), 10 mM phos-phoenolpyruvate, 5 mM MgCl2, 0 2 mM NADH, and

3 UÆmL)1desalted rabbit muscle lactate dehydrogenase in

a final volume of 1 mL.All assays were initiated by the addition of enzyme preparation and corrected for NADH oxidase activity by omitting phosphoenolpyruvate from the reaction mixture

Phosphatase assay B Acid-washed microtitre plates were used for all kinetic studies.For substrates other than phosphoenolpyruvate, the

Pireleased by the AP reaction was quantified [31].Between 1 and 10 mU of AP (determined using assay A) was incubated in a 96-well microtitre plate in a final volume of

40 lL.Each assay contained 50 mMNa-acetate (pH 5.3),

5 mM MgCl2 and an alternative substrate (5 mM unless otherwise stated).Assays were initiated by the addition of substrate, allowed to progress for 6 min, and terminated by the addition of 200 lL of reagent A which was prepared daily by mixing four parts 10% (w/v) ascorbate with one part 10 mM ammonium molybdate in 15 mM Zn-acetate (pH 5.0) Samples were incubated for 25 min at 40C and the A660 determined using a Spectromax 250 Microplate spectrophotometer (Molecular Devices).Controls were run for background amounts of Pi present at each substrate concentration tested.To calculate activities, a standard curve over the range 1–100 nmol Pi was constructed for each set of assays

Kinetic studies and protein concentration determination

Apparent Vmaxand Km values were calculated from the Michaelis–Menten equation fitted to a nonlinear least-squares regression computer kinetics program [32].The I50

values (concentration of inhibitor producing 50% inhibition

of AP activity) were determined using the aforementioned computer kinetics program.Competitive and uncompetitive inhibition constants are represented as K and K¢ K values

were determined from Dixon plots, whereas Ki¢ values were

determined from plots of [phosphoenolpyruvate]

vs.[inhib-itor] [33].All kinetic parameters are the means of three

separate experiments and are reproducible within ± 10%

SE of the mean value

Protein concentrations were determined with the

Coo-massie Blue G-250 dye-binding method [34] using bovine

c)globulin as the protein standard

Peroxidase assay

A chemiluminescence assay was employed to determine the

capacity of the purified tomato APs to catalyze the

peroxidation of 5-aminophthalhydrazide (luminol) [35]

Chemiluminescence was recorded in a Lmax Microplate

Luminometer (Molecular Devices).The reaction was

initi-ated by the addition of 0.1 mL of 4.4 mM hydrogen

peroxide to 0.1 mL of 0.2MTris/HCl (pH 8.1) containing

300 mMluminol and 5–20 nMof AP in a 96-well microtitre

plate.Photon emission was monitored continuously for 10 s

after H2O2 addition, and expressed as relative light units

with Softmax data analysis software (Molecular Devices)

In control experiments, equimolar concentrations of BSA or

horseradish peroxidase were substituted for purified AP

Buffers used during AP purification

Buffer A: 50 mM Na-acetate (pH 5.7), 1.5 mM MgCl2,

1 mMEDTA, 1 mMdithiothreitol, and 10% (v/v) glycerol

Buffer B: 25 mM Na-acetate (pH 5.7), 1.5 mM MgCl2,

1 mMEDTA, 1 mMdithiothreitol, 100 mMKCl, and 10%

(v/v) glycerol.Buffer C: 25 mM Na-acetate (pH 5.7),

100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1.5 mM

MgCl2, 0 2 mM CaCl2, 0 2 mM MnCl2, and 10% (v/v)

glycerol

AP purification

All steps were carried out at 0–4C.Cell culture media from

–Picells (4 L) was concentrated approximately 20-fold via

tangential ultrafiltration (Millipore Mini-tan) using 10

30 kDa MWCO regenerated cellulose plates.Concentrated

media was clarified by centrifugation at 40 000 g for

20 min.Pre-chilled ()20 C) acetone (3 parts) was

com-bined with concentrated culture media (1 part), stirred

gently overnight, and centrifuged at 12 000 g for 25 min

The supernatant was decanted and pellets dried under a

stream of air for 7–8 h.Acetone pellets were resuspended in

50 mMNa-acetate (pH 5.8) containing 1 mMMgCl2using a

Polytron (3–5 s pulses).The solution was stirred for 60 min,

clarified by centrifugation at 40 000 g for 20 min, and

loaded at 1.5 mLÆmin)1 onto a column (1.6· 5 cm) of

Fractogel EMD SO3650 (S) cation-exchange resin that had

been connected to an A¨KTA FPLC system and

pre-equilibrated with buffer A.The column was washed with

buffer A until the A280 decreased to baseline, and then

developed with a linear gradient (150 mL) of 0–500 mM

KCl in buffer A.AP activity resolved as two peaks (SAP1

and SAP2) at approximately 250 and 390 mM KCl,

respectively (Fig.1).Fractions (6 mL) containing greater

than 20% of peak activity were pooled and concentrated

separately to 2 mL by ultrafiltration over a YM-30

mem-brane (Amicon).Both samples were further concentrated to

0.25 mL using an Amicon Centricon-30 ultrafilter, and separately applied at 0.2 mLÆmin)1onto a Superose 12 HR 10/30 column, which had been connected to an FPLC system and pre-equilibrated in buffer B.Fractions (0.5 mL) containing AP activity were pooled and concentrated to 0.2–0.5 mL as described previously Concentrated SAP1 was divided into 25-lL aliquots, frozen in liquid N2 and stored at)80 C.The final SAP1 preparation was stable for

at least 4 months when stored frozen.The concentrated SAP2 was absorbed at 0.5 mLÆmin)1 onto a column (1· 1.2 cm) of Concanavalin A-Sepharose that had been connected to the FPLC and pre-equilibrated in buffer C The column was washed with 5 mL of buffer C and developed with a linear gradient (20 mL) of 0–500 mM

methyl a)D-mannopyranoside in buffer C.AP eluted as a single peak at approximately 120 mMmethyl a-D -manno-pyranoside.Fractions (1 mL) were pooled and concentrated

to 0.5 mL using an Amicon Centricon-30 ultrafilter The retentate was divided into 25-lL aliquots, frozen in liquid

N2and stored at)80 C.The final SAP2 preparation was stable for at least 5 months when stored frozen

Estimation of native molecular mass by gel filtration FPLC

This was performed during AP purification by Superose 12 FPLC as described above.Native Mrwas calculated from a plot of Kd(partition coefficient) against log Mrusing the following protein standards: catalase (232 kDa), aldolase (158 kDa), alcohol dehydrogenase (150 kDa), BSA (67 kDa), ovalalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa)

Electrophoresis SDS/PAGE (10 and 12% separating gels) was performed as described previously [36].For the determination of subunit

Mrby SDS/PAGE, a plot of relative mobility vs.the log Mr was constructed using the following standard proteins: myosin (200 kDa), b-galactosidase (116 kDa), phosphory-lase B (97.5 kDa), BSA (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).AP was also visualized in acrylamide gels by staining for AP activity.SDS/PAGE was

Fig 1 Separation of secreted AP isozymes from culture media of –P i

tomato suspension cells via SO 3 -Fractogel cation-exchange FPLC The column was developed with a linear KCl gradient (0–0.5 M ) and fractions of 6 mL were collected.AP activity eluted as two distinct peaks (SAP1 and SAP2).

performed as described above except that the samples were

not boiled.To detect AP activity, the gel was washed for 2 h

(three changes every 30 min) at 25C in a casein–EDTA

buffer [37] in order to remove SDS.The gel was then

incubated for 1 h at 25C in 100 mMNa-acetate (pH 5.3),

followed by incubation in the same buffer containing 10 mM

MgCl2, 1 mgÆmL)1Fast Garnet GBC salt, and 0.03% (w/v)

b-naphthyl-P.Carbohydrate staining was performed using

a periodic-acid Schiff staining procedure [38]

Amino acid sequencing

Purified SAP1 (2 lg) was subjected to SDS/PAGE as

described above.Following PAGE the gel was incubated

for 10 min in transfer buffer (10 mM Caps, pH 11,

containing 10% (v/v) methanol) and electroblotted onto a

Bio-Rad poly(vinylidene difluoride) membrane for 40 min

at 250 mA.The membrane was washed in Milli-Q H2O,

stained for 10 min with 0.1% (w/v) Coomassie Blue R-250

in 40% (v/v) methanol and 1% (v/v) acetic acid, destained

for 10 min in 50% (v/v) methanol and 10% (v/v) acetic acid,

rinsed (5· 2 min) with 25 mL of Milli-Q H2O, and

air-dried In situ trypsin digestion and separation of tryptic

peptides was performed at the NRC Protein and Peptide

Sequencing Facility (Montreal, Quebec).SAP2 (2 lg) was

subjected to SDS/PAGE as described above, digested in situ

with trypsin, and tryptic peptides separated at the

Labor-atory for Macromolecular Structure (Purdue University)

Sequencing of SAP1 and SAP2 tryptic peptides was

performed by automated Edman degradation.Similarity

searches were conducted with theBLASTProgram using the

short but nearly exact option available on the National

Center for Biotechnology Information website [39].Further

similarity searches were conducted using the Patmatch

BLASTProgram available on the Arabidopsis Information

Resource website (http://www.arabidopsis.org/cgi-bin/

patmatch/nph-patmatch.pl)

Peptide mapping by CNBr cleavage

Polypeptides were excised individually from SDS/PAGE

minigels and cleaved in situ with CNBr, and the degradation

products analyzed on a SDS/PAGE 14% (w/v) minigel

[40], followed by protein staining with SYPRO Red

(Amersham Biosciences).Fluorescence imaging was

per-formed using a Typhoon 9400 Imager Workstation

(Amer-sham Biosciences)

R E S U L T S

Influence of Pistarvation on growth and secreted

AP activity of tomato suspension cells

Tomato cells cultured for 8 days in the absence of

exogen-ous Pihad only approximately 40% of the fresh weight of

the 8-day-old +Picells (approximately 63 and 25 g of cells

were obtained per 500 mL culture of 8-day-old +Piand –Pi

cells, respectively).Eight days following subculture of the

tomato cells into –Piculture media, secreted (culture media)

AP activity increased from undetectable levels to a

maxi-mum of 7.5 UÆmg protein)1.Secreted AP activity decreased

to undetectable levels within two days of an 8-day-old –Pi

cell culture being resupplied with 2.5 mMP.All subsequent

studies were performed using cell culture media filtrate from 8-day-old –Picells.AP activity staining of 8-day-old –Picell culture filtrate proteins resolved by PAGE indicated the presence of two Pi-starvation inducible APs with Mrvalues

of approximately 84 and 57 kDa (Fig.2A)

AP purification Concentration of culture media proteins secreted by –Picells was facilitated by Mini-Tan ultrafiltration followed by acetone precipitation.This also eliminated pectic substances that otherwise interfered with subsequent column chroma-tography.AP activity resolved as two peaks of activity (SAP1 and SAP2, respectively) during Fractogel cation-exchange FPLC (Fig.1).As outlined in Table 1, SAP1 was purified approximately 30-fold to a final phosphoenolpru-vate-hydrolyzing specific activity of 222 UÆmg)1 and a recovery of approximately 3%, whereas SAP2 was purified 50-fold to a final phosphoenolpruvate-hydrolyzing activity

of 370 UÆmg)1and a yield of 5%.Although SAP2 specific activity was not increased following affinity chromato-graphy on Concanavalin A-Sepharose (Table 1), this step eliminated several minor impurities present following Superose 12 FPLC (results not shown), and resulted in a homogeneous SAP2 preparation (Fig.2B)

Gel electrophoresis and physical properties When the final preparations of SAP1 and SAP2 were denatured and subjected to SDS/PAGE, single Coomassie

Fig 2 PAGE analyses of purified SAP1 and SAP2 (A) Non-dena-turing PAGE on a 10% (w/v) separating gel of AP from the media of tomato suspension cells.Lanes 1 and 2, respectively, contain 3 lg of culture media proteins from 8-day-old +P i and –P i cells.The gel was incubated in a casein/EDTA wash buffer for SDS removal [37] and stained for AP activity using Fast Garnet GBC salt and b-naphthyl-P

as described under Materials and methods.(B–D) PAGE analyses using 12% (w/v) separating gels of purified SAP1 and SAP2.(B) Lanes

1 and 2 of the denaturing SDS gel contain 1 lg of the final prepara-tions of SAP1 and SAP2, respectively.The gel was stained with Coomassie Blue R-250.The migration of various M r standards is shown on the left.TD, tracking dye front.(C) Lanes 1 and 2 of this nondenaturing gel contain 0.5 lg of the final preparation of SAP1 and SAP2, respectively.The gel was stained for AP activity following SDS removal as described above.(D) Lanes 1 and 2 of this denaturing SDS gel contain 2 lg of the final preparations of SAP1 and SAP2, respectively.Glycoprotein staining was performed using a periodic acid-Schiff procedure [38].

Blue-staining polypeptides of 84 and 57 kDa, respectively,

were observed (Fig.2B).For both purified APs, nonboiled

samples resolved by SDS/PAGE followed by SDS removal

generated single protein staining polypeptides (not shown)

that comigrated with AP activity (Fig.2C).SAP1 and SAP2

were identified as glycoproteins by periodic acid-Schiff

staining (Fig.2D).The native Mrof SAP1 and SAP2 was

determined by analytical gel filtration FPLC to be 82 and

60 kDa, respectively.This indicates that both APs are

monomeric

Both APs were relatively heat stable, losing no activity

when the respective final preparations were incubated for

5 min at 70C

The concentrated final SAP1 (1 mgÆmL)1) and SAP2

(200 lgÆmL)1) preparations were pink in color and

dis-played an Amax at 518 and 538 nm, respectively.This

suggests that both APs are PAPs [1,18]

Peptide mapping and amino acid sequencing

The structural relationship between the two purified APs

was investigated by peptide mapping of their CNBr cleavage

fragments.The CNBr cleavage patterns of SAP1 and SAP2

were highly dissimilar (Fig.3)

N-terminal microsequencing of SAP1 and SAP2 was

impossible owing to N-terminal blockage of both

polypep-tides.A tryptic fragment of SAP1 and SAP2 was purified by

reverse phase HPLC and sequenced using automated

Edman degradation.A BLAST search of the Arabidopsis

Genome Index revealed that the respective SAP1 and SAP2

peptide sequence exhibited significant similarity to a

differ-ent pair of putative Arabidopsis PAPs (Fig.4)

Kinetic properties

Unless otherwise stated, all kinetic studies were performed

using assay A.SAP1 and SAP2 both showed a relatively

narrow pH-phosphatase activity profile with maximal

activity occurring at approximately pH 5.3 (Fig 5) All

subsequent AP kinetic studies were carried out at pH 5.3

Effect of divalent cations

SAP1 and SAP2 were activated in the presence of saturating

(5 mM) MgCl by approximately 135% and 180%,

respect-ively.When the reaction mixture contained 5 mM EDTA and no added divalent cations, SAP2 activity was reduced

by approximately 71%, whereas SAP1 activity was unaf-fected.SAP1 and SAP2 were also differentially inhibited by various divalent metal cations.In particular, SAP2 was potently inhibited by Co2+, Ba2+, and Ca2+(Table 2) Substrate specificity

AP activity was determined using assay B and a broad range

of phosphorylated compounds, tested at a concentration of

5 mMunless otherwise specified.Neither enzyme exhibited

Table 1 Purification of secreted AP isozymes from 4-L of culture media of 8-day-old –P i tomato suspension cells.

Step

Volume (mL)

Activity (U)

Protein (mg)

Specific activity (UÆmg)1)

Purification (fold)

Yield (%)

Fractogel SO 3 FPLC

Superose 12 FPLC

Concanavalin-A Sepharose

Fig 3 Electrophoretic patterns of CNBr cleavage fragments of SAP1 and SAP2 CNBr cleavage fragments were prepared from gel slices containing 3 lg of SAP1 (lane 1) and SAP2 (lane 2) and analyzed on

an SDS/14% PAGE minigel as previously described [40].Peptides were stained with SYPRO Red and image analysis performed using a Typhoon Imaging workstation.The migration of various M r stand-ards is shown on the left.

phosphatase activity when tested with dihydroxyacetone-P,

P-choline, or phytate, and no phosphodiesterase activity

was observed with bis-pNPP.SAP2 showed a broader

substrate specificity profile when compared to SAP1.Unlike

SAP2, SAP1 showed no or much lower activity with the

hexose-P, triose-P, or P-amino acids that were tested

(Table 3 and results not shown)

Table 3 lists kinetic parameters of SAP1 and SAP2 for

those compounds that were identified as being the most

effective substrates.Both APs were relatively unspecific,

with maximal specificity constant (Vmax/Km) obtained with

phosphoenolpyruvate and pNPP for SAP1 and SAP2,

respectively.The apparent Vmaxfor similar substrates was

150–300% greater for SAP2 when compared to SAP1.Of

the physiologically relevant substrates,

phosphoenolpyru-vate, tetrapoly-P, and phenyl-P were utilized most

effi-ciently by SAP1, whereas the most efficient substrates for

SAP2 were phosphoenolpyruvate, P-Tyr, PPi, and tetrapoly

P (Table 3).Hyperbolic substrate saturation kinetics was

always observed.Identical apparent Vmaxand Kmvalues for

phosphoenolpyruvate were obtained when assay A was

substituted for assay B

Metabolite and ion effects

A wide variety of compounds were examined for effects on the activity of SAP1 and SAP2 with subsaturating (approximate Km) concentrations of phosphoenolpyruvate (2.1 and 1.5 mM, respectively).The following compounds exerted no influence (± 10% control velocity) on the AP activity of either isozyme: L-tartrate, L-Glu, L-Asp, and phosphite (5 mM each); KCl, NaCl, or dithiothreitol (125 mM each).Significant inhibition was exerted by molybdate, Pi, fluoride, and vanadate (Table 4).Inhibition

by these compounds was further characterized, and the patterns of inhibition and inhibition constants are presented

in Table 4.For SAP1, inhibition by Piwas mixed, whereas for SAP2 the pattern of inhibition by Piwas competitive Inhibition of SAP1 and SAP2 by molybdate and fluoride was competitive and mixed, respectively (Table 4).More-over, I50and Kivalues of SAP2 for molybdate, fluoride, and

Pi were generally lower than the corresponding values determined for SAP1 (Table 4)

Peroxidase activity The ability of tomato SAP1 and SAP2 to catalyze the peroxidation of luminol was investigated using a chemi-luminescence assay.In the presence of luminol and H2O2,

nM amounts of SAP1 and SAP2 induced striking chemi-luminescence when compared to a BSA control (results not shown).SAP2 produced approximately 1.5-fold greater chemiluminescence than equimolar amounts of SAP1 Photon emission induced by SAP1 and SAP2 peroxidase activity was proportional to their respective concentration SAP1 and SAP2 both showed a fairly broad pH/peroxi-dase activity profile in the alkaline range with maximal activity occurring at approximately pH 8.4 (Fig 5) Calib-ration of the luminometer with known amounts of horseradish peroxidase (200 UÆmg protein)1) allowed us

to estimate the specific peroxidase activities of approxi-mately 10 and 14 UÆmg protein)1 for SAP1 and SAP2, respectively.MgCl2, EDTA, ZnCl2and molybdate (5 mM

each) exerted no influence on the peroxidase activity of SAP1 or SAP2

Fig 4 Comparison of SAP1 and SAP2 tryptic peptide sequences with a

portion of the deduced amino acid sequence for several putative PAPs

from Arabidopsis thaliana The sequence of the SAP1 and SAP2 tryptic

peptide was obtained by automated Edman degradation.Other

sequences were derived from the translation of putative PAP

nucleo-tide sequences.Swiss-Prot accession numbers are shown in

paren-theses.Numbering is relative to the first amino acid of the deduced AP

sequence, and colons indicate an amino acid residue identical to that of

the respective tomato PAP peptide sequence.

Fig 5 Phosphatase vs peroxidase activities of purified SAP1 and SAP2

as a function of assay pH Assays were buffered by a mixture of 25 m M

Na-acetate, 25 m M Mes and 25 m M Bis-Tris-propane.All values

rep-resent the means ± SE of n ¼ 3 separate determinations.

Table 2 Effect of various divalent metal cations and EDTA on the activity of SAP1 and SAP2 The standard assay A was used except that the phosphoenolpyruvate concentration was subsaturating (4 m M ) Enzyme activity in the presence of metal cations or EDTA (5 m M ) is expressed relative to the control set at 100.

Addition (5 m M )

Relative activity

D I S C U S S I O N

Purification and physical properties of SAP1 and SAP2

SAP1 and SAP2 were resolved by cation-exchange FPLC

and purified to final pNPP-hydrolyzing specific activities of

246 and 940 UÆmg protein)1, respectively (Fig.1, Table 3)

These values are in the range reported for other

homo-genous plant APs [2,4–6,15,22,23], including PAPs from

soybean and sweet potato [20].PAGE followed by protein

and AP activity staining confirmed that both SAPs were

purified to homogeneity (Fig.2B,C).Analytical gel

filtra-tion FPLC, SDS/PAGE (Fig.2B), and periodic acid-Schiff

staining (Fig.2D) indicated that SAP1 and SAP2,

respect-ively, exist as 84 and 57 kDa monomeric glycoproteins

SAP2 may be identical to the partially purified 57 kDa AP

from 3-day-old Pitomato suspension cell cultures [10]

SAP1 and SAP2 exhibited an Amaxat 518 and 538 nm,

respectively, and were insensitive to tartrate inhibition [2],

indicating that they are PAPs [1,18].All plant PAPs that

have been studied thus far are homodimers of 55 kDa

subunits [20,22].Thus, SAP1 and SAP2 appear to be the first

monomeric plant PAPs characterized to date.The subunit

Mr (57 kDa) of SAP2 is within the range for previously

characterized plant PAPs, whereas SAP1 exists as an

unusual 84 kDa monomeric PAP.To our knowledge, no

other PAP having a similar subunit M has been described

A nine-amino acid tryptic peptide sequence of SAP1 was highly similar to a portion of the deduced amino acid sequence of two putative Arabidopsis PAPs (Fig.4).Simi-larly, a seven-amino acid sequence of a SAP2 peptide was found to be highly similar to portions of two other Arabidopsis PAPs (Fig.4) As the analysis of the SAP1 peptide sequence identified similarity with two putative ArabidopsisPAPs that were not identified in searches con-ducted with the SAP2 peptide sequence, this suggests that the two tomato PAP isoforms are structurally unrelated This was corroborated by the highly dissimilar CNBr peptide maps of SAP1 and SAP2 (Fig.3).CNBr fragmen-tation patterns depend on the position and number of methionine residues in the protein [40].The results imply that the two tomato PAPs are structurally dissimilar isozymes encoded by separate genes

Kinetic properties of SAP1 and SAP2

A pH-phosphatase activity profile centered at approxi-mately pH 5.3 (Fig 5) is consistent with the designation of both isozymes as an AP.SAP1 and SAP2 were activated by

5 mM Mg2+ (Table 2), which has also been shown for various plant APs [4–6,15].SAP1 and SAP2 were potently inhibited by Zn2+ and Cu2+.Inhibition by Zn2+ was observed for APs isolated from red kidney beans [23], potato tuber [4], banana fruit [5], and –P B nigracells [6]

Table 3 Substrate saturation kinetics of SAP1 and SAP2 Kinetic parameters were determined using assay B as described in Materials and methods N.A., No activity detected with up to 5 m M of this metabolite.

Substrate

V max

(UÆmg)1)

K m

(m M )

V max /K m

(UÆmg)1Æm M )1 )

V max

(UÆmg)1)

K m

(m M )

V max /K m

(UÆmg)1Æm M )1 )

Table 4 The effect of inhibitors, inhibition pattern and enzyme-inhibitor dissociation constants for selected inhibitors of SAP1 and SAP2 I 50 values, patterns of inhibition and K i and K i ¢ values (representing competitive and uncompetitive inhibition constants, respectively) were determined using assay A as described under Materials and methods.

Inhibitor

I 50

(m M ) Inhibition type

K i

(m M )

K i ¢ (m M )

I 50

(m M ) Inhibition type

K i

(m M )

K i ¢ (m M )

EDTA exerted no effect on SAP2, but caused a 71%

inhibition of SAP2 (Table 2).This suggests that only SAP2

requires divalent metal cations to be fully active.However,

our results indicate that both tomato AP isozymes are

PAPs, APs with a binuclear metallic center.Complete

removal of metal ions from the active site of kidney bean

PAP requires prolonged dialysis against EDTA at elevated

temperatures [41].Plant PAPs containing different binuclear

metal centers have been reported [20,41,42].It is possible

that the differential effect of divalent metal cations on the

activity of SAP1 and SAP2 is due to differing metal contents

at the active site of each isozyme

Similar to other APs [2], SAP1 and SAP2 were subject to

potent competitive inhibition by molybdate (Table 4)

However, differing patterns of inhibition of by Piindicate

that some structural and/or conformational differences may

exist between SAP1 and SAP2.AP inhibition by Pisuggests

a potential control mechanism through product inhibition

[2]

Extracellular APs usually display broad substrate

speci-ficity, whereas APs showing restricted substrate specificity

tend to be intracellular and may play a more specific role in

plant metabolism [2,4,6,15].Although SAP1 and SAP2

demonstrated relatively nonspecific substrate selectivities, a

broader range of substrates utilization was evident in SAP2,

and this isozyme was more active with any substrate as

compared to SAP1 (Table 3).SAP1 and SAP2 were

particularly efficient at catalyzing the hydrolysis of Pifrom

substrates with good leaving groups (i.e

phosphoenolpyru-vate, ATP, GTP, and tetrapoly P; Table 3), whereas

P-esters with poorer leaving groups (i.e hexosephosphates)

(DG¢ < 5000 calÆmol)1) [43] were not as effectively utilized

Their nonspecific substrate specificities are consistent with

SAP1 and SAP2 playing a role in Pi scavenging from

extracellular P-esters when external Pilevels are depleted

Studies defining the temporal and spatial expression pattern

of SAP1 and SAP2 during Pi-starvation (or pathogen

infection; see below) will help to confirm their precise

physiological role(s).The data presented here, in

combina-tion with studies on the regulacombina-tion of other Pi-starvation

inducible proteins including high-affinity Pitransporters [8],

and secreted ribonuclease and phosphodiesterases [29,44]

indicate the presence of a highly coordinated response in –Pi

tomato.Cloning of the genes encoding SAP1 and SAP2 will

facilitate studies of their overexpression and molecular

regulation in an effort to increase Pi acquisition during

Pi-limited tomato growth

Peroxidase activity was recently reported for

Arabid-opsisand recombinant human PAPs [24,35,45].SAP1 and

SAP2 also displayed peroxidase activity at alkaline pH

(Fig.5), and this activity was unaffected by potent

inhibitors of AP activity.This is reminiscent of a

mammalian PAP, where site-directed mutagenesis of

conserved residues within its active site revealed that its

AP and peroxidase activities are functionally independent

[46].In mammals, the involvement of PAP peroxidase

activity in the generation of ROS appears to be pivotal in

processes linked to bone resorption or macrophage killing

of invading microbes [21,35,46].Similarly, the production

of extracellular ROS is closely associated with the

oxidative burst that occurs during the hypersensitive

response of plants to pathogen attack [46].It is notable

that the oxidative burst in plants is associated with

extracellular alkalinization [46].Moreover, several plant PAPs have been reported to be induced in response to pathogen attack or elicitor treatment [47,48].An A thali-ana PAP displaying peroxidase activity has been sugges-ted to be involved in ROS metabolism during senescence [24].Future studies are required to determine whether SAP1 and SAP2 are induced and/or play a role in ROS production during the oxidative burst that accompanies pathogen infection of tomato

A C K N O W L E D G E M E N T S

This work was supported by research and equipment grants from the Natural Sciences and Engineering Research Council of Canada to WCP, and U.S Department of Agriculture-National Research Initiat-ive CompetitInitiat-ive Grants Program (grant no.590 1165–2614) to KGR.

We are grateful to William Turner for helpful discussions.

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