Purification and characterization of two secreted purple acid
phosphatase isozymes from phosphate-starved tomato
(
Lycopersicon esculentum
) cell cultures
Gale G. Bozzo
1
, Kashchandra G. Raghothama
3
and William C. Plaxton
1,2
Departments of
1
Biology and
2
Biochemistry, Queen’s University, Kingston, Ontario, Canada;
3
Department of Horticulture and
Landscape Architecture, Purdue University, Indiana, USA
Two secreted acid phosphatases (SAP1 and SAP2) were
markedly up-regulated during P
i
-starvation of tomato
suspension cells. SAP1 and SAP2 were resolved during cat-
ion-exchange FPLC of culture media proteins from 8-day-
old P
i
-starved cells, and purified to homogeneity and final
p-nitrophenylphosphate hydrolyzing specific activities of 246
and 940 lmol P
i

i
, respectively. Inter-
estingly, both SAPs exhibited significant peroxidase activity,
which was optimal at approximately pH 8.4 and insensitive
to Mg
2+
or molybdate. This suggests that SAP1 and SAP2
may be multifunctional proteins that operate: (a) PAPs that
scavenge P
i
from extracellular phosphate-esters during P
i
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 P
i
,
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 M

is a critical macronutrient that
limits plant growth in many natural ecosystems [7,8]. Soil P
i
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 P
i
status [2,7]. An
increase in secreted AP and P
i
-uptake activity is believed to
assist in the acquisition of limiting P
i
from the environment
by P
i
-deficient (–P
i
) plants [7,8]. Secreted APs are likely
involved in P
i
scavenging from extracellular organic
P-monoesters [7–9]. P
i
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,

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 by
L
-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 M
r
PAPs 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 –P
i
tomato plants or
suspension cell cultures [10,11,27]. Tadano and Sakai [28]
reported the secretion of significant AP activity from the
roots of –P
i
tomato and lupin, which was greater than most

MATERIALS AND METHODS
Chemicals and plant material
Fractogel EMD-SO
3
–
650 (S) cation exchange resin, and KCl
were from BDH Chemicals. A Superose 12 HR10/30 column
and gel filtration M
r
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 m
M
P
i
as described previously
[30]. P deficiency treatments were initiated 7 days after
subculturing the cells into fresh P
i
-sufficient (+P
i
)media.
A50-mLportionof+P

M
MgCl
2
,0.2m
M
NADH, and
3UÆmL
)1
desalted 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
P
i
released 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 m
M
Na-acetate (pH 5.3),
5m
M
MgCl
2
and an alternative substrate (5 m
M

Michaelis–Menten equation fitted to a nonlinear least-
squares regression computer kinetics program [32]. The I
50
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
i
and K
i
¢. K
i
values
Ó FEBS 2002 Secreted acid phosphatases of P
i
-starved tomato cells (Eur. J. Biochem. 269) 6279
were determined from Dixon plots, whereas K
i
¢ 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].

1m
M
EDTA, 1 m
M
dithiothreitol, and 10% (v/v) glycerol.
Buffer B: 25 m
M
Na-acetate (pH 5.7), 1.5 m
M
MgCl
2
,
1m
M
EDTA, 1 m
M
dithiothreitol, 100 m
M
KCl, and 10%
(v/v) glycerol. Buffer C: 25 m
M
Na-acetate (pH 5.7),
100 m
M
KCl, 1 m
M
EDTA, 1 m
M
dithiothreitol, 1.5 m
M

2
using 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 · 5cm) of
Fractogel EMD SO
3
–
650 (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 A
280
decreased to baseline, and then
developed with a linear gradient (150 mL) of 0–500 m
M
KCl in buffer A. AP activity resolved as two peaks (SAP1
and SAP2) at approximately 250 and 390 m
M
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

N
2
andstoredat)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 M
r
was calculated from a
plot of K
d
(partition coefficient) against log M
r
using 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
M
r
by SDS/PAGE, a plot of relative mobility vs. the log M
r
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

described above. Following PAGE the gel was incubated
for 10 min in transfer buffer (10 m
M
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 H
2
O,
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 H
2
O, 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 the
BLAST
Program using the
Ôshort but nearly exactÕ option available on the National
Center for Biotechnology Information website [39]. Further
similarity searches were conducted using the Patmatch
BLAST

culture 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 –P
i
cell culture being resupplied with 2.5 m
M
P
i
. All subsequent
studies were performed using cell culture media filtrate from
8-day-old –P
i
cells. AP activity staining of 8-day-old –P
i
cell
culture filtrate proteins resolved by PAGE indicated the
presence of two P
i
-starvation inducible APs with M
r
values
of approximately 84 and 57 kDa (Fig. 2A).
AP purification
Concentration of culture media proteins secreted by –P
i
cells
was facilitated by Mini-Tan ultrafiltration followed by

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].
Ó FEBS 2002 Secreted acid phosphatases of P
i
-starved tomato cells (Eur. J. Biochem. 269) 6281
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 M
r
of SAP1 and SAP2 was
determined by analytical gel filtration FPLC to be 82 and
60 kDa, respectively. This indicates that both APs are

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 m
M
)MgCl
2
by approximately 135% and 180%, respect-
ively. When the reaction mixture contained 5 m
M
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 Co
2+
,Ba
2+
,andCa
2+
(Table 2).
Substrate specificity
AP activity was determined using assay B and a broad range
of phosphorylated compounds, tested at a concentration of
5m

SAP1 3.5 40 0.18 222 30 3
SAP2 5 70 0.19 368 49 6
Concanavalin-A Sepharose
SAP2 2 59 0.16 370 49 4
Fig. 3. Electrophoretic patterns of CNBr cleavage fragments of SAP1
and SAP2. CNBr cleavage fragments were prepared from gel slices
containing 3 lgofSAP1(lane1)andSAP2(lane2)andanalyzedon
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-
ardsisshownontheleft.
6282 G. G. Bozzo et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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,orP-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 (V
max
/K
m
)obtainedwith
phosphoenolpyruvate and pNPP for SAP1 and SAP2,

-tartrate,
L
-Glu,
L
-Asp, and
phosphite (5 m
M
each); KCl, NaCl, or dithiothreitol
(125 m
M
each). Significant inhibition was exerted by
molybdate, P
i
, 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 P
i
was mixed, whereas
for SAP2 the pattern of inhibition by P
i
was competitive.
Inhibition of SAP1 and SAP2 by molybdate and fluoride
was competitive and mixed, respectively (Table 4). More-
over, I
50
and K
i
values of SAP2 for molybdate, fluoride, and
P

respectively. MgCl
2
,EDTA,ZnCl
2
and molybdate (5 m
M
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 t haliana. 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
asafunctionofassaypH.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

98
EDTA 114 29
Ó FEBS 2002 Secreted acid phosphatases of P
i
-starved tomato cells (Eur. J. Biochem. 269) 6283
DISCUSSION
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 P
i
tomato suspension cell cultures [10].
SAP1 and SAP2 exhibited an A
max
at 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

5m
M
Mg
2+
(Table 2), which has also been shown for
various plant APs [4–6,15]. SAP1 and SAP2 were potently
inhibited by Zn
2+
and Cu
2+
. Inhibition by Zn
2+
was
observed for APs isolated from red kidney beans [23],
potato tuber [4], banana fruit [5], and –P
i
B. nigra cells [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
SAP1 SAP2
V
max
(UÆmg
)1
)
K
m

M
)1
)
pNPP 246 4.5 55 940 3.3 285
b-Naphthyl-P 227 3.9 58 964 4.5 214
a-Naphthyl-P 162 6.4 25 460 5.3 87
Phosphoenolpyruvate 241 2.1 115 384 1.4 274
ATP 264 4.1 64 437 5.5 80
Phenyl-P 180 2.1 86 545 5.3 103
Tetrapoly-P 173 1.9 91 423 2.9 146
GTP 258 8.6 30 500 5.4 93
PP
i
N.A. – – 688 4.6 150
P-Tyr N.A. – – 496 2.1 236
6-phosphogluconate N.A. – – 333 4.5 74
3-phosphoglycerate N.A. – – 414 6.0 69
Glycerol-3-phosphate N.A. – – 252 5.0 50
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
SAP1 SAP2
I

)
Molybdate 0.0028 Competitive 0.0002 – 0.0015 Competitive 0.0002 –
Fluoride 2.2 Mixed 0.95 3.4 0.55 Mixed 0.48 1.6
P
i
4.5 Mixed 1.3 2.1 3.7 Competitive 1.2 –
6284 G. G. Bozzo et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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
attheactivesiteofeachisozyme.
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 P
i
indicate
that some structural and/or conformational differences may
exist between SAP1 and SAP2. AP inhibition by P
i
suggests
a potential control mechanism through product inhibition
[2].

tion with studies on the regulation of other P
i
-starvation
inducible proteins including high-affinity P
i
transporters [8],
and secreted ribonuclease and phosphodiesterases [29,44]
indicate the presence of a highly coordinated response in –P
i
tomato. Cloning of the genes encoding SAP1 and SAP2 will
facilitate studies of their overexpression and molecular
regulation in an effort to increase P
i
acquisition during
P
i
-limited tomato growth.
Peroxidase activity was recently reported for Arabid-
opsis and 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

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