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 producedÆmin )1 mgÆprotein )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 V max of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 m M Mg 2+ , and demon- strated potent competitive inhibition by molybdate, but mixed and competitive inhibition by P 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 r , 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 P i -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 P i -stress [6,7]. The induction of AP is a universal response of vascular plants to P i starvation [2]. P i 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, 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) Eur. J. Biochem. 269, 6278–6286 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03347.x and roots [10,11], white-lupin proteoid roots [12], and Arabidopsis thaliana seedlings [13]. Cell wall localized APs are associated with P i -starvation of white clover [14], Brassica nigra suspension cells [15], and duckweed [16]. The cell wall-localized AP from –P i B. nigra suspension 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 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 crop species tested under similar conditions. A 130-kDa homodimeric secreted AP was purified from –P i L. esculen- tum roots, and was found to have comparable kinetic properties to a homodimeric AP secreted from –P i lupin roots [11]. Recently, Baldwin and coworkers [27] isolated a cDNA sequence encoding a putative 30 kDa AP (LePS2) from –P i tomato roots. The subcellular localization and physiological role of LePS2 have yet to be elucidated. Bosse and Ko ¨ ck [29] reported similar P i -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 –P i tomato 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 –P i tomato suspension cells. 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 i cell suspension was centrifuged axenically at 4000 g for 12 min at 25 °C. The cells were washed with –P i media, and used to inoculate 500 mL of fresh –P i media. After 8 days the –P i cells 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 25 °C. 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 25 °C by monitoring the oxidation of NADH at 340 nm using a Gilford 260 recording spectrophotometer. Standard AP assay condi- tions were 50 m M Na-acetate (pH 5.3), 10 m M phos- phoenolpyruvate, 5 m 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 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 m M ammonium molybdate in 15 m M Zn-acetate (pH 5.0). Samples were incubated for 25 min at 40 °Cand the A 660 determined using a Spectromax 250 Microplate spectrophotometer (Molecular Devices). Controls were run for background amounts of P i present at each substrate concentration tested. To calculate activities, a standard curve over the range 1–100 nmol P i was constructed for each set of assays. Kinetic studies and protein concentration determination Apparent V max and K m values were calculated from the 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]. 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 m M hydrogen peroxide to 0.1 mL of 0.2 M Tris/HCl (pH 8.1) containing 300 m M luminol and 5–20 n M of AP in a 96-well microtitre plate. Photon emission was monitored continuously for 10 s after H 2 O 2 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 m M Na-acetate (pH 5.7), 1.5 m M MgCl 2 , 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 MgCl 2 ,0.2m M CaCl 2 ,0.2m M MnCl 2 , and 10% (v/v) glycerol. AP purification All steps were carried out at 0–4 °C. Cell culture media from –P i cells (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 m M Na-acetate (pH 5.8) containing 1 m M MgCl 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 )1 onto 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 N 2 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 m M methyl a) D -mannopyranoside in buffer C. AP eluted as a single peak at approximately 120 m M methyl 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 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 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). 6280 G. G. Bozzo et al.(Eur. J. Biochem. 269) Ó FEBS 2002 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 25 °Cinacasein–EDTA buffer [37] in order to remove SDS. The gel was then incubated for 1 h at 25 °C in 100 m M Na-acetate (pH 5.3), followed by incubation in the same buffer containing 10 m M MgCl 2 ,1mgÆmL )1 Fast 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)wassubjectedtoSDS/PAGEas 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 Program 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). RESULTS Influence of P i starvation on growth and secreted AP activity of tomato suspension cells Tomato cells cultured for 8 days in the absence of exogen- ous P i had only approximately 40% of the fresh weight of the 8-day-old +P i cells (approximately 63 and 25 g of cells were obtained per 500 mL culture of 8-day-old +P i and –P i cells, respectively). Eight days following subculture of the tomato cells into –P i 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 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 )1 and 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 lgof 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]. 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 monomeric. Both APs were relatively heat stable, losing no activity when the respective final preparations were incubated for 5minat70°C. The concentrated final SAP1 (1 mgÆmL )1 ) and SAP2 (200 lgÆmL )1 ) preparations were pink in color and dis- played an A max 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 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 M unless 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 (%) Cell culture filtrate 4000 1200 160 7.5 1 100 Mini-tan concentration 270 900 120 7.5 1 100 Acetone precipitation 120 670 90 7.5 1 56 Fractogel SO 3 – FPLC SAP1 30 90 1.1 80 11 7 SAP2 42 141 1.4 100 13 12 Superose 12 FPLC 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, respectively. The apparent V max for 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, PP i , and tetrapoly P (Table 3). Hyperbolic substrate saturation kinetics was always observed. Identical apparent V max and K m values 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 K m ) concentrations of phosphoenolpyruvate (2.1 and 1.5 m M , 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 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 i 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 H 2 O 2 , n M 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. 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 ). 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 SAP1 SAP2 MgCl 2 136 181 MnCl 2 113 74 CoCl 2 102 5 CaCl 2 104 40 ZnCl 2 00 BaCl 2 115 50 CuSO 4 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 subunits [20,22]. Thus, SAP1 and SAP2 appear to be the first monomeric plant PAPs characterized to date. The subunit M r (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 r 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 Arabidopsis PAPs 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 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 M ) V max /K m (UÆmg )1 Æm M )1 ) V max (UÆmg )1 ) K m (m M ) V max /K m (UÆmg )1 Æm 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 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 ) 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]. 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 P i from 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 SAP1andSAP2playingaroleinP i scavenging from extracellular P-esters when external P i levels are depleted. Studies defining the temporal and spatial expression pattern of SAP1 and SAP2 during P i -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 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 Ôoxidative burstÕ 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. 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Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato ( Lycopersicon esculentum ) cell cultures Gale. only approximately 40% of the fresh weight of the 8-day-old +P i cells (approximately 63 and 25 g of cells were obtained per 500 mL culture of 8-day-old