Acid phosphatases from beet root (Beta vulgaris) plasma membranes

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Purificación y caracterización de fosfatasas de membrana plasmática de betabel

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Acid phosphatases from beet root (Beta vulgaris) plasma membranes

  1. 1. PHYSIOLOGIA PLANTARUM 121: 223–230. 2004 DOI: 10.1111/j.1399-3054.2004.00331.x Printed in Denmark – all rights reserved Copyright # Physiologia Plantarum 2004 Acid phosphatases from beet root (Beta vulgaris) plasma membranes ´ Eduardo Armienta-Aldana and Luis E. Gonzalez de la Vara* Departamento de Biotecnologı´a y Bioquı´mica, Unidad Irapuato, Centro de Investigacio´n y de Estudios Avanzados del IPN. Apartado Postal 629, 36500 Irapuato Gto, Me´xico *Corresponding author, e-mail: lgonzale@ira.cinvestav.mx Received 25 November 2003; revised 28 January 2004 Several acid phosphatases (EC 3.1.3.2) were found in beet root basic protein (phospho-MBP). This phosphatase presented two (Beta vulgaris L.) plasma membranes. Two of them were par- polypeptides with molecular masses of 36 and 65 kDa and was tially purified by an extraction of plasma membranes with 83% inhibited by 2 nM okadaic acid, which suggests it is a octylglucoside and successive gel-filtration and anion-exchange PP2A protein phosphatase. As the phosphatase activity was chromatographies. With p-nitrophenyl-phosphate (pNPP) as high in soluble (non-membrane) fractions, the possibility that substrate, most of the phosphatase activity was found in a frac- phosphatases in plasma membranes were soluble contaminants tion containing an 82-kDa protein. This phosphatase showed an ´ was assessed. Following the method of Berczi and Møller (Plant optimum pH of 5.4 and was inhibited by Cu21, Zn21, molybdate Physiol. 116:1029, 1998), it was found that about 45% of both or vanadate. The other phosphatase had a lower specific activity acid and protein phosphatase activities could be due to soluble with pNPP, but was able to dephosphorylate phospho-myelin enzymes trapped inside membrane vesicles. Introduction Acid phosphatases or orthophosphoric-monoester phos- the plant. Among acid phosphatases, purple acid phos- phohydrolases (EC 3.1.3.2) are abundant in plant cells. phatases (PAPs) are probably the most thoroughly stud- These enzymes appear to be important in the production, ied. PAPs are metallo-phosphatases that catalyse the transport, and recycling of Pi (reviewed in Duff et al. hydrolysis of several phosphate esters and anhydrides. 1994). Acid phosphatases are widely distributed in In the Arabidopsis genome, 29 genes for PAPs have been plants, and have been found in seeds (Park and Van predicted. The expression of some of them increases in Etten 1986, Biswas and Cundiff 1991, Kawarasaki et al. response to phosphate deficiency (Li et al. 2002). Some 1996, Granjeiro et al. 1999), roots (Panara et al. 1990, acid phosphatases could be involved in protein dephos- Penheiter et al. 1997), leaves (Staswick et al. 1994) and phorylations and therefore in signalling pathways (Duff fruits (Turner and Plaxton 2001). However, no acid et al. 1994). phosphatases have been found in plasma membranes On the other hand, protein phosphatases are grouped from plant cells, although their presence in them has in three distinct families. The PPP and PPM families been suggested by the discovery of a glycosylphosphati- include enzymes that remove phosphate groups from dylinositol-anchored acid phosphatase in Spirodela oli- Ser-P and Thr-P residues; whereas members of the pro- gorrhiza (Nakazato et al. 1998). tein tyrosine phosphatase (PTP) family include Tyr-P- Plant acid phosphatases are involved in responses to specific and dual-specificity phosphatases. The PPP phosphate deficiency (Duff et al. 1991, Tadano et al. family includes protein phosphatases belonging to the 1993), salt stress (Pan 1987) and water deficit (Barrett- PP1 and PP2A classes [which can be distinguished by Lennard et al. 1982). These enzymes break down organic its sensitivity to the inhibitor okadaic acid (OA)], as well molecules to liberate phosphate, making it available to as the PP2B group. The PPM family includes protein Abbreviations – Brij 58 P, polyethylene glycol hexadecyl ether; CHAPS, 3-[ (3-cholamidopropyl) dimethylammonio]-1-propanesulphonate; EDTA, ethylendinitrile tetraacetic acid; EGTA, ethylenglycol-bis (ether b-aminoethyl) N,N,N0 ,N0 -tetraacetic acid; HEDTA, N-hydroxyethylethylenediamine- triacetic acid; MBP, myelin basic protein; pNPP, p-nitrophenyl phosphate; PAP, purple acid phosphatase. Physiol. Plant. 121, 2004 223
  2. 2. phosphatases 2C (PP2C) (Luan 2000). In the Arabidopsis 19 400 Â g for 30 min at 4 C. The supernatant (2 ml) was thaliana genome, 112 protein phosphatase genes have loaded onto a Sephacryl S-300-HR gel-filtration column been predicted (Kerk et al. 2002). Of these, 69 belong (50 Â 1 cm), equilibrated with 30 mM HEPES, 1 mM to the PP2C group, 23 are classified as PPP (including 8 DTT, 1 mM EDTA and 100 mM NaCl (pH 7.0) and PP1 and 5 PP2A phosphatases) and 18 as dual-specificity eluted with the same buffer. The fractions with highest PTPs. phosphatase activity from the Sephacryl column were There are several enzymes whose activities are con- pooled and loaded onto a DEAE-Sepharose column trolled by phosphorylation in plasma membranes. For (6 Â 0.7 cm), equilibrated with 30 mM HEPES (pH 7.0), instance, the H1-transporting ATPase is an enzyme 1 mM DTT and 1 mM EDTA (column buffer). Proteins phosphorylated in vivo (Desbrosses et al. 1998). Its were eluted first with 1 ml column buffer with 0.1 M phosphorylation status, which reflects the activity of NaCl, then with 4 ml of a 0.1–0.8 M NaCl gradient in endogenous kinases and phosphatases, regulates its column buffer. Phosphatase activity (using pNPP as sub- association with 14-3-3 proteins and, thereby, its activity strate) and protein content were measured in all (0.5 ml) (Morsomme and Boutry 2000). In plasma membranes fractions collected. from tomato leaves, the H1-ATPase activity increased To analyse the phosphatase activity not extracted with when they were exposed to a fungal elicitor. This stimu- n-octylglucoside, the pellet obtained in this extraction lation was correlated with the dephosphorylation of the was treated with 3% Triton X-114 (v/v) in column buffer H1-ATPase by a membrane-bound phosphatase (Vera- (2 ml) for 30 min at 4 C with moderate shaking. To allow Estrella et al. 1994). More recently, Camoni et al. (2000) a phase separation, this mixture was centrifuged at showed that the activity of a PP2A phosphatase in maize 19 400 Â g for 3 min at room temperature. The upper roots reduced drastically the association of 14-3-3 pro- (least hydrophobic) phase was collected, and the lower teins with the plasma membrane H1-ATPase. phase was extracted with 1 ml of upper phase (prepared Herein we report the purification and some properties without membranes). Upper phases were pooled of two plasma membrane-associated acid phosphatases. (2 ml) and loaded onto a DEAE-Sepharose column One of them appears to be a typical acid phosphatase, (4.5 Â 1.2 cm), equilibrated with column buffer. Phos- whereas the other is probably a PP2A protein phospha- phatases were eluted with 5 ml of column buffer with tase. To assess the possibility that these phosphatases 0.1 M NaCl. The fraction (1 ml) with highest phospha- were soluble contaminants, plasma membrane phospha- tase activity was loaded onto a CM-Sepharose column ´ tases were sequentially extracted as proposed by Berczi (6 Â 0.7 cm), equilibrated with column buffer. Proteins and Møller (1998). We found that an important fraction were then eluted with a 4-ml 0–0.6 M NaCl gradient in of the phosphatase activity could be due to soluble the same buffer. enzymes trapped inside membrane vesicles. Phosphatase activity measurements Materials and methods Phosphatase activity was routinely measured observing the conversion of p-nitrophenyl phosphate (pNPP) into Chemicals and plant material p-nitrophenol in a reaction mixture (0.5 ml) containing Beet roots (Beta vulgaris L.) were obtained at a local 15 mM BTP/succinic-acid buffer (at the indicated pH), market and stored at 4 C, until used, usually 1 or 2 days 150 mM KCl, and 5 mM pNPP, unless otherwise indi- later. Unless indicated otherwise, chemicals were obtained cated. Reactions were started by adding the enzyme- from Sigma Chemical Co (St Louis, MO, USA). containing sample and reading the absorbance at 405 and 466 nm (control wavelength) at 0, 15, 30 and 60 min. To obtain the p-nitrophenol concentration pro- Plasma membrane preparation duced, a differential millimolar extinction coefficient Plasma membranes from beet roots were prepared by (e(405À466)) of 8.317 (in 0.1 N NaOH) was used. This two-phase aqueous partitioning as described (Lino et al. coefficient was obtained hydrolysing a solution of 1998) and kept at À70 C in 25 mM Tris/MES (pH 7.5), pNPP with alkaline phosphatase and determining the 2 mM EDTA, 1 mM DTT (United States Biochemical phosphate concentration and the A405–A466 value in it. Corp. (USB), Cleveland, OH) and 45% (v/v) glycerol Extinction coefficients at different pH values were until used. The supernatant obtained with the total mem- calculated using a pKa for p-nitrophenol of 7.15. brane (microsomal) pellet was used as a source of soluble Alternatively, phosphatase activities were determined proteins. by measuring the inorganic phosphate released according to Ames (1966). Reaction mixtures (120 ml) contained 30 mM BTP/succinic-acid buffer (at the indicated pH), Purification of acid phosphatases 5 mM pNPP (or other substrate at the indicated concen- Plasma membranes (with 8 mg of protein) were extracted tration) and 150 mM KCl. Reactions were started by with n-octylglucoside (1 g per g of protein) in 30 mM adding 8–10 ml of the sample with phosphatase activity. HEPES, 1 mM DTT, 1 mM EDTA for 1 h at 4 C with After 60 min at 30 C, the reaction was stopped with moderate shaking. The homogenate was centrifuged at 1.88 ml of the molybdate reagent (0.02 g ascorbic acid, 224 Physiol. Plant. 121, 2004
  3. 3. 0.42% ammonium heptamolybdate in 1 N H2SO4) used Electrophoresis to measure the released phosphate. The amount of phos- The electrophoretic separation of proteins was performed phate released was measured by reading the A820 30 min in 7.5% (w/v) polyacrylamide gels (SDS-PAGE) following later. This method was used to determine phosphatase the method described by Schagger and von Jagow (1987). ¨ activities at pH values lower than 6.0 (where the p-nitro- Electrophoreses were run at room temperature. After phenol absorbance is too low), or with substrates other electrophoresis, gels were fixed in 50% methanol and than pNPP. 10% acetic acid for 30 min, and stained with Coomassie blue (Serva blue G, SERVA Electrophoresis GmbH, Heidelberg, Germany) (Schagger and von Jagow 1987). ¨ Phosphoprotein phosphatase activity measurements Protein phosphatase activity was determined using 32 Kinetic studies P-labelled myelin basic protein (32P-MBP) as substrate. 32 P-MBP was prepared by phosphorylating MBP [Gibco Activity versus substrate concentration curves were (Invitrogen, Carlsbad, CA) or Sigma] with a 63-kDa Ca21- obtained for pNPP (0.1–10 mM) and for 32P-MBP (0.05– dependent protein kinase purified from beet root plasma 2 mg mlÀ1). When pNPP was used as substrate, the reac- ´ membranes (Carrillo, Lino and Gonzalez de la Vara; tions were run in a 30-mM MES/Tris (pH 6.2) buffer. 32P- unpublished results). The phosphorylation mixture (75– MBP was dephosphorylated in 30 mM BTP/succinic acid 90 ml) contained 30 mM HEPES/Tris (pH 7.0), 1.5 mM (pH 6.6), 150 mM KCl, 1 mM EDTA and 5 mM MgSO4. EGTA, 1.5 mM HEDTA, 2.4 mM CaCl2, 3.3 mM MgSO4 Activity versus pH curves were obtained using 30 mM (calculated to obtain free ion concentrations of 10 mM and BTP/succinic acid (at the indicated pH, from 4.2 to 7.5). 2.5 mM for Ca21 and Mg21, respectively), 0.1 mM For the 82- and 95-kDa acid phosphatases, the phos- [g-32P]ATP (37 TBq molÀ1) and 5 mg mlÀ1 MBP. The phate released was measured as described by Ames kinase (with an activity of about 20 pmol minÀ1, using (1966). For the 36-kDa acid phosphatase we measured MBP as substrate) was added to start the phosphorylation the dephosphorylation of 32P-MBP with the above buffer reaction. After an overnight incubation at room tempera- at pH values in the same range. To calculate best-fitting ture, low-molecular mass compounds were removed with a parameters, all curves were analysed with the program 1-ml spin gel-filtration (Sephadex G25) column, equili- ORIGIN version 6.1 (OriginLab, Northampton, MA). brated with 5 mM HEPES/Tris (pH 7.0) and 100 mM KCl. The phosphatase assay mixture (90 ml total volume) contained 30 mM BTP/succinic acid (pH 6.6), 150 mM Sequential extraction of plasma membrane phosphatases KCl, 1 mM EDTA and 0.5 mg mlÀ1 32P-MBP. The To estimate if the phosphatases were bound to plasma mem- dephosphorylation reaction was started by adding 50 ml branes or were simply trapped inside the vesicles formed by of the phosphatase preparation, and a 20-ml aliquot was them (which would suggest that these phosphatases could be immediately withdrawn from this mixture and spotted soluble contaminants), proteins were sequentially extracted onto a 2 Â 2 cm Whatman P81 phosphocellulose paper ´ from plasma membranes following the method of Berczi and piece. After a 30-min incubation at room temperature, Møller (1998) with some modifications. Frozen plasma three 20-ml aliquots were withdrawn and spotted onto membranes (with 5 mg of protein) were thawed and diluted P81 paper pieces. These pieces were washed with 75 mM to 5 ml in 0.2 M sucrose, 20 mM Tris/MES (pH 7.0) and H3PO4 (3 Â 10 min), rinsed in ethanol (1 Â 5 min), air- 2 mM DTT, and centrifuged at 200 000 Â g at 4 C for dried, placed in vials with scintillation liquid and counted 30 min in a Beckman NVT90 rotor (Beckman Coulter, Full- for radioactivity. To calculate the phosphate released erton, CA). This first supernatant (SN1) contains trapped or from 32P-MBP, the radioactivity in the aliquots taken weakly bound proteins in plasma membrane vesicles that are at 30-min time was subtracted from that in the aliquot released by thawing. The pellet (P1) was re-suspended and taken at zero time. diluted in 5 ml of 20 mM Tris/MES (pH 7.0), 2 mM DTT and 0.3 M KI. After a 5-min incubation, this suspension was centrifuged as above. The supernatant (SN2, which contains Estimation of native molecular masses proteins bound to the outside of membrane vesicles by weak Native molecular masses of acid phosphatases were esti- ionic or hydrophobic forces) was collected. The pellet (P2) mated by gel filtration in the same Sephacryl S-300-HR was re-suspended in 5 ml of 0.2 M sucrose, 20 mM Tris/MES column used in the purification process. One-millilitre (pH 7.0), 2 mM DTT, and 0.05% (w/v) polyethylene glycol fractions were eluted with 30 mM HEPES, 1 mM DTT, hexadecyl ether (Brij 58 P). After a 15-min incubation, the 1 mM EDTA and 100 mM NaCl (pH 7.0) at a flow rate homogenate was centrifuged as above. The detergent Brij 58 of 0.25 ml minÀ1. Native relative molecular masses (Mr) P opens membrane vesicles and changes their sidedness were calculated from a plot of Kd (partition coefficient) (Johansson et al. 1995), so that this third supernatant against log (Mr) using the following protein standards: (SN3) contains mainly proteins trapped inside them. The urease hexamer (545 kDa), urease trimer (272 kDa), pellet (P3) was re-suspended and diluted (to 5 ml) in 20 mM b-amylase (200 kDa), alcohol dehydrogenase (150 kDa) Tris/MES (pH 7.0), 2 mM DTT and 0.3 M KI, to release the and carbonic anhydrase (29 kDa). proteins weakly bound to the inner surface of the membrane Physiol. Plant. 121, 2004 225
  4. 4. vesicles. After a 5-min incubation, this suspension was cen- in a single peak in the DEAE-Sepharose ion-exchange trifuged as above. The supernatant (SN4) was collected, and column. Fractions in this peak contained few polypep- the pellet (P4) was re-suspended in 5 ml of 0.2 M sucrose, tides, the most conspicuous among them was an 82-kDa 20 mM Tris/MES (pH 7.0), 2 mM DTT and 15 mM 3-[(3- one (Fig. 2). Because this polypeptide appeared in all cholamidopropyl)dimethylammonio]-1-propanesulphonate preparations with acid phosphatase activity, we attribu- (CHAPS). This suspension was incubated for 20 min and ted the phosphatase activity to it, even though we have centrifuged as indicated. The supernatant (SN5), which no further evidence to support this. contains solubilized membrane proteins, was collected. The 82-kDa phosphatase was purified 129 times The last pellet (P5) was re-suspended in 0.5 ml of 0.2 M from plasma membranes, to a specific activity of sucrose, 20 mM Tris/MES (pH 7.0) and 2 mM DTT. Acid 3726 nmol mg À1minÀ1 (Table 1). Its optimum pH value phosphatase activity in supernatants and P5 was measured was 5.6 Æ 0.2 (with pK1 and pK2 values of 4.5 and 6.6, with pNPP as substrate. Protein phosphatase activity was respectively). With pNPP, the 82-kDa acid phosphatase measured with 32P-MBP as described. presented Michaelis–Menten kinetics; the Km calculated for this substrate was 7.7 Æ 2.0 mM. The activity of the purified 82-kDa phosphatase was Protein determination assayed using a variety of phosphorylated substrates at a Protein concentration was determined by a modification 5-mM concentration. This phosphatase was able to dephos- of Stoscheck (1990) method, using bovine serum albumin phorylate phosphoaminoacids (Tyr-P, Ser-P and Thr-P), (BSA) as standard. ATP, GTP, PPi, a- and b-naphtyl phosphates, in addition to pNPP. The 82-kDa phosphatase was unspecific: its high- est (observed with sodium pyrophosphate) and lowest (with Ser-P) activities were only 113 and 92%, respectively, Results of those obtained with pNPP. This phosphatase was Partial purification of plasma membrane phosphatases unable to dephosphorylate 32P-MBP (data not shown). As with most acid phosphatases, the 82-kDa one was Acid phosphatase activity is conspicuous in beet root inhibited by 1 mM ammonium heptamolybdate (63%), plasma membranes. To partially purify some of these 1 mM sodium orthovanadate (55%) or 5 mM NaF phosphatases, the procedure described in Materials and (36%). This phosphatase was also strongly inhibited by methods was followed. As shown in Table 1, treating 1 mM CuSO4 and by 5 mM ZnSO4: 72 and 58%, res- plasma membranes with n-octylglucoside extracted about pectively. The only significant activation (82%) was 60% of the acid phosphatase activity in them. Extracted observed with 150 mM KCl. Divalent cations such as proteins were separated in a Sephacryl S-300-HR gel- Mg21, Mn21 (at 5 mM), Ca21 (1 mM), or chelators like filtration column, from which most of the acid phospha- EDTA, EGTA or HEDTA (at 5 mM) did not signifi- tase activity was recovered in a peak corresponding to cantly affect the activity of this acid phosphatase. proteins with native Mr about 85 kDa (Fig. 1A). This peak contained a low amount of protein (Fig. 1A) and showed few protein bands in SDS-PAGE (Fig. 2). In the The 36-kDa protein phosphatase following purification step (a DEAE-Sepharose column), The phosphatase peak eluting with about 0.5 M NaCl two phosphatase activity peaks were obtained (Fig. 1B). from the DEAE-sepharose column (Fig. 1B), unlike the The fraction with highest activity (using pNPP as sub- main acid phosphatase peak, presented phosphoprotein strate) showed an 82-kDa polypeptide (among others) in phosphatase activity. Fractions with this activity showed SDS-PAGE gels (Fig. 2). The minor activity peak (eluted two conspicuous polypeptides with Mr values of 65 and with about 0.5 M NaCl. Fig. 1B) presented two main poly- 36 kDa in SDS-PAGE (Fig. 2, lane 5). Using 32P-MBP as peptides with molecular masses of 36 and 65 kDa (Fig. 2). substrate, the highest specific activity obtained was 6845 pmol mg À1 minÀ1, 73.9 times the activity shown by plasma membranes (Table 2). This protein phosphatase The 82-kDa acid phosphatase presented maximal activity at pH 6.6 Æ 0.7, with pK1 and As seen in Fig. 1B, most of the phosphatase activity, pK2 values of 6.2 and 7.1, respectively. Activity versus 32 measured at pH 6.3 with pNPP as substrate, appeared P-MBP concentration curves showed a linear trend up Table 1. Purification of an 82-kDa acid phosphatase from beet root plasma membranes. Phosphatase activity was measured at pH 6.3 with 5 mM p-nitrophenyl phosphate as substrate. Protein Total activity Specific activity Purification Yield Step (mg) (nmol minÀ1) (nmol mgÀ1 minÀ1) (fold) (%) Plasma membranes 5.51 159.5 28.9 1.0 100 Octylglucoside extract 1.96 96.7 49.4 1.7 60.7 Gel filtration 0.0499 23.5 470.2 16.2 14.7 Ion-exchange 0.0018 6.71 3726 129 4.2 226 Physiol. Plant. 121, 2004
  5. 5. Fig. 1. Purification of two acid phosphatases from beet root plasma membranes. The acid phosphatase activity (*) and the protein concentration (*) of the eluted fractions are shown. Solid arrows Fig. 2. Protein patterns of fractions obtained in the purification of point to peaks with highest acid phosphatase activity. (A) Elution acid phosphatases. Proteins were separated by SDS-PAGE, and the profile of a Sephacryl S-300 HR chromatography column loaded with gels were stained with Coomassie blue. The electrophoresis gel was an n-octylglucoside extract of beet root plasma membranes. One- loaded with plasma membrane proteins (10 mg of protein mixed with millilitre fractions were collected. Broken-line arrows on the top point n-octylglucoside; lane 1), proteins extracted with octylglucoside to the fractions in which urease hexamer (545 kDa), urease trimer (5 mg; lane 2), the fraction with highest acid phosphatase activity (272 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa) from a Sephacryl S-300 column (2 mg; lane 3), a fraction from a and carbonic anhydrase (29 kDa) were eluted. (B) DEAE-Sepharose DEAE-Sepharose column with the 82-kDa acid phosphatase (1 mg; chromatography of the fraction with highest acid phosphatase activity lane 4), and a fraction with the 36-kDa phosphatase from the same from the Sephacryl column. Fraction volume was 0.5 ml. Proteins were column (1 mg; lane 5). Positions of molecular mass markers (values eluted with 0.1 M NaCl (1 ml), a linear 0.1–0.8 M NaCl gradient (4 ml) in kDa) are shown at the left margin. The solid arrow point to the and 1 M NaCl (1 ml), as indicated by the broken line without symbols. 36-kDa polypeptide (probable catalytic subunit of a PP2A phosphatase), the broken-line arrow to the 82-kDa acid phosphatase, and the dotted-line one to the 65-kDa polypeptide (probably, a regulatory subunit of a PP2A phosphatase). to 2 mg mlÀ1, which did not allow us to calculate any Km value for this substrate (data not shown). The effects of various possible inhibitors or activators The polypeptide composition of this phosphatase (Fig. 2, on this phosphatase are shown in Table 3. The best inhi- lane 5) also suggests it could be a PP2A: the 36-kDa bitor tested was okadaic acid, which caused an inhibition polypeptide could be the catalytic subunit and the 65- greater than 80% at a concentration of 2 nM (100% at kDa one, a ‘type-A’ regulatory subunit (Luan 2000). 2 mM). This phosphatase was also inhibited by 5 mM fluoride. As expected, it was not inhibited by vanadate Sequential extraction of plasma membrane phosphatases (1 mM), a known inhibitor of phospho-tyrosine phos- phatases. The high sensitivity to okadaic acid shown by During the preparation of plasma membranes, a high this phosphatase suggests it belongs to the PP2A class. acid-phosphatase activity (measured with pNPP) was Table 2. Purification of a phosphoprotein phosphatase from beet root plasma membranes. Phosphatase activity was measured at pH 6.6 with 0.5 mg mlÀ1 32P-MBP as substrate. Protein Total activity Specific activity Purification Yield Step (mg) (pmol minÀ1) (pmol mgÀ1 minÀ1) (fold) (%) Plasma membranes 7.58 701.7 92.6 1 100 Octylglucoside extract 1.98 232.7 117.3 1.27 33.2 Gel filtration 0.0544 83.01 1526 16.5 11.8 Ion-exchange 0.0013 9.15 6845 73.9 1.3 Physiol. Plant. 121, 2004 227
  6. 6. Table 3. Effects of various compounds on the activity of the purified 36-kDa acid phosphatase. Phosphatase activity was measured with 0.5 mM 32P-MBP as substrate, in media containing 150 mM KCl (with the exception of the NaCl-containing reaction medium). Activities are means of two measurements, and are expressed as a percentage of the activity with KCl only (2863 pmol mg À1 minÀ1). Activity Compound Concentration (%) Okadaic acid 2 nM 16.6 Okadaic acid 2 mM 0 Ammonium heptamolybdate 1 mM 55.1 Sodium orthovanadate 1 mM 99.3 NaF 5 mM 10.7 MgSO4 5 mM 31.2 MnSO4 5 mM 51.7 FeSO4 5 mM 80 KCl 150 mM 100 NaCl 150 mM 105 found in the supernatant containing soluble proteins. When this supernatant was processed in the same way as the octylglucoside extract from plasma membranes, the phosphatase activity was found in the same chromatogra- phy fractions (the fractions with native Mr about 85 kDa in the gel filtration column and the fractions eluting with 0.1 M NaCl in the ion-exchange one) where the plasma- Fig. 3. Sequential extraction of acid phosphatase activity in plasma membrane vesicles using pNPP (A) or 32P-MBP (B) as substrate. membrane phosphatases were found. Phosphoprotein Plasma membranes kept frozen were thawed and centrifuged as phosphatase activity was also found in the ion-exchange indicated in Materials and Methods to get the first supernatant column fractions eluting with about 0.5 M NaCl (data not (SN1). The resulting pellet was extracted with 0.3 M KI to get the proteins weakly bound to the outer surface of plasma membranes in shown). These observations suggested that the purified the second supernatant (SN2). The pellet was treated with 0.05% phosphatases from plasma membranes could be contam- (w/v) Brij 58 P to remove soluble proteins trapped inside vesicles ´ inating soluble proteins (Berczi and Asard 2003). (SN3). Membranes were then extracted with 0.3 M KI to get the To evaluate this possibility, phosphatases were proteins weakly bound to the inner surface in SN4. Finally, the resulting pellet was extracted with 15 mM CHAPS to get the extracted sequentially from plasma membranes as sug- solubilized membrane proteins in SN5, and the proteins resisting ´ gested by Berczi and Møller (1998). With this method, this extraction procedure in the last pellet (P5). Acid (A) and protein proteins that are released just by freezing and thawing (B) phosphatase activities were measured as described in Materials and Methods. the membrane vesicles are found in the first supernatant (SN1). Later on, proteins weakly bound to the outside of the vesicles (SN2), trapped inside them (SN3), weakly phosphatase activity, some (27%) protein phosphatase bound to the inside of the vesicles (SN4) and strongly activity was extracted with KI, which showed the pres- bound to membranes (SN5) are extracted in sequence. In ence of protein phosphatases that were weakly bound to Fig. 3A, it can be seen that a great proportion of the acid plasma membranes. phosphatase activity (measured with pNPP) was released by only opening the membrane vesicles (in SN1 and SN3), but a strong detergent was needed to extract a Phosphatases not extracted with octylglucoside significant percentage of it (in SN5). Less than 15% of the total phosphatase activity was released with the KI As shown in Table 1, about 40% of the acid phosphatase treatments (SN2 and SN4), which suggests that very few activity was not extracted from the plasma membranes acid phosphatases were weakly bound to plasma mem- with octylglucoside. Proteins in these membranes were brane surfaces. All these results showed that more than extracted with Triton X-114, and the phosphatases in 45% of the acid phosphatase activity found in plasma this new extract were purified partially by two ion- membranes could be due to soluble phosphatases exchange chromatography steps as described in Mater- trapped inside the membrane vesicles. ials and Methods. Several peaks with acid phosphatase The phosphoprotein phosphatase activity in the frac- activity were observed, and one acid phosphatase (opti- tions of this sequential extraction is shown in Fig. 3B. mum pH: 6.0) was purified to near homogeneity. Frac- About 45% of this activity was extracted from plasma tions with this phosphatase presented a protein band membranes with treatments that only open membrane with a Mr of 95 kDa in SDS-PAGE. This phosphatase vesicles, and a further 24% needed a strong detergent hydrolysed pNPP, but not 32P-MBP (data not shown). treatment to be extracted. However, unlike the acid We are actually characterizing it. 228 Physiol. Plant. 121, 2004
  7. 7. Discussion the maize and beet phosphatases, respectively) are of the same order of magnitude, and reflect their degrees of In this article, we show the presence of three acid phospha- purification. On the other hand, the protein yield of the tases in beet root plasma membranes. With one extraction 36-kDa phosphatase from beet root plasma membranes and two chromatography steps, two of them were purified. (1.3 mg) was much lower than that of the maize root After the first chromatographic step: a gel filtration, most of enzyme (25 mg). This low protein yield could make uncer- the activity appeared in a peak with proteins having mol- tain the estimation of specific activities. ecular masses about 85 kDa. In the second chromatographic ´ Berczi and Asard (2003) have pointed out that the step (ion-exchange), the fraction with highest specific activ- possibility of contamination of membrane preparations ity was obtained eluting the DEAE-Sepharose column with with soluble proteins is often overlooked. To find out a low salt concentration. This fraction contained a conspic- if the phosphatases in plasma membranes were only uous 82-kDa protein, a molecular mass similar to that soluble enzymes trapped inside membrane vesicles, the estimated in a gel-filtration column; which suggests that ´ sequential extraction procedure proposed by Berczi and this phosphatase is a monomer. This phosphatase presented Møller (1998) was followed. With this procedure, we an acid pH optimum: 5.4, and was able to hydrolyse many found that important fractions of the acid and protein substrates. Its Km value for pNPP (7.7 mM) is higher than phosphatase activities appear to be soluble contami- most of the values reported for acid phosphatases (Duff et al. nants. Particularly, it is possible that the 82-kDa phos- 1994). This phosphatase was inhibited most strongly by phatase were only a trapped soluble enzyme, since a Cu21 and Zn21 ions, and by phosphate analogues such as phosphatase with its chromatographic properties is molybdate and vanadate. The inhibitions by Cu21 and abundant in the soluble fraction obtained during the vanadate suggest the participation of SH groups in the preparation of plasma membranes, and almost no acid catalytic mechanism (Granjeiro et al. 1999). phosphatase activity weakly bound to these membranes The 82-kDa phosphatase is, most probably, different was found. The phosphatase activity strongly bound to from purple acid phosphatases (PAPs). PAPs from Ara- plasma membranes (solubilized with CHAPS) could be bidopsis thaliana (Coello 2002, Li et al. 2002) or from due to phosphatases not extracted by octylglucoside, Lupinus albus (Wasaki et al. 1999, 2000) have monomer such as the 95-kDa phosphatase. molecular masses lower than 82 kDa. Taking into In contrast, a significant fraction of the protein phos- account its molecular mass and its sensitivity to Cu21 phatase activity was found to be weakly bound to plasma and Zn21 ions, this phosphatase could be similar to those membranes. This suggests that soluble phosphatases isolated from white clover root cell walls, one of which could bind to, and dephosphorylate, plasma membrane presents a Mr of 113 kDa (Zhang and McManus 2000). phosphoproteins. In fact, the PP2A phosphatase from Although phosphoprotein phosphatase activities have maize, able to dephosphorylate the plasma membrane been described for some plant acid phosphatases (Duff H1-ATPase, was purified from a soluble (cytosolic) frac- et al. 1994, Gellatly et al. 1994), and the 82-kDa acid tion (Camoni et al. 2000). phosphatase purified in this work was able to dephos- In conclusion, three acid phosphatases were purified phorylate Ser-P, Thr-P and Tyr-P; it is, most probably, from beet root plasma membranes. The 82-kDa one not a protein phosphatase involved in signal transduc- shows biochemical properties similar to those observed tion, as it was unable to dephosphorylate MBP phos- with other plant acid phosphatases, and could be a solu- phorylated on Ser or Thr residues. ble contaminating enzyme trapped inside plasma mem- In contrast to the 82-kDa phosphatase, the 36-kDa brane vesicles. The 36-kDa phosphatase is probably a APase showed a very low specific activity with pNPP PP2A-type protein phosphatase. It will be interesting to as substrate. However, it was able to dephosphorylate study if it is able to dephosphorylate plasma membrane phospho-MBP. The fractions with highest specific activity phosphoproteins. Finally, the 95-kDa phosphatase, presented two protein bands in SDS-PAGE with mol- which was not extracted with octylglucoside, is worthy ecular masses of 65 and 36 kDa. This protein pattern, the of further characterization. sensitivity of this enzyme to nanomolar concentrations of okadaic acid, its insensitivity to vanadate and its lack of ´ Acknowledgements – We thank Barbara Lino for providing us with activation with divalent cations (PP2B and PP2C phospha- the kinase needed to phosphorylate MBP. This work was supported tases are activated by these cations), suggest that it could by a grant from Conacyt, Mexico. 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