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Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAM plant Aptenia cordifolia
Plant Science 164 (2003) 95 /102 www.elsevier.com/locate/plantsci
Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAM plant Aptenia cordifolia Marı´a Lorena Falcone Ferreyra, Carlos S. Andreo, Florencio E. Podesta´ * Centro de Estudios Fotosinte´ticos y Bioquı´micos, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina Received 18 June 2002; received in revised form 12 September 2002; accepted 30 September 2002
Abstract Two isoforms of NADP-dependent malic enzyme (NADP-ME) with the same molecular mass 72 kDa and different isoelectric points, 6.1 and 6.4, were found in crude extracts from the leaves of Aptenia cordifolia , a constitutive CAM plant. In the roots, only one isoform of 72 kDa was found, with a pI of 6.1. The isoform of pI 6.4 was partially purified from leaves to a final specific activity of 30.14 U mg 1, a value similar to the photosynthetic isozymes. This enzyme showed a native mass of 264 kDa, suggesting a homotetramer. An optimal pH of 7.3 and Km values for NADP and L-malate 13 mM and 1.1 mM, respectively, were determined. The enzymatic activities and the level of immunoreactive protein did not vary with the day/night cycle. The enzyme was strongly and competitively inhibited by oxaloacetate (OAA), L-aspartate and phosphoenolpyruvate (PEP) and to a lesser degree by citrate, suggesting that NADP-ME activity might be subject to metabolite control. At night, high levels of OAA, L-aspartate and citrate might inhibit NADP-ME, avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of L-malate. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Crassulacean acid metabolism; Aptenia cordifolia ; NADP-malic enzyme (EC 1.1.1.40); Isoforms
1. Introduction NADP-malic enzyme (NADP-ME; L-malate: NADP oxidoreductase, oxaloacetate decarboxylating; EC 1.1.1.40) catalyses the decarboxylation of L-malate to yield pyruvate, CO2 and NADPH [1]. NADP-ME is present in C3, C4 and CAM species, fulfilling different metabolic roles [2]. Accordingly, the different isoforms have been classified as photosynthetic and non-photosynthetic based on their function. Photosynthetic isoforms of the enzyme are present in some C4 and CAM plants.
Abbreviations: DPDS, dipyridyl disulfide; 2-ME, 2mercaptoethanol; NADP-ME, NADP-dependent malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PMSF, phenylmethylsulfonyl fluoride. * Corresponding author. Tel.: /54-341-437-1955; fax: /54-341437-0044 E-mail address: [email protected] (F.E. Podesta´).
The photosynthetic isoform of NADP-ME-type C4 plants is localized in bundle sheath chloroplasts and plays a key role as a decarboxylating enzyme that provides CO2 for fixation by RuBisCO. Similarly, certain CAM plants possess an NADP-ME involved in malate decarboxylation, although in this case it is found in the cytosol [3]. In addition, non-photosynthetic NADP-ME isoforms are widely distributed among different tissues in C3, C4 and CAM plants [1,2]. Its ubiquitous presence in many different tissues suggest a housekeeping function, acting as an anaplerotic enzyme that either provides NADPH and pyruvate to biosynthetic pathways or participates in the respiratory metabolism [1]. Although the C4 photosynthetic isoform has been extensively studied [4,5], information about NADP-ME in CAM plants is scarce. In Mesembryanthemum crystallinum , a facultative CAM plant, a cDNA for a cytosolic NADP-ME has been cloned [6]. The enzyme from this
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plant was purified from CAM-induced leaves and kinetically and structurally characterized [7]. Later, a different isoform, which possessed distinct antigenic properties and electrophoretic mobility in non-denaturing gels, was found in C3 mode leaves. Both isoforms have been reported to be differentially expressed during induction of CAM [8]. Aloe arborescens , an obligate monocot CAM plant of the phosphoenolpyruvate (PEP) carboxykinase type, possesses three isoforms of NADPME: a non-photosynthetic isoform, expressed at low level in all tissues [9]; a leaf-specific isozyme, which probably bears a photosynthetic role; and a root-specific form, which belongs to the non-photosynthetic group [10]. The purpose of the current study was to identify and characterize the isoform(s) of NADP-ME involved in CAM photosynthesis in Aptenia cordifolia, an obligate CAM plant categorized as ME type ([11]; Trı´podi and Podesta´, unpublished results) and investigate the possible activity control mechanisms of the enzyme during the night/day cycle.
2. Materials and methods 2.1. Plant material Plants of A. cordifolia were grown in a greenhouse with natural light supplemented with fluorescent light bulbs (Philips TLD 36W/79, The Netherlands) to achieve a regime of 16:8 h light:dark. Temperature was maintained at 25/15 8C day/night. Plants were watered every 5 days. Leaf samples (third to fifth leaf pair, numbered from the shoot tip) were taken after 5 h into the light period. After collection, the leaves were immediately frozen in liquid N2 and stored at /80 8C. 2.2. Reagents Substrates, phenyl-agarose, Q-Sepharose, Sephadex G-50, molecular weight markers for gel filtration and SDS-PAGE were from Sigma Chemical Co., St. Louis. All other reagents were of analytical grade purchased from Merck, Buenos Aires, Argentina. 2.3. Buffers used in NADP-ME purification Buffer A: 400 mM Tris /HCl, pH 8.2, 10 mM MgCl2, 5 mM EDTA, 20% (v/v) glycerol, 10 mM 2-mercaptoethanol (2-ME), 2.5% (w/v) polyvinylpyrrolidone, 0.5% (v/v) Triton X-100, 0.5% (w/v) ascorbate, 1 mM phenylmethylsulfonyl fluoride (PMSF). Buffer B: 50 mM Tris /HCl, pH 7.3, 5 mM MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol, 10 mM 2-ME. Buffer C: 50 mM Pi, pH 7.3, 2 mM 2-ME, 1 mM EDTA, 40% saturation (NH4)2SO4. Buffer D: 20 mM Tris /HCl, pH 7.3, 5% (v/
v) glycerol, 2 mM 2-ME, 1 mM EDTA. Buffer E: buffer B plus 100 mM NaCl and 1 mM NADP. Buffer F: 50 mM Tris /HCl, pH 7.3, 5 mM MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol, 10 mM 2-ME, 20 mM KNO3. 2.4. Purification of the enzyme As starting material, 200 g of A. cordifolia leaves were homogenized in a commercial blender at maximal speed in 2 vol of extraction buffer (buffer A) at 4 8C. The homogenate was filtered through cheesecloth and centrifuged at 9000/g for 20 min. The supernatant was brought to 30% saturation with solid ammonium sulfate, stirred for 30 min at 4 8C and subsequently centrifuged for 30 min at 9000/g . The supernatant fraction was made 70% saturated with ammonium sulfate, stirred for 40 min and centrifuged for 45 min at 15,000 /g. The resulting precipitate was resuspended in a minimal volume of buffer B and passed through a 200 ml column of Sephadex G-50 equilibrated with the same buffer. Fractions containing NADP-ME activity were collected and applied to a 10 ml column of Q-Sepharose previously equilibrated with buffer B. After washing with 3 vol of buffer B at 0.5 ml min 1, the enzyme was eluted by increasing the NaCl concentration linearly from 0 to 400 mM over 10 bed vol at 2 ml min 1. NADP-ME was eluted between 84 and 240 mM NaCl. These fractions were pooled and brought to 70% saturation with solid ammonium sulfate. The precipitate collected by centrifugation was dissolved in buffer C and loaded at 0.5 ml min1 into a 3 ml phenyl agarose column pre-equilibrated with buffer C. After washing the column, the elution was performed at 1 ml min1 by linearly increasing the amount of buffer D to 100%. The fractions containing enzyme activity were pooled, concentrated, diluted in buffer B and reconcentrated in Centricon 30 tubes (Amicon) before being applied to a 2 ml Matrex Blue (Amicon) affinity column equilibrated with buffer B. After washing the column, the elution was carried out at 1 ml min1 with buffer E. After concentration of pooled fractions, NADP-ME was fractionated into aliquots and stored at /80 8C until use. All chromatographic procedures were carried out at 4 8C using a Pharmacia FPLC system (AmershamPharmacia, Sweden). 2.5. Protein extraction Frozen leaves (:/1 g) were powdered with liquid N2 in a mortar and then homogenized with 2 vol of buffer A, 5 mg ml 1 chymostatin, 5 mg ml 1 leupeptin, 1 mM 4,4?-dipyridyl disulfide (DPDS) and 1 mM tosylphenylalanine chloromethylketone. The homogenates were centrifuged for 15 min at 4 8C in an Eppendorf microcentrifuge at maximum speed.
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For the assay of NADP-ME activity from A. cordifolia roots, total soluble protein of roots (2.5 ml buffer per gram tissue) was extracted using the procedure described for leaves with the following modifications: extraction buffer: 100 mM Tris /HCl, pH 7.5; 10 mM MgCl2, 1 mM EDTA, 20% (v/v) glycerol, 10 mM 2-ME, 1 mM PMSF, 1 mM DPDS, 1 mM p -aminobenzamidine; 5 mg ml1 chymostatin; 5 mg ml1 leupeptin. The homogenates were centrifuged for 15 min at 4 8C in an Eppendorf microcentrifuge at maximum speed. For activity assays, the crude extracts were desalted in a Sephadex G-50 column, previously equilibrated with 50 mM Tris /HCl, pH 7.3, 10 mM MgCl2 and 10% glycerol, according to Ref. [12]. For Western blots, total protein from A. cordifolia roots and leaves were extracted according to Ref. [13].
2.6. Assay of activity and kinetic studies NADP-ME activity was determined spectrophotometrically at 30 8C by following NADPH production at 340 nm. The standard assay medium contained 50 mM Tris /HCl, pH 7.3, 10 mM MgCl2, 0.5 mM NADP and 10 mM L-malate. Enzyme activity (1 U) is defined as the amount of enzyme resulting in the production of 1 mmol of NADPH per minute. Prior to activity measurements, the enzyme was desalted as described in Ref. [12]. Since metal-ligand chelates are not reactants for the NADPME reaction [14], the concentration of each substrate was corrected for the concentration of the metal bound substrates [15]. The following dissociation constants were used in the correction: Mg-malate 28.2 mM and Mg-NADP 19.1 mM [15]. For the pH-dependence studies, a mixture of 25 mM bis-Tris propane and 25 mM MES was used and substrate concentrations were saturated at the different pH value tested. Reactions were started by the addition of the enzyme preparation. The Km for L-malate was calculated by non-linear fitting of the initial rate to the Michaelis /Menten equation using a least squares-regression computer kinetic program [16]. Changes in absorbance were linear during the time taken for the assay and the activity was proportional to the amount of enzyme used. An initial concentration of 50 mM was used to determine the Km for NADP, while L-malate and Mg2 were kept constant at 10 mM. The reaction was left to proceed until completion and data were analyzed using the integrated Michaelis /Menten equation [17]. The enzyme was stable during the assay period and no product inhibition was recorded (NADPH). I50 values were obtained using the Job plot option included in the kinetics program [16]. Activity values are the average of three replicates differing by B/10%.
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2.7. Protein measurement Protein concentration was determined according to the method described in Ref. [18] using BSA as standard. 2.8. Native molecular weight determination The molecular weight of native NADP-ME was determined using a BioSep-SEC-S3000 column equilibrated with buffer F and connected to a Waters HPLC system. Column calibration was performed with the following standards: thyroglobulin (669 kDa); apoferritin (443 kDa); b-amylase (220 kDa); alcohol dehydrogenase (150 kDa), BSA (132 and 66 kDa) and carbonic anhydrase (29 kDa). The elution volume of the Blue Dextran ( /2000 kDa) was taken as the column void volume. 2.9. Electrophoresis, immunoblotting and isoelectrofocusing SDS-PAGE was performed according to Ref. [19]. The final acrylamide concentration was 10% (w/v) for the separating gel and 5% (w/v) for the stacking gel. The following standard proteins were included for the determination of the subunit molecular mass: myosin (205 kDa); b-galactosidase (116 kDa); phosphorylase b (97.4 kDa); BSA (66 kDa) and ovalbumin (45 kDa). Immunoblotting was performed according to Ref. [20]. Crude extracts from A. cordifolia or purified NADPME were run in denaturing polyacrylamide gels and transferred to nitrocellulose membranes. Affinity-purified anti-maize 62 kDa NADP-ME antibodies were used for detection [21]. Denaturing and native isoelectrofocusing were carried out in a Mini-Protean II cell, as outlined in Ref. [20]. Nondenaturing gels were electrophoresed at 200 V for 1.5 h and then at 400 V for another 1.5 h at 4 8C. The enzyme was detected using the assay for malic enzyme activity by incubating the gel in a solution containing 100 mM Tris /HCl, pH 7.3, 20 mM MgCl2, 20 mM L-malate, 1 mM NADP, 0.1 mg ml 1 nitroblue tetrazolium and 5 mg ml 1 phenazine methosulfate at 30 8C. Rinsing the gel with cold distilled water stopped the reaction. Denaturing IEF gels were subjected to Western blot, using affinitypurified anti-maize 62 kDa NADP-ME antibodies.
3. Results 3.1. Isoforms of NADP-ME in crude extracts of A. cordifolia Total protein was extracted from A. cordifolia leaves and roots and separated by SDS-PAGE. Immunoblots
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3.2. Purification of NADP-ME from leaves and structural features
Fig. 1. Western blot analysis of A. cordifolia NADP-ME. Lane 1: crude extracts of maize leaves (10 mg); lane 2: crude extracts of A. cordifolia leaves (30 mg); and lane 3: crude extracts of A. cordifolia root (30 mg). For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used.
of NADP-ME with anti-maize NADP-ME antibodies showed one immunoreactive band of 72 kDa (Fig. 1, lanes 2 and 3). Denaturing isoelectrofocusing of crude extracts from leaves showed the presence of two immunoreactive bands with pI of 6.4 and 6.1 (Fig. 2B, lane 2); root extracts contained a single immunoreactive band with a pI of 6.1 (Fig. 2C, lane 2), which is probably related to the isoform found in leaves with the same pI and molecular weight.
Fig. 2. (A) Native IEF of purified NADP-ME from A. cordifolia leaves; and (B) denaturing IEF analyzed by Western blot. For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used. Lane 1: purified A. cordifolia leaf NADP-ME; lane 2: crude extracts of A. cordifolia leaves. (C) Denaturing IEF analyzed by Western blot. Lane 1: purified NADP-ME from A. cordifolia leaves; lane 2: crude extracts from A. cordifolia roots.
The extractable activity of NADP-ME after 5 h of illumination was :/0.23 U g1 fresh weight. The enzyme was partially purified (:/513-fold) from A. cordifolia leaves to a final specific activity of 30.1 U mg 1, with a recovery of 9%. Partially purified NADPME subjected to SDS-PAGE showed a molecular mass of 72 kDa (Fig. 3A) and a Western blot of the purified enzyme showed one immunorreactive band of the same molecular mass as the enzyme from crude extracts, indicating that no degradation occurred during purification (Fig. 3B, lanes 1 and 3). The pI of the native A. cordifolia NADP-ME was 6.2 (Fig. 2A), while an IEF under denaturing conditions of the purified enzyme indicated a pI of 6.4 (Fig. 2B, C; lane 1). The difference in pI between the native and denatured enzyme could be due to a methodological difference between both types of gels or to a change in the net charge in the native enzyme due to oligomerization [22]. The native molecular mass, assessed by gel filtration, was 264 kDa (not shown). According to these data, the A. cordifolia leaf NADP-ME appears to be a homotetramer. 3.3. Kinetic characterization The NADP-ME from A. cordifolia leaves displays optimal activity at a pH around 7.3 (Fig. 4). The saturation curves obtained when the rate of the reaction was studied as a function of free L-malate concentration
Fig. 3. (A) Denaturing PAGE analysis of purified NADP-ME (5 mg). Numbers indicate standard molecular weight in kDa. (B) Immunodetection of A. cordifolia leaf NADP-ME. Lane 1: A. cordifolia leaf crude extract obtained under denaturing condition (30 mg); lane 2: Zea mays green leaf crude extract (10 mg); lane 3: NADP-ME purified from A. cordifolia leaf (6 mg). For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used.
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Table 1 Effect of metabolites on A. cordifolia NADP-ME activity Metabolite
Fig. 4. pH dependence on A. cordifolia leaf NADP-ME activity. Activity was determined in the presence of saturating concentrations of the substrates and cofactor (Mg2 ).
in the presence of saturating concentrations of NADP (0.5 mM) and Mg2 (10 mM) were hyperbolic, with a Km value of 1.109/0.08 mM. For NADP, the Km value determined applying the integrated Michaelis /Menten equation was 139/0.9 mM. No activity was recorded when NAD (up to 4 mM) was used as a cofactor. 3.4. Modulation of NADP-ME activity by metabolites An investigation was conducted to survey potential regulatory mechanisms of A. cordifolia NADP-ME. Activity was measured through the day/night cycle and the response of the purified enzyme to several metabolites was also tested. NADP-ME activity (in terms of units g1 fresh weight or units mg 1 Chl) in crude extracts from leaves collected in light and dark period, and Western blots for NADP-ME of these samples probed with purified anti-maize 62 kDa NADP-ME IgG showed no significant differences, suggesting that NADP-ME activity does not have a diurnal rhythm of activity or protein turnover (data no shown). Several metabolites were assayed as potential enzyme modulators at sub-saturating conditions (0.4 mM Lmalate). Table 1 summarizes the results obtained. No significant effects were observed with the following glycolytic and Krebs cycle intermediates: glucose-6phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, pyruvate and 2-ketoglutarate, L-glutamine, L-alanine, UDP and Pi. NADPME activity was slightly inhibited (28%) by 5 mM succinate, although this Krebs cycle intermediate has been reported to activate NADP-ME in Ricinus communis cotyledons, potato tuber, sugarcane leaves and various fruits [1,23]. ADP, ATP and AMP acted as potent inhibitors of the enzymatic activity when assayed at 2 mM. Citrate also inhibited NADP-ME, although to a lesser degree. A major effect on NADP-ME was caused by oxaloacetate (OAA, 70% inhibition, 2 mM), PEP (82%, 2 mM) and L-aspartate (94%, 10 mM), all of which strongly inhibited the activity of the NADP-malic enzyme. I50 values for OAA, PEP and L-aspartate were
Relative activity (%)
Succinate 2 mM 5 mM
81 72
2-Ketoglutarate 2 mM 5 mM
72 66
Citrate 2 mM 5 mM
64 39
PEP 1 mM 2 mM
47 18
OAA 1 mM 2 mM
52 30
ADP 2 mM
17
ATP 2 mM
17
AMP 2 mM
25
UDP-Glucose 2 mM
132
Phosphate 5 mM
65
Pyruvate 2 mM
78
Glutamate 10 mM 20 mM
100 138
Glutamine 10 mM 20 mM
94 62
L-Alanine 10 mM 20 mM
78 72
L-Aspartate 10 mM
6
Activity of a control without additions 100%.
1.069/0.06, 0.949/0.08 and 4.059/0.30 mM, respectively. Only UDP-glucose (2 mM) and glutamate (20 mM) had an activating effect on malic enzyme activity, albeit restricted to 132 and 138%, respectively. The saturation curves for reaction rates as a function of free L-malate at pH 7.3, in the presence of OAA (1 mM) and saturating concentration of NADP (0.5 mM) and Mg2 (10 mM) were also hyperbolic, but the Km value for L-malate was 2.059/0.10 mM. Little or no effect on the maximum velocity was observed. This result suggests a competitive-type inhibition of NADPME by OAA. The same result was obtained with Laspartate and PEP.
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4. Discussion CAM plants have been classified in two subgroups, ME and PEP carboxykinase-types, indicating the main decarboxylation system present [24] Actually, NADand NADP-ME are present in both subgroups, while PEP carboxykinase is absent from the first. Little effort has been directed to elucidating the properties of the enzyme in these plants, despite its crucial role in CAM and evidence that NADP-ME may differ in a number of important properties when compared to its counterpart from C3 and C4 plants, such as the intracellular location (cytosolic in CAM and chloroplastic in C4 and several C3 plants). So far, the only NADP-ME characterized from a CAM plant is that from M. crystallinum , a saltor drought-inducible CAM plant belonging to the Aizoaceae family [7]. A. cordifolia is a constitutive CAM plant which also belongs to the same family (formerly classified as M. cordifolium ) and lacks PEP carboxykinase, being thus classified among the MEsubgroup ([11], Trı´podi and Podesta´, unpublished results). We have partially purified a NADP-ME from leaves of A. cordifolia and fully characterized its kinetic, regulatory and physical properties. Results indicate that substantial differences exist between this enzyme and that from M. crystallinum leaves. NADP-ME from A. cordifolia leaves has been purified 513-fold to a final specific activity of 30.14 U mg 1. This relatively high value positions this enzyme as being photosynthetic (rather than housekeeping), along with those found in C3 leaves [4,5,7]. In this way, we suggest that the purified NADP-ME isoform from A. cordifolia might be involved in the decarboxylation of malic acid during deacidification in the light period. The optimum pH determined is consistent with the values reported for NADP-ME from C3 and CAM plants, that are more acidic than the values for C4 plants [4,5,7,25,26]. The difference in the optimum pH of photosynthetic NADP-ME between C4 and CAM species goes along with the fact mentioned above that they have a different subcellular location. Binding kinetics for NADP yielded values in the low micromolar range, typical for most malic enzymes studied so far, regardless of the species. This observation suggests that the high affinity and the binding site for NADP may have been conserved during evolution [2,27]. The hyperbolic response to increasing NADP contrasts with the sigmoidal kinetics demonstrated by the M. crystallinum enzyme [7]. Malate binding kinetics have been reported to present negative, positive or no cooperativity, depending on the source of the enzyme and the pH of the assay [1]. For NADP-ME from A . cordifolia, a hyperbolic response was obtained with a Km value of 1.109/0.08 mM at pH 7.3. This result, as a more acidic pH optimum, also suggests that this enzyme is more related to that of C3
and CAM plants than C4-type plants, which tend to present a greater affinity for malate [1,7]. With regard to the structure of A. cordifolia NADPME, SDS-PAGE followed by Western blot and native molecular weight determination indicate that A. cordifolia NADP-malic enzyme is a tetramer of identical, 72 kDa subunits. It is interesting to note that this is the first apparently photosynthetic NADP-ME isoform with this rather large subunit composition. Structural and functional characterization of NADP-ME in plants shows that the 72 kDa-subunit isoforms found in the leaves and roots of CAM, C4 or C3 plants or etiolated tissue of C4 plants belong to the non-photosynthetic, housekeeping subgroup of ME [10,28,29]. Photosynthetic isoforms of NADP-ME have been reported to range in size from 62 to 67 kDa in C4 plants [4,14] and from 64 to 65 kDa in CAM plants [7,10]. Immunoblot analysis of crude extracts from roots indicate that A. cordifolia also possess an isoform of 72 kDa. However, it differs from the 64 kDa NADP-ME isoform from M. crystallinum roots [8]. The root isozyme of A. cordifolia is probably related to one of the two bands that can be visualized in crude leaf extracts by denaturing IEF which possesses a pI of 6.1. NADP-ME activity or protein did not vary with the day/night cycle. Assays made with several metabolites as possible modulators on NADP-ME indicated a marked and competitive-type inhibition for OAA, PEP and Laspartate. The competitive-type inhibition of NADPME activity by OAA was also observed with the purified NADP-ME from M. crystallinum leaves in CAM mode [7]. Although the actual cytosolic concentration of OAA is not known, it has been reported that K . daigremontiana, K. pinnata, Ananas comosus [30,31] and Bryophyllum crenatum [32] accumulate OAA during the night. Moreover, in K. daigremontiana the levels of OAA and aspartate are maximum during the night and minimum in the light period [30,31]. Assuming that the same occurs in A. cordifolia and considering that (a) the leaf density is 1 g ml 1; (b) the cytosol represents only 5% of the total cellular volume [33]; (c) a concentration of 0.5 mg Chl g1 fresh weight; and (d) that :/70% of the leaf tissue is photosynthetic in K. daigremontiana leaves (our observations), we estimate that values of OAA and aspartate at night are around 4.3 and 13.6 mM, respectively. These concentrations suggest that these metabolites are probably effective in vivo as negative effectors of NADP-ME. Although PEP also presents competitive inhibition towards NADP-ME, the effect could be largely suppressed due to the high diurnal levels of L-malate during the decarboxylation phase (1000-fold greater than that of PEP) [30]. In addition, citrate also acts as an inhibitor of the NADP-ME activity, but causes less inhibition than L-aspartate, PEP and OAA. The accumulation of citrate in vacuoles at night has been reported in A. cordifolia , where the nocturnal
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accumulation of citrate (D-citrate) is 24% that of Dmalate. During drought, the ratio of D-citrate to Dmalate increases to almost 0.5 [11]. These results suggest that A. cordifolia NADP-ME might be subject to metabolic regulation. At night, the high levels of OAA and L-aspartate in the cytosol and, to a lesser degree citrate, might inhibit NADP-ME avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of L-malate. The relatively high degree of inhibition caused by ATP, ADP and AMP is expected to be due to the structural analogy of these compounds with NADP, but probably lacks any physiological meaning [34]. Further biochemical studies will be required to determine whether the inhibition pattern observed in vitro has relevance in the regulation in vivo. 4.1. Concluding remarks A NADP-ME has been purified from the CAM plant A. cordifolia . The enzyme presents novel characteristics for a photosynthetic isozyme, namely a higher molecular mass compared to its counterpart from C4 and other CAM plants and a regulatory mechanism based on metabolite control by key compounds present in the cytosol of CAM plants.
Acknowledgements This work was supported by grant BID 1201/OC-AR PICT No. 1-03397 from ANPCyT, Argentina. MLFF is a Fellow under this grant. CSA and FEP are Career Investigators of CONICET, Argentina. The authors would like to thank Dr M. Fabiana Drincovich for her assistance and advice.
References [1] G.E. Edwards, C.S. Andreo, NADP-malic enzyme from plants, Photosynth. Res. 31 (1992) 1845 /1857. [2] M.F. Drincovich, P. Casati, C.S. Andreo, NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways, FEBS Lett. 490 (2001) 1 /6. [3] K. Winter, J.G. Foster, G.E. Edwards, J.A.M. Holtum, Intracellular localization of enzymes of carbon metabolism in Mesembryanthemum crystallinum exhibiting C3 photosynthetic characteristics or exhibiting crassulacean acid metabolism, Plant Physiol. 69 (1982) 300 /309. [4] P. Casati, C.S. Andreo, G.E. Edwards, Characterization of NADP-malic enzyme from two species of Chenopodiaceae: Haloxylon persicum (C4) and Chenopodium album (C3), Phytochemistry 52 (1999) 985 /992.
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[5] M.F. Drincovich, A.A. Iglesias, C.S. Andreo, Interaction of divalent metal ions with the NADP-malic enzyme from maize leaves, Physiol. Plant 81 (1991) 462 /466. [6] J.C. Cushman, Characterization and expression of a NADP-malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, M. crystallinum , Eur. J. Biochem. 208 (1992) 259 /266. [7] K. Saitou, W. Agata, M. Asakura, F. Kubota, Structural and kinetic properties of NADP-malic enzyme from the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum L, Plant Cell Physiol. 33 (1992) 595 /600. [8] K. Saitou, W. Agata, Y. Masui, M. Asakura, F. Kubota, Isoforms of NADP-malic enzyme from M. crystallinum that are involved in C3 photosynthesis and crassulacean acid metabolism, Plant Cell Physiol. 35 (1994) 1165 /1171. [9] H. Honda, H. Shimada, H. Akagi, Isolation of cDNA for an NADP-malic enzyme from Aloe arborescens , DNA Res. 4 (1997) 397 /400. [10] H. Honda, H. Akagi, H. Shimada, An isozyme of the NADPmalic enzyme of a CAM plant, Aloe arborescens , with variation on conservative amino acid residues, Gene 243 (2000) 85 /92. [11] W.B. Herppich, K. Peckmann, Responses of gas exchange, photosynthesis, nocturnal acid accumulation and water relations of Aptenia cordifolia to short-term drought and rewatering, J. Plant Physiol. 150 (1997) 467 /474. [12] H.S. Penefsky, Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase, J. Biol. Chem. 252 (1977) 2891 / 2899. [13] F.S. Wu, M.Y. Wang, Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues, Anal. Biochem. 139 (1984) 100 /103. [14] A.A. Iglesias, C.S. Andreo, Kinetic and structural properties of NADP-malic enzyme from sugar cane leaves, Plant Physiol. 92 (1990) 66 /72. [15] S.D. Grover, P.F. Canellas, R.T. Wedding, Purification of NAD malic enzyme from potato and investigation of some physiological and kinetic properties, Arch. Biochem. Biophys. 209 (1981) 396 / 407. [16] S.P.J. Brooks, A simple computer program with statistical tests for the analysis of enzyme kinetics, Biotechniques 13 (1992) 906 / 911. [17] M. Dixon, E.C. Webb, Enzymes, Academic Press, New York, 1979, pp. 65 /67. [18] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265 /275. [19] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 /685. [20] D.M. Bollag, S.J. Edelstein, Protein Methods, Wiley-Liss, New York, 1991, pp. 1 /229. [21] V.G. Maurino, M.F. Drincovich, C.S. Andreo, NADP-malic enzyme isoforms in maize leaves, Biochem. Mol. Biol. Int. 38 (1996) 239 /250. [22] C.P. Spampinato, C.P. Casati, C.S. Andreo, Factors affecting the oligomeric state of NADP-ME from wheat and maize tissue: a chemical crosslinking study, Biochim. Biophys. Acta 1383 (1998) 245 /252. [23] S.L. Colombo, C.S. Andreo, F.E. Podesta´, Carbon metabolism in germinating Ricinus communis cotyledons. Purification, characterization and developmental profile of NADP-dependent malic enzyme, Physiol. Plant 101 (1997) 821 /826. [24] P. Dittrich, W.H. Campbell, C.C. Black, Phosphoenolpiruvate carboxykinase in plants exhibiting crassulacean acid metabolism, Plant Physiol. 52 (1973) 357 /361.
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[25] P. Casati, C.P. Spampinato, C.S. Andreo, Characteristics and physiological function of NADP-malic enzyme from wheat, Plant Cell Physiol. 38 (1997) 928 /934. [26] P. Casati, A. Fresco, C.S. Andreo, M.F. Drincovich, An intermediate form of NADP-malic enzyme from the C3 /C4 intermediate species Flaveria floridana , Plant Sci. 147 (1999) 101 /109. [27] B.A. Rothermel, T. Nelson, Primary structure of the maize NADP-dependent malic enzyme, J. Biol. Chem. 264 (1989) 19857 /19892. [28] M.F. Drincovich, P. Casati, C.S. Andreo, V. Franceschi, G.E. Edwards, M.S.B. Ku, Evolution of C4 photosynthesis in Flaveria species: isoforms of NADP-malic enzyme, Plant Physiol. 117 (1998) 733 /744. [29] V.G. Maurino, M.F. Drincovich, P. Casati, C.S. Andreo, C.S.G.E. Edwards, M.S.B. Ku, S.K. Gupta, V.R. Franceschi, NADP-malic enzyme: immunolocalization in different tissues of the C4 plant maize and the C3 plant wheat, J. Exp. Bot. 48 (1997) 799 /811.
[30] W.K. Kenyon, A.S. Holaday, C.C. Black, Diurnal changes in metabolite levels and crassulacean acid metabolism in Kalanchoe¨ daigremontiana leaves, Plant Physiol. 68 (1981) 1002 /1007. [31] L.-S. Chen, Q. Lin, A. Nose, A comparative study on diurnal changes in metabolite levels in the leaves of three Crassulacean acid metabolism (CAM) species, Ananas comosus , Kalanchoe¨ daigremontiana and K. pinnata , J. Exp. Bot. 53 (2002) 341 /350. [32] T.R. Milburn, D.J. Pearson, N.A. Ndegwe, Crassulacean acid metabolism under natural tropical conditions, New Phytol. 67 (1968) 883 /897. [33] J.A.C. Smith, J. Ingram, M.S. Tsiantis, B.J. Barkla, D.M. Bartholomew, M. Bettey, O. Pantoja, A.J. Pennington, Transport across the vacuolar membrane in CAM plants, in: K. Winter, J.A.C. Smith (Eds.), Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution, Springer-Verlag, Berlin, 1996, pp. 53 /71. [34] C.P. Spampinato, P. Paneth, M.H. O’Leary, C.S. Andreo, Analogues of NADP as inhibitors and coenzymes for NADP malic enzyme from maize leaves, Photosynth. Res. 28 (1991) 69 / 76.
Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAM plant Aptenia cordifolia Marı´a Lorena Falcone Ferreyra, Carlos S. Andreo, Florencio E. Podesta´ * Centro de Estudios Fotosinte´ticos y Bioquı´micos, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina Received 18 June 2002; received in revised form 12 September 2002; accepted 30 September 2002
Abstract Two isoforms of NADP-dependent malic enzyme (NADP-ME) with the same molecular mass 72 kDa and different isoelectric points, 6.1 and 6.4, were found in crude extracts from the leaves of Aptenia cordifolia , a constitutive CAM plant. In the roots, only one isoform of 72 kDa was found, with a pI of 6.1. The isoform of pI 6.4 was partially purified from leaves to a final specific activity of 30.14 U mg 1, a value similar to the photosynthetic isozymes. This enzyme showed a native mass of 264 kDa, suggesting a homotetramer. An optimal pH of 7.3 and Km values for NADP and L-malate 13 mM and 1.1 mM, respectively, were determined. The enzymatic activities and the level of immunoreactive protein did not vary with the day/night cycle. The enzyme was strongly and competitively inhibited by oxaloacetate (OAA), L-aspartate and phosphoenolpyruvate (PEP) and to a lesser degree by citrate, suggesting that NADP-ME activity might be subject to metabolite control. At night, high levels of OAA, L-aspartate and citrate might inhibit NADP-ME, avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of L-malate. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Crassulacean acid metabolism; Aptenia cordifolia ; NADP-malic enzyme (EC 1.1.1.40); Isoforms
1. Introduction NADP-malic enzyme (NADP-ME; L-malate: NADP oxidoreductase, oxaloacetate decarboxylating; EC 1.1.1.40) catalyses the decarboxylation of L-malate to yield pyruvate, CO2 and NADPH [1]. NADP-ME is present in C3, C4 and CAM species, fulfilling different metabolic roles [2]. Accordingly, the different isoforms have been classified as photosynthetic and non-photosynthetic based on their function. Photosynthetic isoforms of the enzyme are present in some C4 and CAM plants.
Abbreviations: DPDS, dipyridyl disulfide; 2-ME, 2mercaptoethanol; NADP-ME, NADP-dependent malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PMSF, phenylmethylsulfonyl fluoride. * Corresponding author. Tel.: /54-341-437-1955; fax: /54-341437-0044 E-mail address: [email protected] (F.E. Podesta´).
The photosynthetic isoform of NADP-ME-type C4 plants is localized in bundle sheath chloroplasts and plays a key role as a decarboxylating enzyme that provides CO2 for fixation by RuBisCO. Similarly, certain CAM plants possess an NADP-ME involved in malate decarboxylation, although in this case it is found in the cytosol [3]. In addition, non-photosynthetic NADP-ME isoforms are widely distributed among different tissues in C3, C4 and CAM plants [1,2]. Its ubiquitous presence in many different tissues suggest a housekeeping function, acting as an anaplerotic enzyme that either provides NADPH and pyruvate to biosynthetic pathways or participates in the respiratory metabolism [1]. Although the C4 photosynthetic isoform has been extensively studied [4,5], information about NADP-ME in CAM plants is scarce. In Mesembryanthemum crystallinum , a facultative CAM plant, a cDNA for a cytosolic NADP-ME has been cloned [6]. The enzyme from this
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 3 4 0 - 0
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plant was purified from CAM-induced leaves and kinetically and structurally characterized [7]. Later, a different isoform, which possessed distinct antigenic properties and electrophoretic mobility in non-denaturing gels, was found in C3 mode leaves. Both isoforms have been reported to be differentially expressed during induction of CAM [8]. Aloe arborescens , an obligate monocot CAM plant of the phosphoenolpyruvate (PEP) carboxykinase type, possesses three isoforms of NADPME: a non-photosynthetic isoform, expressed at low level in all tissues [9]; a leaf-specific isozyme, which probably bears a photosynthetic role; and a root-specific form, which belongs to the non-photosynthetic group [10]. The purpose of the current study was to identify and characterize the isoform(s) of NADP-ME involved in CAM photosynthesis in Aptenia cordifolia, an obligate CAM plant categorized as ME type ([11]; Trı´podi and Podesta´, unpublished results) and investigate the possible activity control mechanisms of the enzyme during the night/day cycle.
2. Materials and methods 2.1. Plant material Plants of A. cordifolia were grown in a greenhouse with natural light supplemented with fluorescent light bulbs (Philips TLD 36W/79, The Netherlands) to achieve a regime of 16:8 h light:dark. Temperature was maintained at 25/15 8C day/night. Plants were watered every 5 days. Leaf samples (third to fifth leaf pair, numbered from the shoot tip) were taken after 5 h into the light period. After collection, the leaves were immediately frozen in liquid N2 and stored at /80 8C. 2.2. Reagents Substrates, phenyl-agarose, Q-Sepharose, Sephadex G-50, molecular weight markers for gel filtration and SDS-PAGE were from Sigma Chemical Co., St. Louis. All other reagents were of analytical grade purchased from Merck, Buenos Aires, Argentina. 2.3. Buffers used in NADP-ME purification Buffer A: 400 mM Tris /HCl, pH 8.2, 10 mM MgCl2, 5 mM EDTA, 20% (v/v) glycerol, 10 mM 2-mercaptoethanol (2-ME), 2.5% (w/v) polyvinylpyrrolidone, 0.5% (v/v) Triton X-100, 0.5% (w/v) ascorbate, 1 mM phenylmethylsulfonyl fluoride (PMSF). Buffer B: 50 mM Tris /HCl, pH 7.3, 5 mM MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol, 10 mM 2-ME. Buffer C: 50 mM Pi, pH 7.3, 2 mM 2-ME, 1 mM EDTA, 40% saturation (NH4)2SO4. Buffer D: 20 mM Tris /HCl, pH 7.3, 5% (v/
v) glycerol, 2 mM 2-ME, 1 mM EDTA. Buffer E: buffer B plus 100 mM NaCl and 1 mM NADP. Buffer F: 50 mM Tris /HCl, pH 7.3, 5 mM MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol, 10 mM 2-ME, 20 mM KNO3. 2.4. Purification of the enzyme As starting material, 200 g of A. cordifolia leaves were homogenized in a commercial blender at maximal speed in 2 vol of extraction buffer (buffer A) at 4 8C. The homogenate was filtered through cheesecloth and centrifuged at 9000/g for 20 min. The supernatant was brought to 30% saturation with solid ammonium sulfate, stirred for 30 min at 4 8C and subsequently centrifuged for 30 min at 9000/g . The supernatant fraction was made 70% saturated with ammonium sulfate, stirred for 40 min and centrifuged for 45 min at 15,000 /g. The resulting precipitate was resuspended in a minimal volume of buffer B and passed through a 200 ml column of Sephadex G-50 equilibrated with the same buffer. Fractions containing NADP-ME activity were collected and applied to a 10 ml column of Q-Sepharose previously equilibrated with buffer B. After washing with 3 vol of buffer B at 0.5 ml min 1, the enzyme was eluted by increasing the NaCl concentration linearly from 0 to 400 mM over 10 bed vol at 2 ml min 1. NADP-ME was eluted between 84 and 240 mM NaCl. These fractions were pooled and brought to 70% saturation with solid ammonium sulfate. The precipitate collected by centrifugation was dissolved in buffer C and loaded at 0.5 ml min1 into a 3 ml phenyl agarose column pre-equilibrated with buffer C. After washing the column, the elution was performed at 1 ml min1 by linearly increasing the amount of buffer D to 100%. The fractions containing enzyme activity were pooled, concentrated, diluted in buffer B and reconcentrated in Centricon 30 tubes (Amicon) before being applied to a 2 ml Matrex Blue (Amicon) affinity column equilibrated with buffer B. After washing the column, the elution was carried out at 1 ml min1 with buffer E. After concentration of pooled fractions, NADP-ME was fractionated into aliquots and stored at /80 8C until use. All chromatographic procedures were carried out at 4 8C using a Pharmacia FPLC system (AmershamPharmacia, Sweden). 2.5. Protein extraction Frozen leaves (:/1 g) were powdered with liquid N2 in a mortar and then homogenized with 2 vol of buffer A, 5 mg ml 1 chymostatin, 5 mg ml 1 leupeptin, 1 mM 4,4?-dipyridyl disulfide (DPDS) and 1 mM tosylphenylalanine chloromethylketone. The homogenates were centrifuged for 15 min at 4 8C in an Eppendorf microcentrifuge at maximum speed.
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For the assay of NADP-ME activity from A. cordifolia roots, total soluble protein of roots (2.5 ml buffer per gram tissue) was extracted using the procedure described for leaves with the following modifications: extraction buffer: 100 mM Tris /HCl, pH 7.5; 10 mM MgCl2, 1 mM EDTA, 20% (v/v) glycerol, 10 mM 2-ME, 1 mM PMSF, 1 mM DPDS, 1 mM p -aminobenzamidine; 5 mg ml1 chymostatin; 5 mg ml1 leupeptin. The homogenates were centrifuged for 15 min at 4 8C in an Eppendorf microcentrifuge at maximum speed. For activity assays, the crude extracts were desalted in a Sephadex G-50 column, previously equilibrated with 50 mM Tris /HCl, pH 7.3, 10 mM MgCl2 and 10% glycerol, according to Ref. [12]. For Western blots, total protein from A. cordifolia roots and leaves were extracted according to Ref. [13].
2.6. Assay of activity and kinetic studies NADP-ME activity was determined spectrophotometrically at 30 8C by following NADPH production at 340 nm. The standard assay medium contained 50 mM Tris /HCl, pH 7.3, 10 mM MgCl2, 0.5 mM NADP and 10 mM L-malate. Enzyme activity (1 U) is defined as the amount of enzyme resulting in the production of 1 mmol of NADPH per minute. Prior to activity measurements, the enzyme was desalted as described in Ref. [12]. Since metal-ligand chelates are not reactants for the NADPME reaction [14], the concentration of each substrate was corrected for the concentration of the metal bound substrates [15]. The following dissociation constants were used in the correction: Mg-malate 28.2 mM and Mg-NADP 19.1 mM [15]. For the pH-dependence studies, a mixture of 25 mM bis-Tris propane and 25 mM MES was used and substrate concentrations were saturated at the different pH value tested. Reactions were started by the addition of the enzyme preparation. The Km for L-malate was calculated by non-linear fitting of the initial rate to the Michaelis /Menten equation using a least squares-regression computer kinetic program [16]. Changes in absorbance were linear during the time taken for the assay and the activity was proportional to the amount of enzyme used. An initial concentration of 50 mM was used to determine the Km for NADP, while L-malate and Mg2 were kept constant at 10 mM. The reaction was left to proceed until completion and data were analyzed using the integrated Michaelis /Menten equation [17]. The enzyme was stable during the assay period and no product inhibition was recorded (NADPH). I50 values were obtained using the Job plot option included in the kinetics program [16]. Activity values are the average of three replicates differing by B/10%.
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2.7. Protein measurement Protein concentration was determined according to the method described in Ref. [18] using BSA as standard. 2.8. Native molecular weight determination The molecular weight of native NADP-ME was determined using a BioSep-SEC-S3000 column equilibrated with buffer F and connected to a Waters HPLC system. Column calibration was performed with the following standards: thyroglobulin (669 kDa); apoferritin (443 kDa); b-amylase (220 kDa); alcohol dehydrogenase (150 kDa), BSA (132 and 66 kDa) and carbonic anhydrase (29 kDa). The elution volume of the Blue Dextran ( /2000 kDa) was taken as the column void volume. 2.9. Electrophoresis, immunoblotting and isoelectrofocusing SDS-PAGE was performed according to Ref. [19]. The final acrylamide concentration was 10% (w/v) for the separating gel and 5% (w/v) for the stacking gel. The following standard proteins were included for the determination of the subunit molecular mass: myosin (205 kDa); b-galactosidase (116 kDa); phosphorylase b (97.4 kDa); BSA (66 kDa) and ovalbumin (45 kDa). Immunoblotting was performed according to Ref. [20]. Crude extracts from A. cordifolia or purified NADPME were run in denaturing polyacrylamide gels and transferred to nitrocellulose membranes. Affinity-purified anti-maize 62 kDa NADP-ME antibodies were used for detection [21]. Denaturing and native isoelectrofocusing were carried out in a Mini-Protean II cell, as outlined in Ref. [20]. Nondenaturing gels were electrophoresed at 200 V for 1.5 h and then at 400 V for another 1.5 h at 4 8C. The enzyme was detected using the assay for malic enzyme activity by incubating the gel in a solution containing 100 mM Tris /HCl, pH 7.3, 20 mM MgCl2, 20 mM L-malate, 1 mM NADP, 0.1 mg ml 1 nitroblue tetrazolium and 5 mg ml 1 phenazine methosulfate at 30 8C. Rinsing the gel with cold distilled water stopped the reaction. Denaturing IEF gels were subjected to Western blot, using affinitypurified anti-maize 62 kDa NADP-ME antibodies.
3. Results 3.1. Isoforms of NADP-ME in crude extracts of A. cordifolia Total protein was extracted from A. cordifolia leaves and roots and separated by SDS-PAGE. Immunoblots
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3.2. Purification of NADP-ME from leaves and structural features
Fig. 1. Western blot analysis of A. cordifolia NADP-ME. Lane 1: crude extracts of maize leaves (10 mg); lane 2: crude extracts of A. cordifolia leaves (30 mg); and lane 3: crude extracts of A. cordifolia root (30 mg). For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used.
of NADP-ME with anti-maize NADP-ME antibodies showed one immunoreactive band of 72 kDa (Fig. 1, lanes 2 and 3). Denaturing isoelectrofocusing of crude extracts from leaves showed the presence of two immunoreactive bands with pI of 6.4 and 6.1 (Fig. 2B, lane 2); root extracts contained a single immunoreactive band with a pI of 6.1 (Fig. 2C, lane 2), which is probably related to the isoform found in leaves with the same pI and molecular weight.
Fig. 2. (A) Native IEF of purified NADP-ME from A. cordifolia leaves; and (B) denaturing IEF analyzed by Western blot. For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used. Lane 1: purified A. cordifolia leaf NADP-ME; lane 2: crude extracts of A. cordifolia leaves. (C) Denaturing IEF analyzed by Western blot. Lane 1: purified NADP-ME from A. cordifolia leaves; lane 2: crude extracts from A. cordifolia roots.
The extractable activity of NADP-ME after 5 h of illumination was :/0.23 U g1 fresh weight. The enzyme was partially purified (:/513-fold) from A. cordifolia leaves to a final specific activity of 30.1 U mg 1, with a recovery of 9%. Partially purified NADPME subjected to SDS-PAGE showed a molecular mass of 72 kDa (Fig. 3A) and a Western blot of the purified enzyme showed one immunorreactive band of the same molecular mass as the enzyme from crude extracts, indicating that no degradation occurred during purification (Fig. 3B, lanes 1 and 3). The pI of the native A. cordifolia NADP-ME was 6.2 (Fig. 2A), while an IEF under denaturing conditions of the purified enzyme indicated a pI of 6.4 (Fig. 2B, C; lane 1). The difference in pI between the native and denatured enzyme could be due to a methodological difference between both types of gels or to a change in the net charge in the native enzyme due to oligomerization [22]. The native molecular mass, assessed by gel filtration, was 264 kDa (not shown). According to these data, the A. cordifolia leaf NADP-ME appears to be a homotetramer. 3.3. Kinetic characterization The NADP-ME from A. cordifolia leaves displays optimal activity at a pH around 7.3 (Fig. 4). The saturation curves obtained when the rate of the reaction was studied as a function of free L-malate concentration
Fig. 3. (A) Denaturing PAGE analysis of purified NADP-ME (5 mg). Numbers indicate standard molecular weight in kDa. (B) Immunodetection of A. cordifolia leaf NADP-ME. Lane 1: A. cordifolia leaf crude extract obtained under denaturing condition (30 mg); lane 2: Zea mays green leaf crude extract (10 mg); lane 3: NADP-ME purified from A. cordifolia leaf (6 mg). For immunological detection of NADP-ME, IgG purified from antiserum raised against the 62 kDa subunit of NADP-ME from maize green leaves was used.
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Table 1 Effect of metabolites on A. cordifolia NADP-ME activity Metabolite
Fig. 4. pH dependence on A. cordifolia leaf NADP-ME activity. Activity was determined in the presence of saturating concentrations of the substrates and cofactor (Mg2 ).
in the presence of saturating concentrations of NADP (0.5 mM) and Mg2 (10 mM) were hyperbolic, with a Km value of 1.109/0.08 mM. For NADP, the Km value determined applying the integrated Michaelis /Menten equation was 139/0.9 mM. No activity was recorded when NAD (up to 4 mM) was used as a cofactor. 3.4. Modulation of NADP-ME activity by metabolites An investigation was conducted to survey potential regulatory mechanisms of A. cordifolia NADP-ME. Activity was measured through the day/night cycle and the response of the purified enzyme to several metabolites was also tested. NADP-ME activity (in terms of units g1 fresh weight or units mg 1 Chl) in crude extracts from leaves collected in light and dark period, and Western blots for NADP-ME of these samples probed with purified anti-maize 62 kDa NADP-ME IgG showed no significant differences, suggesting that NADP-ME activity does not have a diurnal rhythm of activity or protein turnover (data no shown). Several metabolites were assayed as potential enzyme modulators at sub-saturating conditions (0.4 mM Lmalate). Table 1 summarizes the results obtained. No significant effects were observed with the following glycolytic and Krebs cycle intermediates: glucose-6phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, pyruvate and 2-ketoglutarate, L-glutamine, L-alanine, UDP and Pi. NADPME activity was slightly inhibited (28%) by 5 mM succinate, although this Krebs cycle intermediate has been reported to activate NADP-ME in Ricinus communis cotyledons, potato tuber, sugarcane leaves and various fruits [1,23]. ADP, ATP and AMP acted as potent inhibitors of the enzymatic activity when assayed at 2 mM. Citrate also inhibited NADP-ME, although to a lesser degree. A major effect on NADP-ME was caused by oxaloacetate (OAA, 70% inhibition, 2 mM), PEP (82%, 2 mM) and L-aspartate (94%, 10 mM), all of which strongly inhibited the activity of the NADP-malic enzyme. I50 values for OAA, PEP and L-aspartate were
Relative activity (%)
Succinate 2 mM 5 mM
81 72
2-Ketoglutarate 2 mM 5 mM
72 66
Citrate 2 mM 5 mM
64 39
PEP 1 mM 2 mM
47 18
OAA 1 mM 2 mM
52 30
ADP 2 mM
17
ATP 2 mM
17
AMP 2 mM
25
UDP-Glucose 2 mM
132
Phosphate 5 mM
65
Pyruvate 2 mM
78
Glutamate 10 mM 20 mM
100 138
Glutamine 10 mM 20 mM
94 62
L-Alanine 10 mM 20 mM
78 72
L-Aspartate 10 mM
6
Activity of a control without additions 100%.
1.069/0.06, 0.949/0.08 and 4.059/0.30 mM, respectively. Only UDP-glucose (2 mM) and glutamate (20 mM) had an activating effect on malic enzyme activity, albeit restricted to 132 and 138%, respectively. The saturation curves for reaction rates as a function of free L-malate at pH 7.3, in the presence of OAA (1 mM) and saturating concentration of NADP (0.5 mM) and Mg2 (10 mM) were also hyperbolic, but the Km value for L-malate was 2.059/0.10 mM. Little or no effect on the maximum velocity was observed. This result suggests a competitive-type inhibition of NADPME by OAA. The same result was obtained with Laspartate and PEP.
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4. Discussion CAM plants have been classified in two subgroups, ME and PEP carboxykinase-types, indicating the main decarboxylation system present [24] Actually, NADand NADP-ME are present in both subgroups, while PEP carboxykinase is absent from the first. Little effort has been directed to elucidating the properties of the enzyme in these plants, despite its crucial role in CAM and evidence that NADP-ME may differ in a number of important properties when compared to its counterpart from C3 and C4 plants, such as the intracellular location (cytosolic in CAM and chloroplastic in C4 and several C3 plants). So far, the only NADP-ME characterized from a CAM plant is that from M. crystallinum , a saltor drought-inducible CAM plant belonging to the Aizoaceae family [7]. A. cordifolia is a constitutive CAM plant which also belongs to the same family (formerly classified as M. cordifolium ) and lacks PEP carboxykinase, being thus classified among the MEsubgroup ([11], Trı´podi and Podesta´, unpublished results). We have partially purified a NADP-ME from leaves of A. cordifolia and fully characterized its kinetic, regulatory and physical properties. Results indicate that substantial differences exist between this enzyme and that from M. crystallinum leaves. NADP-ME from A. cordifolia leaves has been purified 513-fold to a final specific activity of 30.14 U mg 1. This relatively high value positions this enzyme as being photosynthetic (rather than housekeeping), along with those found in C3 leaves [4,5,7]. In this way, we suggest that the purified NADP-ME isoform from A. cordifolia might be involved in the decarboxylation of malic acid during deacidification in the light period. The optimum pH determined is consistent with the values reported for NADP-ME from C3 and CAM plants, that are more acidic than the values for C4 plants [4,5,7,25,26]. The difference in the optimum pH of photosynthetic NADP-ME between C4 and CAM species goes along with the fact mentioned above that they have a different subcellular location. Binding kinetics for NADP yielded values in the low micromolar range, typical for most malic enzymes studied so far, regardless of the species. This observation suggests that the high affinity and the binding site for NADP may have been conserved during evolution [2,27]. The hyperbolic response to increasing NADP contrasts with the sigmoidal kinetics demonstrated by the M. crystallinum enzyme [7]. Malate binding kinetics have been reported to present negative, positive or no cooperativity, depending on the source of the enzyme and the pH of the assay [1]. For NADP-ME from A . cordifolia, a hyperbolic response was obtained with a Km value of 1.109/0.08 mM at pH 7.3. This result, as a more acidic pH optimum, also suggests that this enzyme is more related to that of C3
and CAM plants than C4-type plants, which tend to present a greater affinity for malate [1,7]. With regard to the structure of A. cordifolia NADPME, SDS-PAGE followed by Western blot and native molecular weight determination indicate that A. cordifolia NADP-malic enzyme is a tetramer of identical, 72 kDa subunits. It is interesting to note that this is the first apparently photosynthetic NADP-ME isoform with this rather large subunit composition. Structural and functional characterization of NADP-ME in plants shows that the 72 kDa-subunit isoforms found in the leaves and roots of CAM, C4 or C3 plants or etiolated tissue of C4 plants belong to the non-photosynthetic, housekeeping subgroup of ME [10,28,29]. Photosynthetic isoforms of NADP-ME have been reported to range in size from 62 to 67 kDa in C4 plants [4,14] and from 64 to 65 kDa in CAM plants [7,10]. Immunoblot analysis of crude extracts from roots indicate that A. cordifolia also possess an isoform of 72 kDa. However, it differs from the 64 kDa NADP-ME isoform from M. crystallinum roots [8]. The root isozyme of A. cordifolia is probably related to one of the two bands that can be visualized in crude leaf extracts by denaturing IEF which possesses a pI of 6.1. NADP-ME activity or protein did not vary with the day/night cycle. Assays made with several metabolites as possible modulators on NADP-ME indicated a marked and competitive-type inhibition for OAA, PEP and Laspartate. The competitive-type inhibition of NADPME activity by OAA was also observed with the purified NADP-ME from M. crystallinum leaves in CAM mode [7]. Although the actual cytosolic concentration of OAA is not known, it has been reported that K . daigremontiana, K. pinnata, Ananas comosus [30,31] and Bryophyllum crenatum [32] accumulate OAA during the night. Moreover, in K. daigremontiana the levels of OAA and aspartate are maximum during the night and minimum in the light period [30,31]. Assuming that the same occurs in A. cordifolia and considering that (a) the leaf density is 1 g ml 1; (b) the cytosol represents only 5% of the total cellular volume [33]; (c) a concentration of 0.5 mg Chl g1 fresh weight; and (d) that :/70% of the leaf tissue is photosynthetic in K. daigremontiana leaves (our observations), we estimate that values of OAA and aspartate at night are around 4.3 and 13.6 mM, respectively. These concentrations suggest that these metabolites are probably effective in vivo as negative effectors of NADP-ME. Although PEP also presents competitive inhibition towards NADP-ME, the effect could be largely suppressed due to the high diurnal levels of L-malate during the decarboxylation phase (1000-fold greater than that of PEP) [30]. In addition, citrate also acts as an inhibitor of the NADP-ME activity, but causes less inhibition than L-aspartate, PEP and OAA. The accumulation of citrate in vacuoles at night has been reported in A. cordifolia , where the nocturnal
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accumulation of citrate (D-citrate) is 24% that of Dmalate. During drought, the ratio of D-citrate to Dmalate increases to almost 0.5 [11]. These results suggest that A. cordifolia NADP-ME might be subject to metabolic regulation. At night, the high levels of OAA and L-aspartate in the cytosol and, to a lesser degree citrate, might inhibit NADP-ME avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of L-malate. The relatively high degree of inhibition caused by ATP, ADP and AMP is expected to be due to the structural analogy of these compounds with NADP, but probably lacks any physiological meaning [34]. Further biochemical studies will be required to determine whether the inhibition pattern observed in vitro has relevance in the regulation in vivo. 4.1. Concluding remarks A NADP-ME has been purified from the CAM plant A. cordifolia . The enzyme presents novel characteristics for a photosynthetic isozyme, namely a higher molecular mass compared to its counterpart from C4 and other CAM plants and a regulatory mechanism based on metabolite control by key compounds present in the cytosol of CAM plants.
Acknowledgements This work was supported by grant BID 1201/OC-AR PICT No. 1-03397 from ANPCyT, Argentina. MLFF is a Fellow under this grant. CSA and FEP are Career Investigators of CONICET, Argentina. The authors would like to thank Dr M. Fabiana Drincovich for her assistance and advice.
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