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Keywords:

  • phosphoenolpyruvate carboxylase;
  • plant glycolysis;
  • phosphate starvation response;
  • carbon–nitrogen interactions;
  • Brassica napus (rapeseed)

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (–Pi) cells to electrophoretic homogeneity and final specific activities of 37–40 µmol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and –Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH–activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to l-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and –Pi cells exists mainly in its dephosphorylated (l-malate sensitive) form. Thus, up-regulation of PEPC activity in –Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by l-malate, l-aspartate, l-glutamate, dl-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. l-Aspartate and l-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.

Abbreviations
AAT

aspartate aminotransferase

APase

acid phosphatase

CAM

crassulacean acid metabolism

GS/GOGAT

glutamine synthetase/glutamine 2-oxoglutarate aminotransferase

+Pi and –Pi

cultured in the presence and absence of 2.5 mm KPi, respectively

PEP

phosphoenolpyruvate

PEPC

phosphoenolpyruvate carboxylase

PKc

cytosolic isozyme of pyruvate kinase

PP2A

protein phosphatase type 2A1

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme in vascular plants that is also widely distributed in green algae, cyanobacteria, and bacteria [1]. It catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) in the presence of inline image and Mg2+to yield oxaloacetate and Pi. The enzyme is particularly abundant in the mesophyll cells of C4 and crassulacean acid metabolism (CAM) leaves where it participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2. Both allosteric mechanisms and covalent modification are involved in the regulation of PEPC activity during C4 and CAM photosynthesis [1–3]. Early work determined that C4 and CAM photosynthetic PEPCs were regulated via a diurnal cycle that served to modulate their sensitivity to l-malate inhibition, without affecting Vmax or the amount of PEPC protein. This has been shown to be due to reversible phosphorylation by an endogenous Ca2+-independent PEPC protein kinase and dephosphorylation by a protein phosphatase type 2 A (PP2A) at a key serine residue located near the N-terminus of the 100- to 110-kDa PEPC subunit [1–3].

In contrast to C4 and CAM leaf PEPCs, the properties, regulation and functions for the enzyme from C3 plants and nonphotosynthetic tissues of C4 and CAM plants are less well understood. Proposed roles for the C3 enzyme are diverse and include: (a) regulation of cellular pH and cation balance, (b) production of dicarboxylic acids used as respiratory substrates by nitrogen-fixing bacteroids of legume root nodules, and/or (c) the anaplerotic replenishment of citric acid cycle intermediates consumed in biosynthesis [1]. The enzyme has been partially or fully purified from several nonphotosynthetic plant tissues including germinating castor seeds [4], soybean root nodules [5], and ripening banana fruit [6]. As with the C4 and CAM leaf enzyme, reversible phosphorylation and allosteric effectors have been suggested to be important in the regulation of PEPC from C3 plants [1,4–10]. Radiolabeling studies with 32Pi have demonstrated the in vivo phosphorylation of PEPC in soybean root nodules [5], banana fruit [7], wheat leaves [8], Vicia faba guard cells [9], and germinating wheat seeds [10]. In banana fruit, an endogenous PEPC kinase forms a tight complex with its target enzyme [7]. However, the regulatory properties of plant PEPC that mediate glycolytic flux and PEP partitioning during nutritional Pi deprivation have not been elucidated.

Phosphorous is an essential element for normal growth and metabolism, as it plays a central role in virtually all metabolic processes. Plants preferentially absorb phosphorous from the soil in its fully oxidized anionic form, Pi (H2PO4, orthophosphate). Although widely distributed in the earth's crust, most Pi exists in insoluble mineral forms and, as such, is unavailable to plants [11]. Thus, in many natural environments Pi deficiency is the rule rather than the exception. Plants have evolved to acclimatize to Pi stress to varying degrees through a number of mechanisms, including acid phosphatase (APase) induction [11]. APases can function as intracellular or extracellular Pi salvaging systems that can scavenge Pi from phosphate esters. The plant ‘Pi starvation response’ also involves the induction of PEPC activity, which has been reported for several C3 plants including: Brassica nigra (black mustard) and Catharanthus roseus (periwinkle) suspension cells [12,13], Lupinus albus (lupin), Lycopersicon esculentum (tomato) and Cicer arietinum (chickpea) roots [14,15], and B. napus shoots [16]. PEPC induction has been correlated with increases in in vivo dark 14CO2 fixation and/or levels of PEPC-derived organic acids [13–19]. During Pi deprivation, PEPC (with malate dehydrogenase and NAD-malic enzyme) may provide a metabolic ‘bypass’ to the ADP-limited cytosolic pyruvate kinase (PKc) to facilitate continued pyruvate supply to the citric acid cycle, while concurrently recycling the PEPC by-product Pi for its assimilation into the metabolism of the Pi-starved cells [11,13]. In addition, PEPC induction can promote the synthesis and consequent excretion of large amounts of malic and citric acids from roots during Pi stress. This acidifies the rhizosphere, which therefore increases Pi availability to the plant by solubilizing otherwise inaccessible sources of mineral Pi[11,14,16,19]. Increased levels of PEPC protein and mRNA were correlated with enhanced PEPC activity of proteoid roots of Pi-deficient lupin plants [18]. The possible expression of a separate ‘–Pi inducible’ PEPC isozyme was indicated by the isolation of a PEPC cDNA from the Pi-stressed proteoid lupin roots [19]. In addition, kinetic studies of partially purified PEPC from proteoid lupin roots suggested that during Pi stress the enzyme may be phosphorylated by an endogenous PEPC kinase, resulting in reduced sensitivity of PEPC to inhibition by l-malate [19].

The initial goal of the current study was to investigate the impact of Pi starvation on the PEPC of B. napus suspension cell cultures. Such cultures represent an ideal model system for examining the influence of Pi nutrition on plant metabolism, because they contain a homogeneous population of cells, with each cell in direct contact with the culture medium. Moreover, relatively large quantities of cells at a precise nutritional and developmental state can be amassed for use in enzyme purification. Our second goal was to compare the kinetic and regulatory features of the purified PEPC from Pi-sufficient B. napus with those being concurrently characterized for the homogeneous PKc from the same cells [20]. Coordinate regulation of these two PEP utilizing enzymes plays a critical role in the regulation of plant cytosolic glycolytic flux, particularly with respect to the integration of carbon partitioning with the generation of 2-oxoglutarate needed for nitrogen assimilation by Gln synthetase/Gln 2-oxoglutarate aminotransferase (GS/GOGAT) [21,22]. PEPC has an additional metabolic function during nitrogen assimilation to produce oxaloacetate for l-Asp synthesis by Asp aminotransferase (AAT). PKc and PEPC appear to be activated (or deinhibited) in vivo following nitrogen resupply to nitrogen limited plant tissues and green algae [21]. This not only generates necessary carbon skeletons for AAT and/or GS/GOGAT, but also serves to reduce PEP levels, thereby relieving PEP inhibition of the ATP-dependent phosphofructokinase [22], thus stimulating overall glycolytic flux. Despite the tremendous importance of PEPC and PKc in controlling interactions between plant carbon and nitrogen metabolism, few workers have attempted to characterize both enzymes from the same plant tissue or cell type [4]. Indeed, the simultaneous complete purification and thorough comparative analysis of PEPC and PKc from the same vascular plant source has not been described. This is important because vascular plants express tissue- and developmental-specific isozymes of PKc and PEPC that may display very different physical, kinetic, and regulatory properties [1,21–23]. In particular, the combined results of the present and following [20] papers suggest a pivotal role for l-Asp and l-Glu in the coordinate regulation of B. napus PEPC and PKc.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Chemicals and plant material

PEP, Coomassie Blue R-250, bistris-propane, Mes, and dithiothreitol were from Research Organics, Inc. Tris base and SDS were from Schwartz/Mann Biotech. Ribi adjuvant (product code R-730) was obtained from Ribi Immunochemical Research. Poly(vinylidene difluoride) membranes (Immobilon, 0.45 µm pore size) were obtained from Millipore and all solutions were prepared using Milli-Q-processed water. Other biochemicals, coupling enzymes, SDS molecular mass standards, cell culture reagents and alkaline phosphatase-tagged goat anti-(rabbit IgG) IgG were obtained from Sigma Chemical Co. All other reagents were of analytical grade obtained from BDH Chemicals. Purified bovine heart PP2A (catalytic subunit) was a gift of G. Moorhead, University of Calgary (1 U of PP2A dephosphorylates 1 µmol of bovine glycogen phosphorylase per min at 30 °C).

An embryogenic pollen-derived heterotrophic cell suspension of winter oilseed rape (canola) (B. napus L. cv. Jet Neuf) [24] was provided by R. Weselake, University of Lethbridge, Canada. Cells were maintained on a rotational shaker (125 r.p.m.) at 22 °C in NLN media (pH 7.5) containing 6.5% (w/v) sucrose, 3 mm NO3, 5.5 mm l-Gln, 0.5 mg·L1α-naphthalene acetic acid, 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid and 2.5 mm K2HPO4 (added from a 200-mm sterile stock at the time of subculturing) as described previously [25]. Subculturing was performed by transferring 10 mL of a 7-day-old cell suspension into 40 mL (125 mL flask) of fresh NLN media containing 2.5 mm K2HPO4. Cells used in time-course studies and PEPC purification were obtained by scaling up the culture volume. Briefly, two 7-day-old 50-mL cultures were combined, concentrated to about 60 mL by removing excess media, and used to innoculate 440 mL of fresh NLN media containing either 2.5 mm K2HPO4 (+Pi cells) or 0 mm K2HPO4 (–Pi cells) in 2-L flasks. Cells were harvested on a Buchner funnel fitted with Miracloth, washed with 10 mm CaCl2, frozen in liquid N2, and stored at −80 °C.

Enzyme and protein assays

The PEPC and APase reactions were coupled to the malate dehydrogenase and lactate dehydrogenase reactions, respectively, and assayed at 24 °C by monitoring NADH oxidation at 340 nm using a Gilford 260 recording spectrophotometer, in a final volume of 1 mL. Coupling enzymes were desalted before use. Standard assay conditions for PEPC were: 50 mm bistris-propane/HCl (pH 8.4), 10% (v/v) glycerol, 2 mm PEP, 2.5 mm KHCO3, 12 mm MgCl2, 0.15 mm NADH and 5 U of porcine heart malate dehydrogenase. Assay conditions for APase were: 25 mm bistris-propane/25 mm Mes (pH 5.7), 2 mm PEP, 0.15 mm NADH and 5 U of rabbit muscle lactate dehydrogenase. All assays were: (a) initiated by addition of enzyme preparation, (b) corrected for NADH oxidase activity, and (c) linear with respect to time and concentration of enzyme assayed. To examine, the influence of rutin and quercetin on PEPC activity, a 10-mm stock of each flavonoid was prepared in dimethylsulfoxide and stored at −20 °C. Control assays containing only dimethylsulfoxide were included. One unit of enzyme activity is defined as the amount of enzyme resulting in the production of 1 µmol of product per min at 24 °C.

Protein concentrations were routinely determined using a Dynatech MR-5000 microplate reader and the Coomassie Blue G-250 dye-binding method described by Bollag and Edelstein [26]. Protein concentration of the purified PEPCs was also determined using the bicinchoninic acid method of Hill and Straka [27]. Bovine γ-globulin was used as the protein standard.

Kinetic studies

Kinetic studies were conducted using a Dynatech MR-5000 microplate reader and a final volume of 0.2 mL for the PEPC reaction mixture. Apparent Km values were calculated from the Michaelis–Menten equation fitted to a nonlinear least-squares regression computer kinetics program [28]. Ka and I50 values (concentration of activator and inhibitor producing 50% activation and inhibition of PEPC activity, respectively) were determined using the same computer kinetics program. All kinetic parameters are the means of at least three separate determinations and are reproducible to within ± 10% SE.

Preparation of clarified homogenates used in time-course studies

+Pi or –PiB. napus cells were ground to a powder in liquid N2 and homogenized (1 : 2, w/v) using a mortar and pestle and a small scoop of sand in ice-cold 100 mm imidazole/HCl (pH 7.6) containing 1 mm EDTA, 5 mm MgCl2, 100 mm KCl, 20 mm NaF, 20% (v/v) glycerol, 10 mm thiourea, 1% (w/v) each of insoluble and soluble polyvinylpolypyrrolidone, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 5 µg·mL−1 chymostatin, and 50 nm microcystin-LR. Homogenates were centrifuged at 4 °C and 14 000 g for 15 min, and the resulting clarified extracts prepared for SDS/PAGE and PEPC immunoblotting, and/or assayed for total protein, PEPC and APase activities.

Buffers used during PEPC purification

Buffers were degassed and adjusted to their respective pH values at 24 °C. Buffer A: aforementioned extraction buffer containing 0.1% (v/v) Triton X-100 and 4% (w/v) poly(ethylene glycol) 8000. Buffer B: 50 mm imidazole/HCl (pH 7.1) containing 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 20 mm NaF and 25% (saturation) (NH4)2SO4. Buffer C: 50 mm imidazole/HCl (pH 7.1) containing 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 20 mm NaF and 10% (v/v) ethylene glycol. Buffer D: 100 mm Tris/HCl (pH 8) containing 1 mm EDTA, 5 mm MgCl2, 20% (v/v) glycerol, 20 mm NaF and 1 mm dithiothreitol. Buffer E: 50 mm imidazole/HCl (pH 7.5) containing 15% (v/v) glycerol, 1 mm EDTA, 3 mm MgCl2, 50 mm KCl, 0.02% (w/v) NaN3, 20 mm NaF and 1 mm dithiothreitol.

Purification of PEPC

All procedures were carried out at 4 °C. Identical protocols were employed for the purification of PEPC from 8-day-old +Pi and –PiB. napus suspension cells, henceforth referred to as the ‘+Pi PEPC’ and ‘–Pi PEPC’ preparations, respectively.

Preparation of clarified extract and poly(ethylene glycol) fractionation

Quick-frozen B. napus suspension cells (220 g of +Pi cells or 147 g of –Pi cells) were ground to a powder in liquid N2, homogenized (1 : 2.5, w/v) in buffer A using a Polytron, and centrifuged at 14 000 g for 20 min. Finely ground poly(ethylene glycol) 8000 was added to the supernatant fluid to a final concentration of 24% (w/v). The extract was stirred for 45 min and centrifuged for 20 min 35 000 g. Poly(ethylene glycol) pellets (4–24%; w/v) were stored overnight at −20 °C.

Butyl-Sepharose hydrophobic interaction FPLC

Poly(ethylene glycol) pellets were resuspended in buffer B lacking (NH4)2SO4, but containing 5 µg·mL−1 chymostatin, to yield a protein concentration of about 5 mg·mL−1. Following centrifugation for 20 min at 35 000 g, the extract was adjusted to 25% (saturation) (NH4)2SO4 by the addition of solid (NH4)2SO4. The solution was stirred for 20 min, centrifuged as above, and adsorbed at 4 mL·min−1 onto a column (3 × 6.8 cm) of butyl-Sepharose Fast Flow (Pharmacia) preequilibrated with buffer B. The column was connected to a FPLC system, washed with 100 mL of buffer B, and PEPC activity eluted in a stepwise fashion with 60% buffer C (40% buffer B) (flow rate = 4 mL min−1; fraction size = 5 mL). Pooled peak PEPC activity fractions were diluted with an equal volume of 50% (w/v) poly(ethylene glycol) 8000, stirred for 30 min, and centrifuged as above. The resulting pellets were stored overnight at −20 °C.

Fractogel EMD DEAE-650 (S) anion-exchange FPLC

The poly(ethylene glycol) pellets were solubilized in buffer D to which 5 µg·mL−1 chymostatin was added to yield a protein concentration of about 10 mg·mL−1, centrifuged as above, and loaded at 1.5 mL·min−1 onto a column (1.1 × 6.7 cm) of Fractogel EMD DEAE-650 (S) (Merck) that had been connected to a FPLC system and preequilibrated with buffer D. The column was washed with buffer D until the A280 decreased to baseline, and was PEPC eluted with 80 mL of a linear 0–300 mm KCl gradient in buffer D (fraction size = 5 mL). Pooled peak activity fractions were adjusted to contain 5 µg·mL−1 chymostatin and concentrated to about 1 mL using an Amicon XM-50 ultrafilter.

Superdex 200 gel filtration FPLC

The concentrated anion-exchange fractions were passed through a 0.45-µm syringe filter and applied at 0.3 mL·min−1 onto a column (1.6 × 51 cm) of Superdex 200 Prep Grade (Pharmacia) that had been attached to a FPLC system and preequilibrated with buffer E (fraction size = 1.2 mL).

Mono-Q anion-exchange FPLC

Pooled peak activity fractions from the Superdex 200 column were immediately loaded at 0.5 mL·min−1 onto a Mono-Q HR 5/5 column (Pharmacia) preequilibrated with buffer D. PEPC was eluted using 25 mL of a linear 0–300 mm KCl gradient in buffer D (fraction size = 1 mL). Peak activity fractions were pooled, adjusted to 5 µg·mL−1 chymostatin, concentrated as above to 0.65 mL, divided into 50-µL aliquots, frozen in liquid N2 and stored at −80 °C. The purified PEPC was stable for at least 6 months when stored frozen.

Determination of native molecular mass via Superdex 200 gel filtration

Native molecular mass estimation for the +Pi and –Pi PEPCs was performed during FPLC on the Superdex 200 Prep Grade column as described above. Native molecular masses were estimated from a plot of Kav (partition coefficient) vs. log molecular mass for the following protein standards: ferritin (440 kDa), catalase (232 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Blue Dextran was used to determine the column's void volume.

Antibody production

Purified +Pi PEPC (500 µg) was dialyzed overnight against NaCl/Pi (20 mm NaPi, pH 7.4, 150 mm NaCl), filtered through a 0.2-µm membrane, and emulsified in Ribi adjuvant (1 mL total volume). After collection of preimmune serum, the PEPC was injected (0.6 mL subcutaneously, 0.4 mL intramuscularly) into a 2-kg New Zealand rabbit. A booster injection (250 µg) was administered subcutaneously after 6 weeks. Ten days after the final injection, blood was collected by cardiac puncture. After incubation overnight at 4 °C, the clotted blood cells were removed by centrifugation at 1500 g for 10 min. The crude antiserum was frozen in liquid N2 and stored at −80 °C in 0.04% (w/v) NaN3. For immunoblotting, the anti-(B. napus+Pi PEPC) IgG was affinity-purified against 25 µg of purified B. napus+Pi PEPC as previously described [29].

Immunotitration of PEPC activity

Immunoremoval of enzyme activity was tested by mixing 0.05 units of homogeneous +Pi PEPC with 25 mm Hepes/NaOH (pH 7.5), containing 0.1 mg·mL−1 BSA, 10% (v/v) glycerol, 1 mm dithiothreitol, and various amounts of rabbit preimmune or anti-(B. napus PEPC) immune serum diluted into NaCl/Pi (total volume = 0.1 mL). The mixtures were incubated at 30 °C for 60 min and then for 90 min at 4 °C prior to centrifuging for 5 min at 17 000 g. Residual PEPC activity in the supernatant fraction was determined as described above.

Electrophoresis and immunoblotting

SDS/PAGE was performed according to Laemmli [30] using a Bio-Rad mini-gel apparatus. The acrylamide monomer concentration in the 0.75-mm-thick slab gels was 4% and 9% (w/v) for the stacking and separating gels, respectively. Prior to SDS/PAGE samples were incubated for 3 min at 100 °C in 50 mm Tris/HCl (pH 6.8) containing 1% (w/v) SDS, 10% (v/v) glycerol and 100 mm dithiothreitol. Gels were run at a constant voltage of 200 V for 45 min. To determine subunit molecular masses by SDS/PAGE, a plot of relative mobility vs. the log m was constructed using the following protein standards: α2 macroglobulin (180 kDa), β-galactosidase (116 kDa), phosphofructokinase (84 kDa), pyruvate kinase (58 kDa), fumarase (48.5 kDa), lactate dehydrogenase (36.5 kDa) and triose phosphate isomerase (26.6 kDa).

Nondenaturing PAGE was conducted at 4 °C using the highly porous SDS/PAGE system of Doucet and Trifaró[31] except that SDS was omitted from all buffers, and 20% (v/v) glycerol and 10% (v/v) ethylene glycol were included in the stacking and separating gels which contained acrylamide concentrations of 4% and 6% (w/v), respectively. Gels were run at 200 V for 2 h, and either incubated in a PEPC activity stain, or immunoblotted using affinity-purified anti-(B. napus+Pi PEPC) IgG. To detect in-gel PEPC activity, a gel was incubated for 30 min at 23 °C in 50 mm Tris/HCl (pH 8.4) containing 10% (v/v) glycerol, 12 mm MgCl2, 2.5 mm KHCO3, 0.15 mm NADH, and 5 U·mL of porcine heart malate dehydrogenase. PEP (2 mm) was added to initiate the reaction and PEPC activity was visualized as dark bands in a fluorescent background using a UV transilluminator.

Immunoblotting was performed by transferring protein from SDS or native gels to poly(vinylidene difluoride) membranes by electroblotting for 75 min or 180 min, respectively, at 100 V. Antigenic polypeptides were visualized using an alkaline phosphatase-tagged secondary antibody [29]. The relative amount of PEPC protein in clarified extracts from 8-day-old +Pi vs. –PiB. napus cells was determined by quantification of the antigenic 104-kDa PEPC subunit on immunoblots (in terms of A633) using an LKB Ultroscan XL laser densitometer and gel scan software (version 2.1) (Pharmacia LKB Biotech). Derived A633 values were linear with respect to the amount of the immunoblotted extract. Immunological specificities were confirmed by performing immunoblots in which rabbit preimmune serum was substituted for the affinity-purified anti-(B. napus+Pi PEPC) IgG.

Peptide mapping by CNBr cleavage

Polypeptides corresponding to the 104-kDa subunit of purified +Pi and –Pi PEPCs were excised from SDS/PAGE mini-gels and cleaved in situ with CNBr. The degradation products were analyzed on an SDS/PAGE 14% mini-gel [32], and stained with silver [33].

Phosphatase treatment

Clarified homogenates of –Pi and +Pi cells were prepared in the presence and absence of 20 mm NaF and 50 nm microcystin-LR. Aliquots (0.5 mL) were desalted by centrifugation at 100 g through 5 mL of Sephadex G-50 [34] that had been preequilibrated in 50 mm bistris-propane/HCl (pH 7.3) containing 10% (v/v) glycerol, 5 mm MgCl2, and 1 mm dithiothreitol. The desalted extracts as well as the purified +Pi and –Pi PEPCs (dialyzed free of NaF) were incubated for 1 h at 23 °C in the presence and absence of 0.5 U·mL−1 of bovine heart PP2A or 2 U·mL−1 of bovine intestinal alkaline phosphatase. PEPC activity was determined relative to controls prepared and desalted in the presence of 20 mm NaF and 50 nm microcystin-LR. PEPC assays were conducted at pH 7.3 with subsaturating (0.4 mm) PEP in the presence and absence of 0.1 mm l-malate.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Influence of Pi starvation on growth, and PEPC and APase activities of B. napus suspension cells

B. napus suspension cells cultured for 8 days in the absence of exogenous Pi had only about 50% of the fresh weight of the 8-day-old +Pi cells (approximately 20 and 10 g of cells were obtained per 500 mL culture of 8-day-old +Pi and –Pi cells, respectively). The time-courses for PEPC and APase activities of +Pi and –PiB. napus cells are shown in Fig. 1. PEPC and APase specific activities were increased by about 2.5-fold and fourfold, respectively, in the –Pi cells, whereas the activities of the two enzymes remained relatively low and constant in the +Pi cells. Within 24 h of resupplying 2.5 mm Pi to the 8-day-old –Pi cells, extractable PEPC and APase activities were reduced by at least 50% (Fig. 1). Immunoblotting with rabbit anti-(B. napus+Pi PEPC) IgG was used to estimate the subunit molecular mass and relative amount of PEPC in clarified extracts from the 8-day-old +Pi vs. –PiB. napus cells. In each instance, a single immunoreactive 104-kDa polypeptide was observed (Fig. 1A, inset), identical to that obtained with the respective purified PEPCs (see below). Laser densitometric quantification of the immunoblots revealed that the –PiB. napus extracts contained approximately twofold more of the immunoreactive 104-kDa PEPC subunit, relative to extracts of the +Pi cells.

image

Figure 1. Time-course for extractable activities of PEPC (A) and APase (B) in B. napus suspension cells cultured in 0 or 2.5 mm Pi. Values for the 6-, 8-and 10-day-old –Pi cells represent means ± SE for replicate assays of separate clarified extracts from n = 3 different 500 mL cultures. All other values represent the mean activities of replicate determinations of a single extract. An 8-day-old –Pi culture was resupplied with 2.5 mm Pi and cultured for an additional 1 day as indicated (···). Inset to (A): immunological detection of PEPC from 8-day-old +Pi or –PiB. napus suspension cells. Clarified extracts (each containing 50 µg of protein) were subjected to SDS/PAGE and blot-transferred to a poly(vinylidene difluoride) membrane. Blots were probed with 20-fold diluted affinity-purified anti-(B. napus+Pi PEPC) IgG and immunoreactive polypeptides were detected using an alkaline-phosphatase-linked secondary antibody followed by chromogenic staining as previously described [29].

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PEPC purification

As shown in Table 1, PEPC was purified about 715-fold from 220 g of 8-day-old +PiB. napus cells to a final specific activity of 20 U·mg−1 and an overall recovery of 14%. Using an identical protocol, the PEPC from 147 g of 8-day-old –Pi cells was purified 410-fold to a final specific activity of 20.9 U·mg−1 and an overall yield of 12% (results not shown). As previously documented for banana fruit PEPC [6], the protein concentration of the purified B. napus+Pi and –Pi PEPCs as determined using the bicinchoninic acid-based protein assay was almost 50% of that determined with the Coomassie Blue G-250 dye binding assay (Table 1). Thus, with the bicinchoninic acid protein assay the specific activities of the final +Pi and –Pi PEPC preparations were, respectively, increased to 37.2 and 39.7 U·mg−1 (Table 1, and results not shown). The anomalous color yield of certain proteins with the Coomassie-Blue dye binding assay has been well established [26].

Table 1. Purification of PEPC from 220 g of 8-day-old Pi-sufficient (+Pi) B. napus suspension cells.
StepVolume (mL)Activity (U)Protein (mg)Specific activity (U·mg−1)Purification (fold)Yield (%)
  1. a  Protein determined with Coomassie Blue R-250 dye-binding assay according to the method of Bollag and Edelstein [26]. b Protein determined with the bicinchoninic acid reagent according to the method of Hill and Straka [27]. c Concentrated pooled fractions.

Clarified extract6451134000a0.028  1100
PEG fractionation258 751340a0.055  2.0 66
Butyl Sepharose 34 65 240a0.27  9.6 58
DEAE Fractogelc  1.0 45  16a3.0107 40
Superdex 200  9.4 25   3.7a6.9246 22
Mono-Qc  0.65 16   0.80a    0.43b20.0 37.2714 14

Gel electrophoresis

Denaturation, followed by SDS/PAGE of the final +Pi PEPC preparation resolved a single protein staining band of approximately 104 kDa (Fig. 2A, lanes 2 and 3) that strongly cross-reacted with affinity-purified anti-(B. napus+Pi PEPC) IgG (Fig. 2B, lane 1) or anti-(banana fruit PEPC) IgG (Fig. 2C, lane 1). Identical results were obtained with the final preparation of –Pi PEPC (results not shown). SDS/PAGE and immunoblotting revealed that an polypeptide of approximately 5 kDa was cleaved from the 104-kDa subunit of PEPC during the enzyme's purification from +Pi cells in the absence of added chymostatin (Fig. 2A, lane 4 and Fig. 2C, lane 2) (final specific activity of proteolyzed +Pi PEPC = 11.4 U·mg−1). The repeated inclusion of 5 µg·mL−1 chymostatin at various stages of the purification prevented partial degradation of the enzyme during its purification (Fig. 2).

image

Figure 2. SDS/PAGE and immunoblot analysis of PEPC from Pi-sufficient (+Pi) B. napus suspension cells and developing seeds. (A) SDS/PAGE (9% separating gel) of PEPC purified from 8-day-old +PiB. napus cells. Lane 1 contains 4 µg of various molecular mass standards. Lanes 2 and 3 contain 2.5 and 1 µg, respectively, of the pooled peak fractions from the final purification step (Mono-Q FPLC). Lane 4 contains 5 µg of the +Pi PEPC that was partially purified in the absence of chymostatin. Protein staining was performed with Coomassie Blue R-250. (B) Immunoblot analysis was performed using 20-fold diluted affinity-purified rabbit anti-(B. napus+Pi PEPC) IgG. Lane 1 contains 15 ng of the final preparation of +Pi PEPC. Lane 2 contains 25 µg of protein from a clarified extract prepared from +PiB. napus suspension cells. Lane 3 contains 15 µg of protein from an extract prepared from developing B. napus seed cotyledons. (C) Immunoblot analysis was performed using 20-fold diluted affinity-purified rabbit anti-(banana fruit PEPC) IgG [6]. Lane 1 contains 15 ng of the final preparation of +Pi PEPC. Lane 2 contains 50 ng of +Pi PEPC that was isolated in the absence of chymostatin. Abbreviations: O, origin; TD, tracking dye front.

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Determination of native molecular mass via gel filtration FPLC

The native molecular mass of the purified +Pi and –Pi PEPCs was determined to be 440 ± 20 kDa (mean ± SE; n = 3) as estimated by gel filtration FPLC on a calibrated Superdex 200 column. Thus, the native PEPCs appear to be homotetrameric.

Absorption coefficient

The molar absorption coefficient of B. napus+Pi PEPC is 4.32 × 105m−1·cm−1 at 280 nm (A0.1%280 = 0.918). This value was calculated using the bicinchoninic acid determination of protein concentration (Table 1) and assuming a native molecular mass of 440 kDa.

Immunological characterization

Increasing amounts of rabbit anti-(B. napus+Pi PEPC) immune serum immunoprecipitated 100% of the activity of the purified B. napus+Pi PEPC. Complete immunoremoval of activity occurred at about 900 µL of immune serum per unit of PEPC activity. By contrast, preimmune serum had no effect on the PEPC activity. The affinity-purified anti-(B. napus+Pi PEPC) IgG could detect as little as 5 ng of denatured homogeneous +Pi or –Pi PEPC. Immunoblotting of clarified crude extracts prepared from B. napus suspension cells or developing seed embryos (at mid-cotyledonary stage of development) demonstrated monospecificity of the anti-(B. napus+Pi PEPC) IgG for the 104-kDa PEPC subunit (Fig. 2B, lanes 2 and 3). Likewise, nondenaturing PAGE of clarified extracts from +PiB. napus cells generated a band of PEPC activity that comigrated with a single anti-(B. napus+Pi PEPC) IgG immunoreactive band (results not shown).

Peptide mapping

The structural relationship between the 104 kDa subunits of the +Pi and –Pi PEPCs was examined by peptide mapping of their respective CNBr cleavage fragments (Fig. 3). Indistinguishable peptide maps were obtained indicating that they are likely identical polypeptides.

image

Figure 3. Electrophoretic patterns of CNBr cleavage fragments of the 104 kDa subunit of B. napus+Pi and –Pi PEPCs. CNBr cleavage fragments were prepared from gel slices containing 8 µg of the 104-kDa polypeptide of +Pi (lane 2) or –Pi (lane 3) PEPCs and analyzed on an SDS/14% PAGE mini-gel as previously described [32]. Lane 1 contains 4 µg of the 104-kDa +Pi PEPC polypeptide incubated in the absence of CNBr. Lane 4 contains 4 µg of various molecular mass standards. The gel was stained with silver [33]. O, origin; TD, tracking dye front.

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Kinetic properties

Effect of pH. Similar to other plant PEPCs [1–6,23], the +Pi and –Pi PEPCs exhibited identical and broad pH/activity profiles with a maximum between 8.0 and 9.5 (results not shown). PEPC activity at pH 7.3 was almost 80% of that occurring at pH 8.4 (Table 2), but became almost undetectable below pH 7.0.

Table 2. Influence of glycerol, pH and various metabolites on Vmax and Km(PEP) of PEPC purified from 8-day-old Pi-sufficient (+Pi) and Pi-deprived (– Pi) B. napus suspension cell cultures. The standard spectrophotometric assay [± 10% (v/v) glycerol] was used except that the PEP concentration was varied. Effectors were added individually to +Pi PEPC assays [pH 7.3, 10% (v/v) glycerol] as follows: 0.1 mm Glc-6-P, 0.1 mm l-malate, 1.25 mmdl-isocitrate, 1.5 mm l-Asp, or 5 mm l-Glu. ND, not determined.
 pH 7.3pH 8.4
 VmaxKm (PEP)Vmax (U·mg−1)Km (PEP) (mm)
 – Glycerol+ Glycerol– Glycerol+ Glycerol– Glycerol+ Glycerol– Glycerol+ Glycerol
+ Pi PEPC15.715.70.410.1720.020.00.150.08
+ Glc-6-PND17.0ND0.08NDNDNDND
+ L-malateND14.5ND0.43NDNDNDND
dl-isocitrateND13.7ND0.30NDNDNDND
l-AspND14.7ND1.0NDNDNDND
l-GluND14.7ND0.67NDNDNDND
– Pi PEPC15.315.40.420.1520.921.00.160.08

Cofactor requirements. The activity of B. napus+Pi PEPC showed an absolute dependence for a bivalent metal cation. At pH 8.4, the enzyme's Km(Mg2+) was determined to be 0.084 mm. Mn2+ (12 mm, added as MnCl2) yielded the same Vmax value achieved with saturating Mg2+. These results are analogous to those obtained with other plant PEPCs [1–6].

PEP saturation kinetics.Table 2 summarizes the Vmax and apparent Km(PEP) values of PEPC at pH 7.3 and 8.4 in the presence and absence of 10% (v/v) glycerol. In all instances, identical PEP saturation kinetics were obtained with the purified +Pi and –PiB. napus PEPCs (Table 2). In common with most other plant PEPCs, B. napus PEPC exhibited hyperbolic PEP saturation kinetics. Decreasing the assay pH from 8.4 to 7.3 increased the enzyme's Km(PEP) by more than twofold, whereas the addition of 10% (v/v) glycerol to the assay medium decreased the enzyme's Km(PEP) by about 60 and 50% at pH 7.3 and pH 8.4, respectively (Table 2). Previous workers have cited stabilization of the enzyme's quaternary structure, due to exclusion of solvent molecules, as the rationale for the favorable influence of glycerol on the affinity of plant PEPC for PEP [35]. Glycerol (10%, v/v) was routinely added to the PEPC assay mixture.

Metabolite effects

A wide variety of compounds were tested as possible effectors of +Pi PEPC at pH 7.3 and 8.4 with subsaturating concentrations of PEP (0.15 mm). The following compounds had little or no influence on PEPC activity (± 20% of the control rate) at either pH 7.3 or 8.4: NH4Cl, KPi (10 mm each); 2-oxoglutarate, citrate, l-His, l-Arg, Gly, l-Ala, l-Lys, l-Gln, l-Asn, and sucrose (5 mm each); 2-P-glycerate, and l-Phe (2 mm each); MgADP, MgATP, and shikimic acid (1 mm each); acetyl-CoA, MgPPi, fructose, and NAD+ (0.5 mm each); l-Trp (0.25 mm); fructose 2,6-P2 (20 µm); Triton X-100, Nonidet P-40, and dimethylsulfoxide (2%, v/v, each). Table 3 lists those compounds that were found to significantly activate or inhibit the activity of the purified enzyme.

Table 3. Influence of various metabolites on the activity of PEPC purified from 8-day-old Pi-sufficient (+Pi) B. napus suspension cell cultures. Assays were conducted at pH 7.3 or 8.4 in the presence of 10% (v/v) glycerol using a subsaturating (0.15 mm) concentration of PEP. Enzymatic activity in the presence of effectors is expressed relative to the respective control set at 100%. ND, not determined.
 ConcentrationRelative activity
Addition(mm)pH 7.3pH 8.4
Glc-6-P2230122
Glc-1-P2143108
Fru-6-P2174113
Fru-1-P2130102
Fru-1,6-P25129 92
Glycerol-3-P5171110
3-P-Glycerate2140100
Dihydroxyacetone-P0.5127 99
l-Glu5 52101
l-Asp5 30 91
L-malate5  5 97
dl-isocitrate5 35 26
Succinate5 67 66
Rutin0.1 30ND
Quercetin0.1  5ND

PEPC displayed pH-dependent modulation by several of the metabolites such that they were generally far more effective at pH 7.3 than pH 8.4 (Table 3). Similar observations have been noted for other PEPCs from various plant sources [1,2,4–6,8,23].

Activators. Significant activators of B. napus+Pi PEPC at pH 7.3 were the hexose-Ps (particularly Glc-6-P), and glycerol-3-P (Table 3). Synergistic or additive effects of activators at pH 7.3 were not observed, suggesting that they all interact at a common allosteric site. At 0.1 mm, Glc-6-P significantly decreased the enzyme's Km(PEP) (by about 50%) and slightly increased its Vmax at pH 7.3 (Table 2). Glc-6-P also functions as an activator by effectively relieving the inhibition of PEPC by l-malate, l-Asp, and l-Glu (Table 4; Fig. 4). The addition of 0.1 mm Glc-6-P increased the I50 values of PEPC for these inhibitors by threefold to fourfold (Table 4). Furthermore, the enzyme's fold-activation by saturating Glc-6-P was increased from about twofold to over sixfold in the presence of 0.1 mml-malate, 5 mm l-Glu, or 1.5 mm l-Asp (Fig. 4).

Table 4. Kinetic constants for several effectors of PEPC purified from 8-day-old Pi-sufficient (+Pi) or Pi-starved (– Pi) B. napus suspension cell cultures. The standard spectrophotometric PEPC assay was used except that the assay pH and PEP concentration were suboptimal (pH 7.3; 0.34 mm PEP). I50 values for several inhibitors were determined ± 0.1 mm Glc 6-P. Similarly, Ka(Glc-6-P) values were determined in the presence and absence of approximate I50 concentrations of several inhibitors. ‘Proteolyzed +Pi PEPC’ refers to the partially degraded enzyme isolated in the absence of chymostatin (see Fig. 2A, lane 4; Fig. 2C, lane 2).
EffectorI50 (mm)Ka (mm)
+Pi PEPC
Glc-6-P0.066
 + 0.1 mm L-malate0.17
 + 1.5 mm l-Asp0.26
 + 5 mm l-Glu0.22
L-malate0.085
 + 0.1 mm Glc-6-P0.37
l-Asp1.5
 + 0.1 mm Glc-6-P4.7
l-Glu5.0
 + 0.1 mm Glc-6-P13.0
dl-isocitrate2.5
 + 0.1 mm Glc-6-P2.5
Succinate13.0
Quercetin0.026
 + 0.1 mm Glc-6-P0.028
Rutin0.046
 + 0.1 mm Glc-6-P0.050
Proteolyzed +Pi PEPC
l-malate0.86
–Pi PEPC
l-malate0.081
image

Figure 4. Relationship between +Pi PEPC activity and the concentration of glucose-6-P in the presence and absence of l-Asp, l-Glu, l-malate, and dl-isocitrate. Assays were conducted at pH 7.3 with subsaturating (0.34 mm) PEP in the absence (●) and presence of 1.25 mm dl-isocitrate (○), 0.1 mm l-malate (▾), 5 mm l-Glu (▿), or 1.5 mm l-Asp (▪).

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Inhibitors. At pH 7.3, the B. napus+Pi PEPC was potently inhibited by l-malate, dl-isocitrate, l-Asp, and l-Glu (Tables 3 and 4). Increasing the assay pH to 8.4 nullified the enzyme's inhibition by 5 mm l-malate, l-Asp, or l-Glu (Table 3). These metabolites function as inhibitors at pH 7.3 by markedly reducing the affinity of PEPC for its substrate, PEP, and its activator, Glc-6-P (Tables 2 and 4, Fig. 4). This is reflected by the addition of approximate I50 concentrations of dl-isocitrate and/or l-malate, l-Asp, or l-Glu causing an increase in the Km(PEP) of PEPC by up to sixfold (Table 2), and Ka(Glc-6-P) by at least twofold (Table 4). Of interest is the unique inhibition of B. napus PEPC by dl-isocitrate. In contrast to l-malate, l-Asp, and l-Glu: (a) inhibition by dl-isocitrate was comparable at both pH 7.3 and 8.4, and was not relieved by the addition of 0.1 mm Glc-6-P (Tables 3 and 4), and (b) the presence of 1.25 mm dl-isocitrate almost completely negated PEPC activation by Glc-6-P at pH 7.3 (Fig. 4). As reported for PEPC from leaves of Amaranthus viridus[36], the B. napus PEPC also demonstrated potent inhibition by the flavonoids quercetin and rutin (Table 3). I50 values for both flavonoids were less than 50 µm, and were not influenced by the addition of 0.1 mm Glc-6-P to the reaction mixture (Table 4).

Phosphorylation status of PEPC from +Pi and –PiB. napus suspension cells

Clarified extracts of 8-day-old –Pi and +PiB. napus cells were prepared and desalted in the presence and absence of the phosphatase inhibitors 20 mm NaF and 50 nm microcystin-LR. The desalted extracts were incubated in the presence and absence of 1 U·mL−1 of bovine heart PP2A or 2 U·mL−1 of bovine alkaline phosphatase for 1 h at 23 °C and assayed for PEPC activity at pH 7.3 with subsaturating (0.4 mm) PEP. The subsequent addition of 0.1 mm l-malate resulted in an approximate 50% inhibition of PEPC activity in the +Pi and –Pi cell extracts, irrespective of the treatment. Likewise, an identical incubation of the purified +Pi and –Pi PEPCs with PP2A or alkaline phosphatase failed to influence their sensitivity to l-malate inhibition when assayed at pH 7.3 with subsaturating PEP. Together with the fact that the purified –Pi and +Pi PEPCs exhibited identical I50(l-malate) values at pH 7.3 (Table 4), these results suggest that PEPC mainly exists in its dephosphorylated (l-malate sensitive) form in both +Pi and –PiB. napus cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Influence of Pi starvation on PEPC

Like lupin, B. napus is a ‘nonmycotrophic’ plant whose roots do not form symbiotic associations with mycorrhizal fungi to facilitate Pi uptake from the soil [37]. Recent evidence suggests that, relative to mycorrhizal associating or ‘mycotrophic’ plants (which account for about 90% of terrestrial plant species), the endogenous metabolism of nonmycotrophic plants is geared to allow a more efficient acclimatization to Pi deficiency [11,19,37]. For example, unlike many mycotrophs, roots of Pi-deprived lupin and B. napus plants are highly efficient users of rock Pi[16–19]. This has been ascribed to the PEPC-mediated synthesis and subsequent excretion of malic and citric acids from the roots during Pi deficiency, resulting in decreased rhizosphere pH and the solubilization of mineral-bound Pi. However, until now there has been no report describing the detailed comparison of the physical, immunological and kinetic properties of PEPC purified from the same +Pi and –Pi plant source.

Fresh weight of the 8-day-old –PiB. napus suspension cells was about 50% that of the +Pi cells, indicating that the 8-day-old –Pi and +Pi cells were in fact Pi starved and Pi sufficient, respectively, at the time of harvest. This is corroborated by: (a) the marked reduction in intracellular Pi concentration of the –Pi, but not +Pi, B. napus cells (the Pi concentration of 8-day-old –Pi and +PiB. napus cells was previously determined to be about 0.7 and 3.8 µmol·g−1 fresh wt, respectively) [25], and (b) the significant induction of APase activity, a universal biochemical indicator of plant Pi stress [11], in the –PiB. napus cells relative to the +Pi controls (Fig. 1B). As anticipated, the extractable PEPC specific activity of the –PiB. napus cells significantly increased (by 250%), relative to that of the +Pi cells, which remained low and constant throughout the 9-day time course (Fig. 1A). PEPC and APase were induced in parallel in response to Pi stress, and this was at least partially reversed 1-day after 2.5 mm Pi was resupplied to 8-day-old –Pi cells (Fig. 1A,B). Laser densitometric quantification of PEPC immunoblots revealed a close correlation between extractable PEPC activity and the relative amount of the immunoreactive 104-kDa PEPC subunit in clarified extracts of 8-day-old +Pi and –Pi cells (Fig. 1A). Analogous results have been described for the PEPC activity, concentration and mRNA levels in –Pi proteoid lupin roots relative to +Pi controls [18,19]. Therefore, in both B. napus cell cultures and proteoid lupin roots, the increased PEPC activity that accompanies Pi stress at least partially arises from an increased expression of PEPC protein.

In particular, we wished to determine whether Pi deprivation of B. napus also induced any alteration in the phosphorylation status of PEPC and/or the synthesis of a different PEPC isozyme (having the same subunit size). These goals were provoked by: (a) inhibition studies of partially purified PEPC from proteoid lupin roots indicating that PEPC may be phosphorylated by an endogenous protein kinase during Pi starvation (as reflected by a decreased sensitivity of the enzyme to l-malate inhibition), and (b) the isolation of a PEPC cDNA from proteoid roots of Pi starved lupin plants [19]. Whether this cDNA encodes a separate PEPC isozyme (relative to that expressed in +Pi lupin) has not been reported.

PEPC purification

The specific PP1/PP2A inhibitor microcystin-LR (50 nm) was added to the extraction buffer, and the general phosphatase inhibitor NaF (20 mm) was included in all purification buffers to prevent potential alterations in the phosphorylation status of PEPC during its extraction and isolation from the +Pi and –Pi cells. The protease inhibitor chymostatin was also included in purification buffers to prevent N-terminal truncation and consequent loss of the enzyme's phosphorylation domain, as previously documented for a variety of plant PEPCs [1,2]. Indeed, isolation of the B. napus+Pi PEPC in the absence of chymostatin resulted in a proteolytically clipped enzyme (Fig. 2A, lane 4, and Fig. 2C, lane 2) that was an order of magnitude less sensitive to l-malate inhibition relative to the nondegraded +Pi PEPC isolated in the presence of chymostatin (Table 4). Loss of a ≈ 4-kDa N-terminal phosphorylation domain of maize or sorghum C4 PEPC during their purification in the absence of chymostatin kinetically mimics the effect of phosphorylation of the nonproteolyzed enzymes [e.g. N-terminal proteolysis or phosphoryation of the intact PEPC both elicit a similar increase in the enzyme's I50(l-malate) without affecting Vmax][1].

The final specific activities of the purified +Pi and –PiB. napus PEPCs were about 20 U·mg−1 and compare favorably to the values reported previously for homogeneous PEPCs from various plant sources [1,5,6]. Analysis by SDS/PAGE confirmed that both PEPCs had been purified to apparent homogeneity (Fig. 2A, and results not shown). Similar to most other plant PEPCs, the native +Pi and –Pi PEPCs exist as 440-kDa homotetramers.

Immunological properties

Rabbit anti-(B. napus+Pi PEPC) immune serum immunoprecipitated up to 100% of the activity of the purified +Pi PEPC. Monospecificity of the antibody preparation for PEPC is indicated by the observation that only the 104-kDa PEPC subunit showed a significant cross-reaction when immunoblots of clarified extracts from B. napus developing seed (zygotic) cotyledons and/or suspension cells were probed with the affinity-purified anti-(B. napus+Pi PEPC) IgG (Fig. 2B). An immunoblot of the purified –PiB. napus PEPC cross-reacted with the anti-(B. napus+Pi PEPC) IgG to a similar extent as the purified +Pi PEPC. Similarly, antibodies to banana fruit PEPC cross-reacted strongly with the +PiB. napus PEPC (Fig. 2C). These results are consistent with those of previous studies [4,6,38] and indicate a high degree of structural similarity between PEPCs of vascular plants. In contrast, the anti-(banana or B. napus PEPC) IgGs fail to recognize purified PEPC from green algae or cyanobacteria [38] (J. Rivoal, W. C. Plaxton, and D. H. Turpin, unpublished data).

Peptide mapping

Peptide mapping is a powerful technique for evaluating the structural relationship between polypeptides [32]. We therefore analyzed the fragments generated by CNBr cleavage of the subunits of the purified +Pi and –PiB. napus PEPCs. The cleavage patterns were identical (Fig. 3), demonstrating that the 104-kDa subunit of the +Pi and –Pi PEPCs is probably the same polypeptide.

Kinetic studies

It has been amply demonstrated that phosphorylation of plant PEPCs significantly decreases the enzyme's sensitivity to l-malate inhibition when assayed at subsaturating PEP and suboptimal, but physiological, pH values ranging from about pH 7–7.4 [1–3,5,7–10]. In addition, phosphorylation of plant PEPC may result in an increased Vmax at suboptimal pH (e.g. pH 7.3) and/or a reduced Km(PEP) [7,10]. However, kinetic comparisons of the purified nonproteolyzed +Pi and –PiB. napus PEPCs revealed identical pH–activity profiles, PEP saturation kinetics (Table 2), and sensitivity to l-malate inhibition at pH 7.3 with subsaturating PEP (Table 4). The relatively low I50(l-malate) value of about 0.1 mm obtained for both purified PEPCs (Table 4) suggests that PEPC mainly exists in its dephosphorylated, l-malate sensitive form in the +Pi and –PiB. napus cells. This conclusion was corroborated by the failure of PP2A or alkaline phosphatase treatment of the respective clarified cell extracts or purified PEPCs to alter the enzyme's sensitivity to l-malate inhibition when assayed at pH 7.3 with subsaturating PEP. Thus, phosphorylation does not appear to play a role in regulating B. napus PEPC during Pi deprivation. Although phosphorylation of plant PEPC invariably relieves the inhibitory action of l-malate (and l-Asp [1,7]), this could be unnecessary during severe Pi stress when cellular biosynthetic processes are minimal and the bulk of organic acids produced via PEPC (e.g. l-malate and citrate) do not accumulate within the cytosol, but may either be respired by the mitochondria, sequestered in the vacuole, or excreted from the cell. Overall, our results indicate that the up-regulation of PEPC activity during Pi deprivation of B. napus suspension cells arises solely from the accumulation of the same PEPC isoform as exists in the +Pi cells. Whether this is due to the increased synthesis and/or reduced proteolytic turnover of the enzyme remains to be determined.

Metabolite effectors of B. napus PEPC

PEP is one of the initial substrates for the shikimic acid (aromatic) pathway, and is thus at a major branchpoint between plant primary and secondary metabolism. PEPC from C4 leaves was recently reported to be potently inhibited by several shikimic acid pathway endproducts, notably the flavonoids quercetin and rutin [36]. This was suggested as a possible modulator of the partitioning of PEP between primary and secondary metabolism. B. napus PEPC also displayed potent inhibition by rutin and quercetin (Tables 3 and 4), indicating that this may be a universal response of plant PEPCs. Both flavonoids have been implicated in a number of plant functions, including defense and plant-microbe signaling. However, the precise physiological relevance of rutin and quercetin inhibition of B. napus PEPC (and PKc[20]) will remain obscure until information is obtained as to their respective concentrations in the plant cytosol. Interestingly, shikimic acid (1 mm), a potent inhibitor of A. viridis leaf PEPC [39,40], exerted no influence on the activity of the B. napus PEPC in the presence of 10% (v/v) glycerol.

B. napus PEPC was highly responsive to a number of metabolites involved in carbon and nitrogenmetabolism. Notably, this PEPC was activated by Glc-6-P and potently inhibited by l-malate, dl-isocitrate, l-Asp and l-Glu at pH 7.3 (Tables 24, Fig. 4), whereas sensitivity to these compounds (with the exception of dl-isocitrate) was considerably diminished at pH 8.4 (Table 3). Cytosolic concentrations of Glc-6-P, l-malate, l-Asp and l-Glu in spinach leaves have been estimated to be about 6, 1, 23 and 21 mm, respectively [41]. Thus, the Ka(Glc-6-P) and I50 values for the organic and amino-acid inhibitors of B. napus PEPC are generally well within their probable physiological concentration range, suggesting that these metabolites are important regulators of PEPC activity in vivo. Potent inhibition by l-malate, l-Asp, and l-Glu has also been reported for PEPCs from unicellular green algae [38,42], C3 leaves [8,43], as well as several nonphotosynthetic tissues including soybean root nodules [5,44], germinated castor seeds [4], banana fruit [6], and developing seeds of V. faba[23]. To our knowledge, marked inhibition of a plant PEPC by dl-isocitrate has only been reported for the germinating castor cotyledon enzyme (I50 = 0.7 mm at pH 7.0, 0.1 mm PEP) [4]. In the companion paper [20], the kinetic and regulatory features of the purified B. napus PEPC and PKc are compared and contrasted. A model is presented which highlights the critical role played by l-Asp and l-Glu in the coordinate control of these two key enzymes, particularly as pertains to the regulation of glycolysis and PEP partitioning during nitrogen assimilation.

The activation of C3-leaf PEPCs by protein kinase-mediated phosphorylation in response to nitrogen resupply of nitrogen-limited tissues [1,8,45] raises the possibility that the B. napus PEPC is also subject to this additional form of metabolic control. However, there are other reports that reversible phosphorylation of C3-plant PEPC is of minor importance for its regulation (relative to its control by allosteric effectors) [2,43,46]. Results discussed above and in the following paper clearly emphasize the fundamental role of allosteric effectors in the coordinate control of B. napus PEPC and PKc.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by research and equipment grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). We are also grateful to Drs Florencio Podestá, Jean Rivoal, and David Turpin and for helpful discussions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  1. Enzymes: acid phosphatase (EC 3.1.3.2); aspartate aminotransferase (EC 2.6.1.1), glutamine synthetase (EC 6.3.1.2), glutamine 2-oxoglutarate aminotransferase (EC 1.4.1.13); phosphoenolpyruvate carboxylase (EC 4.1.1.31); pyruvate kinase (EC 2.7.1.40); protein phosphatase type 2A1 (EC 3.1.3.16).

    Note: a web site is available at http://biology.queensu.ca/