Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures

Implications for the integration of glycolysis with nitrogen assimilation

Authors


W. C. Plaxton, Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Fax: + 1613 533 6617, Tel.: + 1613 533 6150, E-mail: plaxton@biology.queensu.ca

Abstract

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 µmol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mm, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. l-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mm, respectively).l-Aspartate functioned as an activator (Ka = 0.31 mm) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by l-glutamate. Reciprocal control by l-aspartate and l-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of l-aspartate and l-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.

Abbreviations
AAT

aspartate aminotransferase

GS/GOGAT

glutamine synthetase/glutamine 2-oxoglutarate aminotransferase

PEP

phosphoenolpyruvate

PEPC

phosphoenolpyruvate carboxylase

PKc and PKp

cytosolic and plastidic isozymes of pyruvate kinase, respectively

Pyruvate kinase (PK) is an important regulatory enzyme of glycolysis that catalyzes the irreversible substrate level phosphorylation of ADP at the expense of phosphoenolpyruvate (PEP), yielding pyruvate and ATP. The majority of nonplant PKs are homotetramers with subunit molecular masses of 55–60 kDa [1–4]. Both allosteric controls and reversible protein kinase-mediated phosphorylation can be used to coordinate the activity of nonplant PKs with the energy and carbohydrate demands of the cell [1–4]. Vertebrate PK is expressed as four tissue-specific isozymes that display catalytic and regulatory properties that reflect the differing metabolic requirements of the respective tissues [1,2,4]. In all eukaryotes, PK is cytosolic, but vascular plant and green algal PK exists as both cytosolic (PKc) and plastid (PKp) isozymes that differ markedly in their respective physical, immunological and kinetic/regulatory characteristics [5–15].

Plant PK is of interest because considerable evidence indicates that it is a primary site of control of glycolytic flux for pyruvate [5]. A reduction in PEP levels, brought about by an enhancement of PK activity, will stimulate ATP-dependent phosphofructokinase because PEP is a potent inhibitor of plant phosphofructokinases. In contrast, phosphofructokinase is generally believed to be the most important control element of nonplant glycolysis, with secondary regulation vested at the level of PK [5]. The biochemical and genetic properties of plant PK are complex. The enzyme may exist as a monomer, homotetramer, heterotetramer or heterohexamer depending upon the species, tissue and intracellular location [6–13]. Moreover, our previous studies indicate that castor oil plant PKc is expressed as tissue-specific isozymes that demonstrate substantial differences in their respective physical and/or kinetic and regulatory properties [10–12,14,15]. In aerobic germinating castor seed endosperm, PKc is inhibited by several metabolite effectors to facilitate the massive gluconeogenic conversion of stored triacylglycerides to hexose phosphates, and stimulated during anaerobiosis in order to partially offset the reduced ATP levels accompanying anoxia [14–16]. Similar to PKc from the green alga Selenastrum minutum[7], the castor leaf PKc (but not castor seed endosperm PKc[14]) demonstrated potent inhibition by l-Glu [12]. The inhibition of some plant PKc enzymes by l-Glu provides a tight feedback control that is believed to balance their overall activity with the production of carbon skeletons required for NH +4 assimilation and transamination reactions in tissues active in amino-acid and protein synthesis [5,11,12,17]. The abnormal growth, carbon partitioning and dark respiration of transgenic tobacco lacking leaf PKc[18,19] underscores the importance of this enzyme in the control and integration of plant carbon and energy metabolism.

With the current interest in the application of genetic engineering for plant oils, there has been considerable attention in the use of nonzygotic embryos of oil-seed crops as a model system for studies of the biochemistry and gene regulation of oil-seed embryogenesis. Cell suspension cultures of embryos derived in vitro from pollen grains of Brassica napus (rapeseed) have been reported to closely resemble their developing zygotic (seed embryo) counterpart with respect to fatty acid and storage lipid composition [20], and expression of PKc and PKp[21]. In this study, we describe the purification to homogeneity of PKc from B. napus suspension cells, and document the physical, immunological and kinetic properties of the purified enzyme. In particular, our results provide further insights into the critical role played by l-Asp and l-Glu in the fine metabolic control of some plant PKc proteins. The preceding paper reported the purification and characterization of PEPC from the B. napus cells [22]. Here, a model is presented for the coordinate regulation of B. napus PKc and PEPC that integrates PEP partitioning with the generation of 2-oxoglutarate needed for NH +4 assimilation by Gln synthetase/Gln 2-oxoglutarate aminotransferase (GS/GOGAT), and oxaloacetate needed for l-Asp production by Asp aminotransferase (AAT).

Materials and methods

Chemicals and plant material

Monospecific rabbit polyclonal antibodies against castor seed endosperm PKc and PKp were obtained as previously described [6,8]. Sources of chemicals and other reagents, as well as protocols for the culture and harvesting of B. napus suspension cells were outlined in the preceding paper [22].

Enzyme and protein assays, and kinetic studies

Unless otherwise stated, standard PKc assay conditions were 25 mm Mes-25 mm Bistris-propane (pH 6.8), 2 mm PEP, 1 mm MgADP, 1 mm dithiothreitol, 5% (w/v) poly(ethylene glycol) 8000, 50 mm KCl, 10 mm MgCl2, 0.15 mm NADH, and 2 U·mL−1 of desalted rabbit muscle lactate dehydrogenase. Poly(ethylene glycol) was routinely added to the reaction mixture because this organic solute significantly enhances the activity of homogeneous castor endosperm PKc by stabilizing the active tetrameric structure of the native enzyme in dilute solutions [23]. Activity was determined at 24 °C by monitoring NADH oxidation at 340 nm using a Gilford recording spectrophotometer or a Dynatech microplate reader. Assays were: (a) initiated by the addition of enzyme preparation, (b) corrected for contaminating PEP phosphatase activity by omitting ADP from the reaction mixture, and (c) linear with respect to time and concentration of enzyme assayed. One unit of PK activity is defined as the amount of enzyme resulting in the utilization of 1 µmol PEP per min at 24 °C.

Protein concentration determination (using the Coomassie Blue G-250 dye-binding method with bovine γ-globulin as the protein standard) and PKc kinetic studies were performed using a Dynatech MR-5000 Microplate reader [22]. All kinetic parameters are the means of at least three separate determinations and are reproducible to within ± 10% SE.

To examine the influence of rutin and quercetin on PKc activity, a 10 mm stock of each flavonoid was prepared in dimethylsulfoxide and stored at −20 °C. Control assays containing only dimethylsulfoxide were included. Reversibility of inhibition by rutin and quercetin was tested by diluting the purified and concentrated PKc twofold into a stabilization buffer [25 mm Hepes/NaOH, pH 7.4, containing 1 mm dithiothreitol, 5 mm MgCl2, 1 mm EDTA, and 20% (v/v) glycerol] in the presence and absence of 0.1 mm of either flavonoid. Following a 5-min preincubation at room temperature, 2 µL of each sample was assayed for PKc activity in 1 mL of the aforementioned reaction mixture, except that the assay pH was 7.4 and PEP and ADP were subsaturating (0.1 mm each).

Buffers used in PKc purification

Buffers were prepared with Milli-Q processed water, degassed, and adjusted to their respective pH value at 24 °C. Buffer A: 100 mm imidazole/HCl (pH 7.6), 1 mm EDTA, 5 mm MgCl2, 100 mm KCl, 20 mm NaF, 10 mm thiourea, 0.1% (v/v) Triton X-100, 4% (w/v) poly(ethylene glycol) 8000, 2% (w/v) insoluble polyvinylpolypyrrolidone, 20% (v/v) glycerol, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, and 1 mm 2,2′-dipyridyl disulfide. Buffer B: 50 mm imidazole/HCl (pH 7.1), 1 mm dithiothreitol, 5 mm MgCl2, 1 mm EDTA, and 25% (saturation) (NH4)2SO4. Buffer C: 50 mm imidazole-HCl (pH 7.1), 1 mm dithiothreitol, 5 mm MgCl2, 1 mm EDTA, and 10% (v/v) ethylene glycol. Buffer D: 100 mm Tris/HCl (pH 8.0), 1 mm dithiothreitol, 1 mm EDTA, 5 mm MgCl2, and 20% (v/v) glycerol. Buffer E: 20 mm imidazole-HCl (pH 7.0), 2 mm dithiothreitol, 5 mm MgCl2, 1 mm EDTA, and 20% (v/v) glycerol.

Purification of PKc

All procedures were carried out at 4 °C, unless otherwise noted.

Preparation of the clarified extract and poly(ethylene glycol) fractionation

Quick-frozen Pi-sufficient B. napus suspension cells (594 g) were ground to a powder under liquid N2, homogenized in 1500 mL of buffer A using a Polytron, and centrifuged at 18 000 g for 20 min. Finely ground poly(ethylene glycol) 8000 was added to the supernatant fluid at a ratio of 0.2 g·mL−1. The extract was stirred for 45 min and centrifuged as above. The poly(ethylene glycol) pellets were stored overnight at −20 °C.

Butyl-Sepharose hydrophobic interaction FPLC

Poly(ethylene glycol) pellets were resuspended with 390 mL of buffer B lacking (NH4)2SO4 and centrifuged at 35 000 g for 20 min. The extract was adjusted to 25% (saturation) (NH4)2SO4 by the addition of solid (NH4)2SO4. The solution was neutralized with NH4OH, stirred for 20 min, centrifuged as above, and adsorbed at 3.6 mL·min−1 onto a column (2.5 × 19 cm) of butyl-Sepharose Fast Flow (Pharmacia) that had been preequilibrated with buffer B. The column was connected to a FPLC system and washed with buffer B (about 400 mL) until the A280 decreased to < 0.05. Adsorbed proteins were eluted at 3 mL·min−1 with a linear gradient (470 mL) of decreasing concentrations of buffer B (100–40%) and simultaneously increasing concentrations of buffer C (0–60%) (fraction size = 12 mL). Pooled peak PK activity fractions were concentrated to 30 mL with an Amicon YM-100 ultrafilter. An equal volume of a 50% (w/v) poly(ethylene glycol) 8000 solution was added to the concentrated fractions, stirred for 30 min, and centrifuged as above. The poly(ethylene glycol) pellets were frozen and stored overnight in liquid N2.

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

The poly(ethylene glycol) pellets were solubilized in 16 mL of buffer D 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 × 5.5 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. PK activity was eluted in two peaks following application of a linear 0–300 mm KCl gradient (90 mL) in buffer D as shown in Fig. 1 (fraction size = 5 mL). The PKc and PKp peak activity fractions were separately pooled and concentrated to about 5 mL with an Amicon YM-100 ultrafilter. The pooled PKp concentrate was divided into 0.5 mL aliquots, frozen in liquid N2, and stored at −80 °C (to be used for analysis of its heat stability and immunological properties). The pooled PKc concentrate was desalted on two Bio-Rad PD-10 columns equilibrated with buffer E.

Figure 1.

Separation of B. napus PKc and PKp via DEAE-Fractogel anion-exchange FPLC. The KCl gradient was plotted on a chart recorder by the gradient programmer of the FPLC system used to run the column. PK activity (●), and protein (○) and KCl (– – –) concentrations.

ADP-agarose affinity chromatography

This step was conducted at room temperature. The desalted PKc sample was loaded at 0.6 mL·min−1 onto a column (0.5 × 6.2 cm) of ADP-agarose (Sigma, cat. A-4398) that had been preequilibrated with buffer E. The column was washed with buffer E until the A280 decreased to baseline, and PKc was eluted in a sharp peak by the application of buffer E containing 2 mm PEP (fraction size = 1.8 mL). Peak activity fractions were pooled, concentrated to 2.5 mL with an Amicon XM-50 ultrafilter, desalted into buffer E using a Bio-Rad PD-10 column, reconcentrated to 0.7 mL, divided into 20-µL aliquots, frozen in liquid N2, and stored at −80 °C until used. The purified PKc was stable for at least 6 months when stored frozen.

Determination of native molecular mass via Superose 6 gel filtration

A Superose 6 HR 10/30 gel filtration column (Pharmacia) was connected to a Waters HPLC system and preequilibrated at room temperature with 20 mm imidazole-HCl (pH 7.1), 50 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm EDTA, and 20% (v/v) glycerol. Purified PKc (sample volume, 200 µL) was chromatographed at a flow rate of 0.2 mL·min−1 and 0.25-mL fractions were assayed for PK activity and A280. The native molecular mass was estimated from a plot of Kav (partition coefficient) vs. log m for the following protein standards: thyroglobulin (669 kDa), ferritin (440 kDa), β-amylase (220 kDa), aldolase (158 kDa), and alcohol dehydrogenase (150 kDa). Blue dextran was used to determine the column's void volume.

Antibody production

After collection of preimmune sera, 200 µg of purified B. napus PKc (dialyzed against NaCl/Pi= 20 mm KPi, pH 7.4, containing 150 mm NaCl) was emulsified in Ribi adjuvant (total volume 1 mL) and injected subcutaneously into a 2-kg New Zealand rabbit. Booster injections of 100 µg were given at 4 and 5 weeks. Seven days after the final injection, blood was collected by cardiac puncture and incubated on ice for 3 h. Clotted cells were removed by centrifugation at 1500 g for 10 min at 4 °C. The crude antiserum was frozen in liquid N2 and stored at −80 °C in 0.04% (w/v) NaN3. For immunoblotting, the antibodies were affinity-purified against 25 µg of purified B. napus PKc, as previously described [6], with the exception that the PKc protein band on the poly(vinylidene difluoride) membrane was visualized by staining for 15 s with 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid. Destaining was performed in distilled H2O prior to band excision and subsequent affinity-purification of the anti-(B. napus PKc) IgG.

The antibody production protocol was in accord with the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care) and was approved by the Queen's University Animal Care Committee.

Immunotitration of PK activity

Immunoremoval of enzyme activity was tested by mixing 0.05 U of homogeneous B. napus PKc or partially purified B. napus PKp 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 PKc, castor PKc, or castor PKp) 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 a 17 000 g. Residual PKc or PKp activity in the supernatant fraction was determined as described above.

SDS/PAGE and immunoblotting

The procedures for SDS/PAGE, determination of subunit molecular mass, and immunoblotting were as described previously [22], except that the acrylamide monomer concentration in the separating gels was 10% (w/v). Immunological specificities were confirmed by performing immunoblots in which rabbit preimmune serum was substituted for the affinity-purified anti-(B. napus or castor PKc) IgG.

Results

Enzyme purification

The maximal activity of PK in B. napus suspension cells was about 3.2 U·g−1 fresh weight. DEAE-Fractogel chromatography resolved major and minor peaks of PK activity eluting at approximately 100 and 200 mm KCl, respectively (Fig. 1). Immunoblotting of the pooled fractions with anti-(castor PKc or castor PKp) IgG established that the early and later eluting peaks of PK activity correspond to PKc and PKp, respectively. This assignment agrees with previous studies that consistently indicate PKc elution prior to PKp during anion-exchange chromatography of plant extracts [6,8,12]. As shown in Table 1, PKc was purified about 200-fold to a final specific activity of 51 U·mg−1 protein and an overall yield of 5%.

Table 1. Purification of PKc from 594 g of B. napus suspension cells. PEG, poly(ethylene glycol).
StepVolume
(mL)
Activity
(U)
Protein
(mg)
Specific activity
(U·mg−1)
Purification
(fold)
Yield
(%)
Clarified extract174519207563 0.25100
PEG fractionation 380 8401839 0.46  1.8 44
Butyl-Sepharose 194 452 161 2.8 11 24
DEAE-Fractogel  40 200  28 7.3 29 10
ADP-agarose   7 103   251201  5

Physical and immunological properties

The purified PKc was relatively heat stable, retaining 100% of its activity following incubation at 65 °C for 3 min. In contrast, the DEAE-Fractogel-purified PKp was completely inactivated following incubation at 60 °C for 3 min. SDS/PAGE of the final PKc preparation resolved a single Coomassie blue staining 56-kDa polypeptide (Fig. 2A, lane 2) that cross-reacted strongly with affinity-purified anti-(B. napus or castor PKc) IgG (Fig. 2B,C, lane 1). The enzyme's native molecular mass as estimated by gel filtration of the final PKc preparation on a calibrated Superose 6 column was 220 ± 10 kDa (mean ± SE, n = 3).

Figure 2.

SDS/PAGE and immunoblot analysisof B. napus PKc. (A) SDS/PAGE (10% separating gel) analysis of purified B. napus PKc. Lane 1 contains 8 µg of various protein standards. Lane 2 contains 2.5 µg of the final PKc preparation. Protein staining was performed with Coomassie Blue R-250. (B and C) Immunoblot analysis was performed using affinity-purified anti-(B. napus PKc) IgG (B) or anti-(castor PKc) IgG [6] (C). Antigenic polypeptides were visualized using an alkaline phosphatase-tagged secondary antibody as described in [6]. Phosphatase staining was for 5–10 min at 30 °C. Lane 1 contained 10 ng of purified B. napus PKc. Lanes 2 and 3 each contain 5 µg of protein from a clarified extract prepared from B. napus suspension cells and developing B. napus seed cotyledons, respectively.

The effect of anti-(B. napus or castor PKc) immune serum on the activities of B. napus PKc and PKp was examined. Complete removal of PKc activity occurred at about 60 and 300 µL of anti-(B. napus PKc) and anti-(castor PKc) immune serum per unit activity, respectively. In contrast, the activity of the partially purified B. napus PKp was unaffected by the anti-(castor seed PKc) immune serum. Similarly, the anti-(castor seed PKp) immune serum effectively immunoprecipitated B. napus PKp activity, but had no influence on the activity of PKc.

The affinity-purified anti-(B. napus PKc) IgG could readily detect 10 ng of denatured homogeneous B. napus PKc that had been electroblotted onto a membrane (Fig. 2B, lane 1). The anti-(B. napus PKc) IgG was monospecific for the 56-kDa subunit of PKc, as only this polypeptide showed a cross-reaction with this IgG when a clarified extract from B. napus suspension cells or developing seed embryos (at mid-cotyledon stage of development) was immunoblotted (Fig. 2B, lanes 2 and 3). Analogous results were obtained when immunoblots of the same extracts were probed with the anti-(castor seed PKc) IgG (Fig. 2C, lanes 2 and 3). In contrast, no cross-reactivity was found when an immunoblot of 0.25 µg of purified B. napus PKc was probed with anti-(castor seed PKp) IgG, or 0.5 µg of partially purified B. napus PKp was probed with anti(castor or B. napus PKc) IgG.

Kinetic Properties

Effect of pH. The B. napus PKc displayed a broad pH/activity profile with a maximum occurring at about pH 6.8. All subsequent PKc kinetic studies were performed at pH 6.8 unless otherwise noted.

Cofactor requirements and substrate saturation kinetics.Table 2 summarizes the Vmax and apparent Km values obtained for metal cation cofactors and substrates of PKc at pH 6.8 and 7.4. The enzyme exhibited hyperbolic saturation kinetics for PEP, ADP, Mg2+ and K+, and its activity showed an absolute dependence for both a monovalent and bivalent cation with K+ and Mg2+ fulfilling this requirement. Increasing the assay pH from 6.8 to 7.4 slightly reduced the enzyme's affinity for K+ while approximately doubling its affinity for Mg2+ (Table 2). Increasing the assay pH to 7.4 led to ≈ 15% reduction in Vmax. However, the enzyme's apparent Km value for PEP was about 35% lower at pH 7.4, than at pH 6.8 (Table 2).

Table 2. Influence of pH and various metabolites on kinetic constants of B. napus PKc for PEP and metal cation cofactors. ND, not determined.
ParameterAdditionpH 6.8pH 7.4
Vmax (U·mg−1) 51 43
10 mm l-Asp 71 60
10 mm l-Glu 28.6 15.4
 0.15 mm Quercetin22.4ND
Km (PEP) (mm)0.120.078
10 mm l-Asp0.0970.074
10 mm l-Glu0.200.28
 0.15 mm Quercetin0.33ND
Vmax/Km (PEP) (U·mg−1·mm−1)  425551
10 mm l-Asp731810
10 mm l-Glu143 55
 0.15 mm Quercetin 75ND
Km (K+) (mm)0.210.36
Km (Mg2+) (mm)0.480.22

B. napus PKc can utilize alternative nucleotide diphosphates as substrates (Table 3). Although the Vmax value obtained with saturating UDP, CDP, or IDP was similar to that obtained with ADP, the apparent Km values for the alternative nucleotide diphosphates were up to 50-fold greater than the Km(ADP) value. Consequently, the maximal specificity constant (Vmax/Km) was achieved with ADP (Table 3), indicating that it is the preferred nucleotide substrate for the enzyme. In contrast to castor endosperm PKc, which demonstrated substrate inhibition by ADP at concentrations greater than 1 mm[14], but similarly to the enzyme from other plant sources [7,11,12], no inhibition of B. napus PKc by nucleotide diphosphate concentrations up to 10 mm was observed.

Table 3. Use of alternate nucleotide diphosphates by B. napus PKc. All assays were conducted at pH 6.8 in the presence of saturating (2 mm) PEP.
NucleotideVmax
(U·mg−1)
Km
(mm)
Vmax/Km
(U·mg−1·mm−1)
ADP51.00.075680
UDP49.20.16307
IDP58.30.68 86
CDP49.24.10 12

Metabolite effects. A wide variety of compounds were tested for effects on PKc at pH 6.8 and 7.4 with subsaturating concentrations of PEP and ADP (0.1 mm each). The following compounds had little or no influence (± 20% control velocity) on PKc activity: 20 mm l-Gln; l-Asn, dl-isocitrate, and KPi (10 mm each); 5 mm l-Arg; Gly, l-Ser, MgAMP, l-Ala, Glc-6-P, and 3-P-glycerate (4 mm each); NaNO3, NH4Cl, ribose-5-P, and 2-P-glycerate (2 mm each); MgPPi and dihydroxyacetone-P (1 mm each); 5 µm Fru-2,6-P2; and 2% (v/v) dimethylsulfoxide. Preincubation of PKc with 50 mm dithiothreitol or 10 mm oxidized glutathione for 30 min at 30 °C also had no effect on enzymatic activity.

Activators. The only activator of B. napus PKc was l-Asp (Tables 4 and 5, Figs 3 and 4). As an activator, l-Asp functions by increasing Vmax by approximately 40%, while slightly lowering the Km(PEP) value (Table 2). Notably, increasing concentrations of l-Asp completely reversed the enzyme's inhibition by l-Glu (Fig. 3). When l-Glu was in excess of 5 mm, l-Asp became an extremely effective PKc activator, such that 5 mm l-Asp caused up to a 500% increase in enzymatic activity (relative to the approximate 50% increase in PKc activity when l-Glu was absent) (Fig. 3). In contrast, 10 mm l-Asp exerted no influence on the inhibition of PKc by quercetin.

Table 4. Effect of various metabolites on the activity of B. napus PKc. Assays were conducted at pH 6.8 or 7.4 using subsaturating (0.1 mm each) concentrations of PEP and ADP. Enzymatic activity in the presence of effectors is expressed relative to the respective control set at 100%.
AdditionConcentrationRelative activity
 (mm)pH 6.8pH 7.4
MgOxalate 0.4 68 38
MgMalate 4 85 67
2-Oxoglutarate 4 78 67
MgCitrate 4 76 66
Fru-1,6-P2 1 65 83
Glycerol-3-P 2 63 79
l-Asp10160160
l-Glu 5 47 30
l-Phe0.2 67 82
l-Trp0.2 65 90
l-Tyr0.2 72 83
Shikimate0.1 73 77
Rutin0.1 54 39
Quercetin0.1 47 35
MgATP 2 75 60
Table 5. Kinetic constants for several effectors of B. napus PKc. Assays were conducted at pH 6.8 with subsaturating concentrations of PEP and ADP (0.1 mm each).
EffectorI50 (mm)Ka (mm)
  1. a These represent I0.5 values (l-Glu concentration required for half-maximal inhibition) under the given assay conditions. b Values in parentheses indicate the fold-activation of PKc by saturating l-Asp under the given assay conditions.

l-Glu4.2
 + 0.3 mm l-Asp6.0a
 + 0.6 mm l-Asp16.8a
 + 0.9 mm l-Asp22.4a
l-Asp0.3 (1.6)b
 + 5 mm l-Glu1.2 (2.7)
 + 20 mm l-Glu3.1 (3.8)
MgOxalate0.52
Rutin0.07
Quercetin0.10
MgATP12
Figure 3.

Influence of l-Asp on the l-Glu saturation kinetics of B. napus PKc. Assays were conducted at pH 6.8 with subsaturating PEP and ADP (0.1 mm each) in the presence or absence of various concentrations of l-Asp.

Figure 4.

Influence of l-Glu on the l-Asp saturation kinetics of B. napus PKc. Assays were conducted at pH 6.8 activity with subsaturating PEP and ADP (0.1 mm each) in the absence (●) and presence of 5 mm (▪) or 20 mm (▴) l-Glu.

Inhibitors. PKc was potently inhibited by l-Glu, Mg-oxalate, and the flavonoids quercetin and rutin (Table 4). Inhibition by these compounds was more pronounced at pH 7.4, relative to that occurring at pH 6.8 (Table 4). l-Glu and quercetin significantly reduced Vmax of PKc, while increasing its Km(PEP) value (Table 2). Preincubation studies (see Materials and methods) indicated that inhibition of PKc by 0.1 mm rutin or quercetin was completely reversible.

l-Glu also functioned as an inhibitor by reducing the enzyme's affinity for its activator, l-Asp (Table 5, Fig. 4). As l-Asn and l-Gln are end-products of NH +4 assimilation, their influence on l-Glu inhibition of PKc was also examined. However, the enzyme's I50(l-Glu) was unchanged by the inclusion of 10 mm l-Gln or 5 mm l-Asn in the assay mixture. Moreover, the addition of 10 mm l-Gln to the 0.6 mm l-Asp curve in Fig. 3 did not alter the shape of the curve suggesting that l-Gln does not influence the inhibition of PKc by l-Glu either alone or in the presence of l-Asp. Under no circumstances was PKc inhibited or activated with varying combinations and concentrations (up to 160 mm) of l-Asn, l-Gln, l-Ser, Gly, or l-Ala, nor were any of these amino acids found to influence l-Glu inhibition of PKc.

Discussion

PKc purification, physical and immunological properties

DEAE-Fractogel anion-exchange chromatography of the pooled butyl-Sepharose fractions resolved two PK activity peaks (Fig. 1). Immunological studies using monospecific rabbit antibodies against castor seed PKc and PKp demonstrated that the early and later eluting peaks of PK activity from the DEAE-column corresponded to PKc and PKp, respectively. This is consistent with the finding that B. napus PKc and PKp were relatively heat stable and heat labile, respectively. All plant and green algal PKc proteins examined to date are more heat stable than the corresponding PKp[6,8,10,24,25]. Heat treatment was used as a purification step in isolating PKc from germinating castor bean endosperm [10], as well as to discriminate between PKc and PKp activity in extracts from developing castor oil seeds [6] and tobacco leaves [18].

The B. napus PKc was purified to homogeneity (Fig. 2A), and a final specific activity of 51 U·mg−1 protein (Table 1), a value that compares favorably with that reported for other homogenous plant PKc proteins [10,12,15]. Gel filtration and SDS/PAGE analyses of the final preparation indicated that B. napus PKc is a 220-kDa tetramer composed of identical 56-kDa subunits. The native molecular mass and subunit composition of the B. napus PKc is similar to castor oil plant PKc which exists as a 240-kDa heterotetramer in both the leaves and germinating endosperm, but as a 240-kDa homotetramer in the developing endosperm and germinating cotyledons [6,10,12]. Similarly, PKc has been reported to be a 224-kDa homotetramer and 590-kDa homodecamer in the green algae Chlamydomonas reinhardtii and S. minutum, respectively [24,25]. Immunoblotting of purified B. napus PKc and clarified extracts of B. napus suspension cells or developing seed embryos with anti-(B. napus PKc)-IgG or anti-(castor PKc)-IgG uniformly resulted in a single 56-kDa antigenic polypeptide (Fig. 2B,C). These results indicate that the anti-(B. napus PKc) IgG is monospecific for PKc, and that the PKc of the embryogenic pollen-derived B. napus suspension cells is also expressed in developing B. napus embryos.

Kinetic studies

Similar to several other plant and algal PKc proteins [11,12,14,15], the activity of the B. napus enzyme exhibited: (a) a broad pH profile centered at about pH 6.8, (b) increased catalytic efficiency (Vmax/Km) for PEP and inhibitor sensitivity at pH 7.4 compared to pH 6.8 (Tables 2 and 4), (c) hyperbolic substrate and cofactor saturation kinetics, (d) an absolute dependence for both a monovalent and bivalent cation, and (e) nonabsolute specificity for ADP, with good use of alternative purine and pyrimidine nucleotide diphosphates, particularly UDP (Table 3). In contrast, the partially purified B. napus PKp displayed a relatively sharp pH activity profile centered at pH 8.0 (C. R. Smith, V. L. Knowles & W. C. Plaxton, unpublished data).

Metabolite effectors

ATP inhibition is widespread among plant and nonplant PKs. However, the I50 of the B. napus enzyme for MgATP is well in excess of estimated levels of ATP in the plant cytosol. This, along with the lack of effect of MgAMP, indicates that energy charge does not play a major role in regulating the in vivo activity of the B. napus PKc. This contrasts with many nonplant PKs, as well as PKc from endosperm of germinating castor oil seeds [14,23], where regulation by energy charge is believed to be a key aspect of the enzyme's in vivo control. The activity of B. napus PKc was quite responsive to several metabolites involved in carbon and nitrogen metabolism. The enzyme demonstrated potent inhibition by oxalate (Tables 4 and 5), a phenomenon common to plant and nonplant PKs. This inhibition is believed to arise from the close structural similarity between oxalate and the enolate form of pyruvate [26]. Plant cells can contain significant amounts of oxalate; although mainly vacuolar, some oxalate may be cytoplasmic [27]. Measurement of the cytosolic oxalate pool is necessary to determine if oxalate inhibition of B. napus and other plant PKc proteins is physiologically relevant.

Similar to B. napus and C4-leaf PEPCs [22,28], B. napus PKc also showed potent inhibition by the flavonoids rutin and quercetin (Tables 2,4 and 5). To our knowledge, this is the first time that any PK has been discovered to be susceptible to flavonoid inhibition. As the inhibition of PKc by 0.1 mm rutin or quercetin inhibition was reversible, this indicates that flavonoids did not induce a permanent structural dislocation in the enzyme. Quercetin or l-Glu both increased the enzyme's Km(PEP) while decreasing Vmax (Table 2), suggesting that they are mixed-type inhibitors that bind to an allosteric inhibitor site. However, unlike l-Glu inhibition (Fig. 3), quercetin inhibition of PKc was not relieved by l-Asp. In contrast to B. napus PKc, the activity of the partially purified B. napus PKp or homogeneous banana fruit PKc is not influenced by 0.1 mm quercetin or rutin (C. R. Smith, W. L. Turner & W. C. Plaxton, unpublished data). Both the evolutionary and physiological relevance of these findings is not yet evident.

As reported for several vascular plant and green algal PKc proteins [5,7,11,12,17,18,25], l-Glu was an effective inhibitor of the B. napus enzyme (Tables 4 and 5, Fig. 3). l-Glu decreased the enzyme's affinity for its substrate PEP (Table 2), and activator l-Asp (Table 5, Fig. 4). As with the castor leaf enzyme [12], l-Asp functioned as a B. napus PKc activator mainly by relieving the enzyme's inhibition by l-Glu (Table 5, Fig. 3). Furthermore, l-Glu increased the activation of PKc by, and its Ka for, l-Asp (Table 5, Fig. 4) indicating a complex interaction between the enzyme and these amino acids. Figure 4 shows that in the absence of l-Glu, no activation of PKc occurred with l-Asp levels in excess of 60 mm, and that very high (> 100 mm) l-Asp concentrations were inhibitory. This inhibition is probably due to l-Asp binding to the l-Glu inhibitor site of PKc, as inhibition by 160 mm l-Asp was not observed in the presence of 5 or 20 mm l-Glu (Fig. 4). Our results demonstrate that the inhibition of B. napus PKc by l-Glu, and the reversal of this inhibition by l-Asp, is specific for these amino acids. It is likely the concentration ratio of cytosolic Asp/Glu, rather than their absolute levels per se, is a major determinant of B. napus PKc activity in vivo. This reciprocal control of B. napus PKc is remarkable considering that l-Glu differs from l-Asp only by having an extra carbon atom in its functional group. As discussed below, the allosteric regulation of B. napus PKc and PEPC [22] by l-Asp and l-Glu indicates their essential role in controlling the provision of carbon skeletons and respiratory substrates during NH +4 assimilation by GS/GOGAT.

Coordinate regulation of B. napus PKc and PEPC by allosteric effectors during N-assimilation

The stimulation of respiration that occurs in vascular plants and green algae during periods of active nitrogen uptake, has been ascribed to an initial activation of PKc and PEPC leading to increased production of citric acid cycle carbon skeletons needed for NH +4 assimilation and transamination reactions [17]. 2-Oxoglutarate is required as an acceptor for NH +4 in the plastid-localized GS/GOGAT pathway, oxaloacetate is needed for l-Asp production via cytosolic AAT, and l-malate and citrate are required as counterions to replace nitrate and prevent alkalinization. l-Malate and oxaloacetate are synthesized from PEP via PEPC, whereas citrate and 2-oxoglutarate are synthesized from PEP via the concerted action of PEPC and PKc(Fig. 5). The conversion of isocitrate to 2-oxoglutarate has been suggested to occur in the cytosol, catalyzed by NADP-isocitrate dehydrogenase [29]. Following incorporation of NH +4 into l-Glu and l-Asp, amino acids such as l-Ala, Gly and l-Ser are generated via transaminations with l-Glu or l-Asp, and l-Asn is formed from l-Asp and l-Gln via Asn synthetase. The minor amino acids are synthesized in longer biosynthetic pathways in which l-Glu (and occasionally l-Gln or l-Asp) act as the NH +4-donor.

Figure 5.

A model for the allosteric regulation of PKc and PEPC in B. napus suspension cells. The coordinate control of PKc and PEPC [22] by allosteric effectors, particularly l-Glu and l-Asp, provides a mechanism for the control of cytosolic glycolytic flux and PEP partitioning during and following NH +4-assimilation as discussed in the text. AAT, Asp aminotransferase; ACON, aconitase; Cit; citrate; CS, citrate synthase; GS, Gln synthetase; GOGAT, Gln 2-oxoglutarate aminotransferase; IC, isocitrate; Mal, malate; MDH, malate dehydrogenase; NADP-ICDH, NADP-dependent isocitrate dehydrogenase; NR, nitrate reductase; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; PDC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase PKc, cytosolic pyruvate kinase; Pyr, pyruvate.

Figure 5 presents a model summarizing the allosteric features of PEPC and PKc that may be important in coordinating carbon and nitrogen metabolism in B. napus suspension cells. The regulatory features of both enzymes appear to be well suited to their central role in providing the mitochondria with respiratory substrates as well as for generating carbon skeletons for NH +4-assimilation via GS/GOGAT and AAT. Hexose-6-P activation of PEPC serves to balance sucrose availability with the flux of PEP carboxylation to dicarboxylic acids via PEPC. l-Malate inhibition of PEPC provides a tight feedback regulation that closely links PEPC activity with the overall rate of l-malate metabolism. PEPC inhibition by l-Asp, isocitrate and l-Glu provides additional feedback controls that balance PEPC activity with the production of carbon skeletons required by GS/GOGAT and AAT. In particular, however, the combined results of the current and preceding [22] manuscripts indicate a central role for l-Asp and l-Glu in the coordinate allosteric control of the PEP branchpoint in vascular plants. Feedback inhibition of B. napus PKc and PEPC by l-Glu provides a rationale for known activation of the two enzymes that occurs in vivo during periods of enhanced nitrogen assimilation (when cellular l-Glu concentrations are reduced) [17]. In contrast to PEPC, l-Asp functions as an allosteric activator of the B. napus PKc by effectively relieving the enzyme's inhibition by l-Glu (Fig. 3). Reciprocal control of B. napus PKc and PEPC by l-Asp provides an intriguing mechanism for decreasing flux from PEP to l-Asp (via PEPC and AAT) while promoting PKc activity when cytosolic l-Asp levels become elevated. This would be expected to occur when the cell's demands for nitrogen are satisfied, and the overall rate of protein synthesis becomes more dependent upon ATP availability, rather than the supply of amino acids. In this instance, respiration (and hence PKc) may assume a more significant function in terms of satisfying a large ATP demand, rather than the generation of biosynthetic precursors. The production of transgenic plants expressing allosteric mutants of PEPC and PKc may help to define the precise roles of l-Asp and l-Glu in coordinating glycolytic flux and PEP partitioning with N-assimilation in vascular plants.

Acknowledgments

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 and Jean Rivoal for helpful discussions.

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); phosphofructokinase (EC 2.7.1.11); pyruvate kinase (EC 2.7.1.40).Note: a web page is available at http://biology.queensu.ca/

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