Phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase protein kinase from developing castor oil seeds: partial purification,characterization, and reversible control by photosynthate supply

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified ∼1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca²⁺-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (K _m = 2.2 μM) activated PEPC1 by ∼80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of ∼pH 8.5, and at pH 7.3 was activated 40–65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.     

Molecular evolution of C4 phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase in the genus<Emphasis Type="Italic"> Flaveria</Emphasis>—a gradual increase from C3 to C4 characteristics

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.     

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C₄ phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro ³²P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH₄Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca²⁺ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca²⁺ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C₄ MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C₄-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C₄ mesophyll cytosol.Abbreviations BS bundle-sheath - CAM Crassulacean acid metabolism - DHAP dihydroxyacetone phosphate - FPLC fast-protein liquid chromatography - Glc6P glucose 6-phosphate - I_0.5 50% inhibition constant - MC mesophyll cell(s) - MP me-sophyll protoplast(s) - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC protein-Ser/Thr kinase - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - Pyr pyruvate - Ser serine The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.     

Physiological implications of the kinetics of maize leaf phosphoenolpyruvate carboxylase

It has been a common practice to assay phosphoenolpyruvate carboxylase (PEPC) under high, nonphysiological concentrations of Mg(2+) and bicarbonate. We have performed kinetic studies on the enzyme from maize (Zea mays) leaves at near physiological levels of free Mg(2+) (0.4 mM) and bicarbonate (0.1 mM), and found that both the nonphosphorylated and phosphorylated enzymes exhibited a high degree of cooperativity in the binding of phosphoenolpyruvate, a much lower affinity for this substrate and for activators, and a greater affinity for malate than at high concentrations of these ions. Inhibition of the phosphorylated enzyme by malate was overcome by glycine or alanine but not by glucose-6-phosphate, either in the absence or presence of high concentrations of glycerol, a compatible solute. Alanine caused significant activation at physiological concentrations, suggesting a pivotal role for this amino acid in regulating maize leaf PEPC activity. Our results showed that the maximum enzyme activity attainable in vivo would be less than 50% of that attainable in vitro under optimum conditions. Therefore, the high levels of PEPC protein in the cytosol of C(4) mesophyll cells might be an adaptation for sustaining the steady-state rate of flux through the photosynthetic CO(2) assimilation pathway despite the limitations imposed by the PEPC kinetic properties and the conditions of its environment.     

Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: An immunological study using specific anti-phosphorylation site-antibodies

A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C₄-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C₄ PEPCs mutated in the N-terminal phosphorylation domain and a C₃-like PEPC isozyme from Sorghum behaved similarly to their C₄ counterpart with respect to these phosphorylation-site antibodies.Abbreviations PEPC phospho enolpyruvate carboxylase - PKA catalytic subunit of the cAMP-dependent protein kinase - KLH Keyhole Limpet Haemocyanin - IgG immunoglobulin G - PEP phospho enolpyruvate - SDS-PAGE sodium dodecyl sulfate, polyacrylamide gel electrophoresis - MDH malate deshydrogenase     

Phosphoenolpyruvate Carboxylase During Development of Crassulacean Acid Metabolism and During a Diurnal Cycle in Mesembryanthemum Crystallinum

Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinumwas induced by transfer of plants from 100 to 400 mM NaCl. Diurnalmalate fluctuations developed slowly; maximum rates of net malatesynthesis in the dark were reached only on the 10th day afterNaCl was increased to 400 mM. In contrast, phosphoenolpyruvatecarboxylase (PEPC) activity, assayed at optimum pH of 8–0,had nearly reached its maximum on the 5th day after plants weretransferred to 400 mM NaCl. Characteristics of PEPC changedduring the first 12 d of exposure of plants to 400 mM NaCl.There were increases in the ratio of PEPC activity at pH 7 0/PEPCactivity at pH 8.0, and decreases in the Km for PEP measuredat pH 7.0, and possibly in the degree of malate inhibition.All further measurements were made once CAM was well established.In vivo rates of malate synthesis were 14–18 times smallerthan PEPC activity at 2 mM PEP, both processes being measuredat 15 °C. It is suggested that the high PEPC levels favourrapid, preferential flow of carbon to malate, by maintainingvery low PEP levels in the cytoplasm. PEPC changed in characteristicsduring the diurnal cycle. During the first few minutes afterisolation, extracts made during the first hours of the day,when malate was consumed, showed very low PEPC activity at pH7.0 but high activity at pH 8.0. The activity of PEPC at pH7.0 rose gradually during storage of the extracts at 0 °C,usually reaching the activity at pH 8.0 after about 30–50min. In contrast, extracts obtained during the first hours ofthe night, when malate was synthesized, showed high PEPC activityat both pH 7.0 and 8–0 within 30–50 s after extraction.The results indicate that PEPC of M. crystallinum, performingdistinct CAM, may exist in two states. One state would favourrapid malate synthesis and transport to the vacuoles and wouldfunction during the night. The second state, with little activitybelow pH 7.5, would occur during the day, thus preventing complicationsof continued synthesis of malate while it is converted to carbohydrates.     

Effects of cadmium on the co-ordination of nitrogen and carbon metabolism in bean seedlings

The effect of cadmium (Cd) was investigated on the in vitro activities of leaf and root enzymes involved in carbon (C) and nitrogen (N) metabolism of bean (Phaseolus vulgaris L. cv. Morgane). Cd induced a high increase in maximal extractable activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). Cd promoted ammonium accumulation in leaves and roots, and a tight correlation was observed between ammonium amount and GDH activity. Changes in GDH activity appear to be mediated by the increase in ammonium levels by Cd treatment. Cd stress also enhanced the activities of phosphoenolypyruvate carboxylase (PEPC, EC 4.1.1.31) and NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH, EC 1.1.1.42) in leaves while they were inhibited in roots. Immuno-titration, the PEPC sensitivity to malate and PEPC response to pH indicated that the increase in PEPC activity by Cd was due to de novo synthesis of the enzyme polypeptide and also modification of the phosphorylation state of the enzyme. Cd may have modified, via a modulation of PEPC activity, the C flow towards the amino acid biosynthesis. In leaves, Cd treatments markedly modified specific amino acid contents. Glutamate and proline significantly accumulated compared to those of the control plants. This study suggests that Cd stress is a part of the syndrome of metal toxicity, and that a readjustment of the co-ordination between N and C metabolism via the modulation of GDH, PEPC and ICDH activities avoided the accumulation of toxic levels of ammonium.     

Changes in properties of phosphoenolpyruvate carboxylase from the CAM plant Sedum praealtum D.C. upon dark/light transition and their stabilization by glycerol

A prenounced decrease in phosphoenolpyruvate earboxylase (PEPC) activity is observed upon dark/light transition in Sedum praealtum D.C., only when glycerol is included in the extraction medium. If glycerol is omitted, the activity extracted in light is initially low, but soon reaches night levels. The stabilization of the light-induced form of the enzyme by glycerol, in crude or desalted extracts, made it possible to study its kinetic properties in comparison to those of the dark form. The behaviour towards substrate (PEP) changes from hyperbolic (dark) to sigmoid (light), S_0.5 is increased and the enzymic activity becomes more sensitive to malate inhibition. Quite different activity/pH profiles are also obtained for the two forms of PEPC.It is inferred that the in vivo regulation of PEPC in CAM is effected by a concerted action of light, malate and pH shifting.     

Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities

Maximum velocity and Km(PEP.Mg) of phosphoenolpyruvate carboxylase (PEPC) from stomatal guard cells of Vicia faba L. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparent in vivo activity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, post-translational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 x 10(-9) g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata. Vmax at pH 7.0 was not significantly different from that at pH 8.5. Neither Vmax nor Km(PEP.Mg) was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However, Km(PEP.Mg) was greater than 4x lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was greater than 3x greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.     

Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C4 plant. When leaves were illuminated, the apparent Km for (HCO3-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in Vmax and 3- to 4-fold increase in Ki for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO3-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent Km (HCO3-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent Km (HCO3-) of the enzyme, in addition increasing Ki (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO3- by illumination in C4 leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO3- in mesophyll cells, the sensitization by light of PEPC to HCO3- could be physiologically quite significant.     

The Interactive Effects of pH,L-Malate,and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase

The interactive effects of pH, L-malate, and glucose-6-phosphate (Glc-6-P) on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. GC pairs dissected from the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P (0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ GC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous concentrations would seem both to activate the enzyme and to diminish the dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on GC PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that GC PEPC is regulated by its cytosolic chemical environment and not by posttranslational modification that is detectable at physiological levels of effectors. It is important to note, however, that we did not determine the phosphorylation status of GC PEPC directly or indirectly (by comparison of the concentration of L-malate that causes a 50% inhibition of GC PEPC).     

Malate content of picoliter samples of Raphanus sativus cytoplasm

Malate, which plays many essential roles in plant metabolism, is a potent in vitro inhibitor of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC). Because PEPC activity leads to malate biosynthesis, malate is assumed to attenuate its own synthesis in situ. To test this hypothesis, we measured directly the malate content of picoliter samples of Raphanus root-hair cytoplasm using quantitative histochemical techniques. We also obtained an estimate for malate accumulation in these cells. These values were compared with the PEPC activity of individual root hairs (less than 2 ng). The results indicate that high cytoplasmic malate concentration does not severely inhibit PEPC in situ. We suggest that the focus for studies on the regulation of organic anion accumulation be on the interactive effects of malate and other PEPC effectors.     

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.     

Phosphorylation of Soybean (Glycine max L.) Nodule Phosphoenolpyruvate Carboxylase in Vitro Decreases Sensitivity to Inhibition by L-Malate

Phosphoenolpyruvate carboxylase (PEPC) from soybean (Glycine max L.Merr.) nodules was purified 187-fold to a final specific activity of 56 units mg-1 of protein. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed one major polypeptide band, with a molecular mass of 110 kD, after the final purification step. Two-dimensional PAGE resolved four isoelectric forms of the purified enzyme. Antibodies raised against the purified enzyme immunoprecipitated PEPC activity from a desalted nodule extract. Two cross-reacting bands were obtained when protein immunoblots of crude nodule extracts subjected to SDS-PAGE were probed with the antiserum. One of these corresponded to the 110-kD subunit of PEPC, and the other had a molecular mass of about 60 kD. PEPC was shown to be activated in a time-dependent manner when desalted soybean nodule extracts were preincubated with Mg.ATP in vitro. Activation was observed when PEPC was assayed at pH 7 in the absence of glycerol but not at pH 8 in the presence of glycerol. When o.5 mM L-malate was included in the assay, activation was much more pronounced than without malate. Maximal activation was 30% in the absence of L-malate and 200% in its presence. The L-malate concentrations producing 50% inhibition of PEPC activity were o.35 and 1.24 mM, respectively, before and after preincubation with Mg.ATP. The antiserum against soybean nodule PEPC was used to immunoprecipitate PEPC from a desalted nodule extract that had been preincubated with Mg.[[gamma]-32P]ATP. The immunoprecipitate was then subjected to SDS-PAGE, followed by autoradiography. The autoradiograph revealed intense labeling of the 110-kD subunit of PEPC following preincubation with [[gamma]-32P]ATP. The data suggest that soybean nodule PEPC becomes phosphorylated by an endogenous protein kinase, resulting in decreased sensitivity of the enzyme to inhibition by L-malate in vitro. The results are discussed in relation to the proposed functions of PEPC in legume nodules.     

Purification and Characterisation of the Light and Dark Forms of Phosphoenolpyruvate Carboxylase from the Dicot Plant Amaranthus viridis L. An Examination of Its Kinetic and Regulatory Properties in the Presence of Water-Alcohol Binary Solvents

The light and dark forms of phosphoenolpyruvate (PEP) carboxylase(PEPC) from the dicot plant Amaranthus viridis L. were purifiedand their kinetic properties were studied in water-based orbinary alcohol-water solvents. At pH 7.3, the specific activityof the purified light form was about 2.7-fold higher than thatpresented by the dark form of PEPC under optimal conditions,while K_m remained virtually unchanged in both forms. The enzyme'slight form was better activated by glucose 6-phosphate and lessinhibited by L-malate than the dark PEPC. From the organic solventsstudied, methanol showed the most important effect, enhancingPEPC activity by two-fold at 20% (v/v). Ethanol, ethylene glycol,tert-butanol and 2-propanol were also activators to a lesserdegree, but at high concentrations (typically greater than 20%,v/v) the effect was reduced or turned to inhibition. K_m (PEP)was reduced by an order of magnitude in the presence of 20%(v/v) methanol (i.e. from 0.32 to 0.022 mM for the light formof the enzyme). The inhibitory effect of malate at low PEP waslessened by methanol for both forms (i.e. I₅₀ 0.25 mM in aqueousmedium to 0.48 mM in binary mixture for the dark form), whileglucose-6-P activation of PEPC was not affected by methanol.The results suggest that the kinetics of PEPC in a medium thatmimics more closely in vivo conditions are different from thoseobserved by standard procedures consisting of aqueous media,and provide a new insight on the properties of PEPC as relatedto its regulation in vivo. (Received June 26, 1995; Accepted August 24, 1995)     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N₂-fixing soybean ( Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phospho enol pyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 m M malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N₂ fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.     

Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C4 plant: decrease in malate sensitivity but no change in the phosphorylation status

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C4 plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by >2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The Ki for malate increased >2.3-fold and >1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in Km for PEP or Ka for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC.