The role of inorganic phosphate in the regulation of C4 photosynthesis

Inorganic phosphate participates in many fundamental processes within the plant cell. Its broad influence on plant metabolism is related to such key operations as metabolite transport, enzyme regulation and carbohydrate metabolism in general. This review discusses these topics with special emphasis on the role assigned to this ubiquitous anion within the C₄ pathway of photosynthesis.Abbreviations DHAP dihydroxyacetone phosphate - Ga3P glyceraldehyde-3-phosphate - NAD(P)-ME-NAD(P) dependent malic enzyme - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PFK and PFP-ATP- and PPi dependent fructose-6-phosphate 1-phosphotransferase - PPDK pyruvate:orthophosphate dikinase - RPPC reductive pentose-phosphate cycle - RuBisCO ribulose bisphosphate carboxylase-oxygenase - SPS sucrose-6-phosphate synthase     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.     

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate,orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the P_i-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for P_i-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.Abbreviations BSA bovine serum albumin - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PCMB p-chloromercuribenzoate - PEP phosphoenolpyruvate - PEPCase phosphoenol-pyruvate carboxylase - PPDK pyruvate, orthophosphate dikinase - PVP polyvinylpyrrolidone     

Enzymic activities of carbohydrate,purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes)

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161^T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PP_i-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PP_i-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P ₂). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P ₂), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PP_i-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.Abbreviations EMP Embden-Meyerhof-Parnas - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PEP phosphoenolpyruvate - PFK phosphofructokinase - PPDK pyruvate, orthophosphate dikinase - TCA cycle tricarboxylic acid cycle Note: Other abbreviations used are as per the instruction to authors, or the reference cited therein (Eur J Biochem 1:259), or Biochem J 120:449 (which supercedes a portion of the first reference)     

Effects of m-Cl-peroxy benzoic acid on glycolysis in Saccharomyces cerevisiae

Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.Abbreviations CPBA m-Chloro-peroxy benzoic acid - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - F-1,6-P₂ frnctose-1,6-bisphosphate - DAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - 2PGA 2-phosphoglycerate - PEP phosphoenol pyruvate - Pyr pyruvate - EtOH ethanol - PFK phosphofructokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - ADH alcohol dehydrogenase Dedicated to Prof. Dr. Wolfgang Gerok at the occasion of his 60th birthday     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.     

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons ofCucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with aKi of 5.9 M. At 25°C the inhibitor caused an increase in the label incorporated from [2-¹⁴C]acetate into CO₂, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (C _PEPCK ^J ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 M at 17°C, 13.3 M at 10°C) and had similar effects on the metabolism of [2-¹⁴C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.Abbreviations PEP Phosphoenolpyruvate - PEPCK PEP carboxykinase The authors thank Dr. Ian Woodrow (University of Melbourne, Australia) for helpful discussions. This work was supported by a grant from the Science and Engineering Research Council, U.K. (GR/F 50978).     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

The role of citrate on the oxidative metabolism of submerged cultures of Claviceps purpurea 129

In submerged cultures of Claviceps purpurea citrate utilization was stopped when the pool phosphate concentration decreased to 0.44–0.55 mmol per g of mycelial protein. The absence of citrate in the medium resulted in a 50% decrease of biomass, cessation of the alkaloid (agro-, elymo-, chanoclavine) synthesis, formation of a central large vacuole, cell enlargement and medium acidification by lactate. Citrate synthase and malate dehydrogenase gradually disappeared. Glucose-6-phosphate dehydrogenase was not detectable. The activities of glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase increased by one or two orders of magnitude higher than in control culture (with 80 mM citrate). Substrate competition on the pyruvate level is discussed.     

Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism

White lupin (Lupinus albus L.) acclimates to phosphorus deficiency (–P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. These specialized plant organs display increased exudation of citric and malic acid. The enhanced exudation of organic acids from P stressed white lupin roots is accompanied by increased in vitro phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity. Here we report the cloning of full-length white lupin PEPC and MDH cDNAs. RNA blot analysis indicates enhanced expression of these genes in –P proteoid roots, placing higher gene expression at the site of organic acid exudation. Correspondingly, macroarray analysis of about 1250 ESTs (expressed sequence tags) revealed induced expression of genes involved in organic acid metabolism in –P proteoid roots. In situ hybridization revealed that PEPC and MDH were both expressed in the cortex of emerging and mature proteoid rootlets. A C₃ PEPC protein was partially purified from proteoid roots of P deficient white lupin. Native and subunit Mr were determined to be 440 kD and 110 kD, respectively. Citrate and malate were effective inhibitors of in vitro PEPC activity at pH 7. Addition of ATP partially relieved inhibition of PEPC by malate but had little effect on citrate inhibition. Taken together, the results presented here suggest that acclimation of white lupin to low P involves modified expression of plant genes involved in carbon metabolism.     

Increases in phosphoenolpyruvate carboxylase activity in iron-deficient sugar beet roots: Analysis of spatial localization and post-translational modification

Root tips of Fe-deficient and Fe-sufficient sugar beet plants grown in hydroponics have been used to study the changes in the amount and activity of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31). Phosphoenolpyruvate carboxylase activity in extracts of the yellow Fe-deficient root tips was, at pH 7.3, 30-fold higher (when expressed on a FW basis) and 7.1-fold higher (when expressed on a protein basis) than that found in the extracts of Fe-sufficient root tips. The amount of phosphoenolpyruvate carboxylase protein determined by immuno-blotting was, on a protein basis, 35-fold larger in the yellow zone of Fe-deficient root tips than in the Fe-sufficient root tips. The inhibition of the phosphoenolpyruvate carboxylase activity by 500 m malate was 41 and 58% in the extracts Fe-deficient and Fe-sufficient roots. The possibility that post-translational regulation of phosphoenolpyruvate carboxylase may occur mediated through phosphorylation, was studied by immunological detection of phosphoserine residues in root tip extracts.     

Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K_m (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K_m (MgPEP) was found to be significantly lower than the respective K_m (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.     

Modification of Maize Leaf Phosphoenolpyruvate Carboxylase with Fluorescein Isothiocyanate

Fluorescein isothiocyanate inactivates phosphoenolpyruvate carboxylasefrom maize leaves, presumably by reacting with lysyl groups.The reaction appears to involve at least two groups of lysineson the enzyme. The more rapid reaction is with groups whichare protected by the substratemagnesium phosphoenolpyruvateand thus probably are located in the active site. In addition,fluorescein isothiocyanate apparently binds more slowly at asite which desensitizes the enzyme to activation by glucose-6-phosphate. Using the fluorescence of the complex of fluorescein isothiocyanatewith phosphoenolpyruvate carboxylase it was shown that bothmagnesium phosphoenolpyruvate and glucoses-6-phosphate causechanges in the conformation of the enzyme and influence thebinding of fluorescein isothiocyanate as well. Light scattering measurements showed that fluorescein isothiocyanateinduced disaggregation of the enzyme, while glucose-6-phosphatecaused aggregation, although less when fluorescein isothiocyanatewas present. ¹Supported in part by National Science Foundation grant no.DMB 88-12484.     

Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays— a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate—resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

Inactivation of maize phosphoenolpyruvate carboxylase by urea

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.     

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-naphalenesulfonate. The effects of ligands on kinetic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.     

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.