Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of <Emphasis Type="Italic">Sorghum vulgare</Emphasis>

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C₄ phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C₄ plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO₂ concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C₄ PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP₃ levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP₃ and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.     

Regulation of phosphoenolpyruvate carboxylase activity in maize leaves

The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C₄ plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S_0.5(phosphoenolpyruvate) but there was no change in V_max. The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6-phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated `dark' cytosol and high activity in a simulated `light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves.     

Characterization of phosphoenolpyruvate carboxylase from mature maize seeds: Properties of phosphorylated and dephosphorylated forms

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to l-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.     

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene

Summary A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.     

Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants

Phosphoenolpyruvate (PEP) carboxylase is regulated by reversible phosphorylation in higher plants. Recently several genes encoding PEP carboxylase kinase have been cloned. The purpose of this article is to assess the contribution that information on the structure and expression of these genes is making to our understanding of the posttranslational control of PEP carboxylase activity.     

Direct gene transfer inPetunia hybrida electroporated protoplasts: Evidence for co-transformation with a phosphoenolpyruvate carboxylase cDNA from sorghum leaf

Summary Co-transformation experiments were carried out onPetunia hybrida protoplasts. The method used was electroporation with two plasmids: one confering kanamycin resistance, and the other harbouring a phosphoenolpyruvate carboxylase (PEPC) cDNA fromSorghum vulgare leaves. Southern blot analysis of the selected lines demonstrated a high co-transformation frequency.Abbreviations PEPC phosphoenolpyruvate carboxylase - NPT neomycin phosphotransferase - ATF absolute transformation frequency - PEG polyethylenglycol - BA 6-benzyl-aminopurine - IAA 3-indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Purification and characterization of phosphoenolpyruvate carboxylase from maize leaves

Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s_20,w) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。     

Changes in protein quantities of phosphoenolpyruvate carboxylase and Rubisco activase in various wheat genotypes

In early seedlings of wheat genotypes two isoforms of Rubisco activase with molecular weights of 42 and 46 kDa are expressed. Amounts of both isoforms significantly increase in early seedlings of the durum wheat genotype Barakatli-95 exposed to salt stress. But at the beginning of the tillering stage, the changes in quantities of both RCA isoforms are different in durum and bread wheat genotypes subjected to a 3-day drought stress. In the leaves of the early seedlings of the studied wheat genotypes exposed to drought stress quantities of PEPC subunits increase compared to the control but they remain relatively stable in early roots and germinating seeds. However, quantities of its subunits decrease sharply in roots and germinating seeds of early seedlings under the influence of 100 mM NaCl. In flag leaves and ear elements of the Barakatli-95 genotype grown under normal water supply conditions protein quantities of PEPC subunits change differently depending on time. Changes in protein quantities of RCA, PEPC and Rubisco enzymes have been studied comparatively in ear elements and flag leaves after the fourth day of anthesis.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants

Phosphoenolpyruvate carboxylase (PEPC) has a variety of functions in plants, including a major anaplerotic role in replenishing the tricarboxylic acid cycle with intermediates to meet the demand of carbon skeletons for synthesis of organic acids and amino acids. Various transgenic C3 plants that overproduce PEPC have been produced and analyzed in detail. The results indicate that foreign PEPC is under the control of the regulatory mechanisms intrinsic to the host plant and down-regulated so as not to cause detrimental metabolic effects, although the anaplerotic reaction is slightly enhanced by the foreign PEPC. By use of foreign PEPCs that can avert such regulation, metabolic flow is largely directed toward synthesis of organic acids and amino acids. Observations with transgenic C3 plants also shed light on the interrelation among various metabolic pathways inside the cell.     

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO₂-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO₂ and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO₂ was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO₂ uptake did not correlate in phase or period.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase - RuBPCase ribulose-1,5-bisphosphate carboxylase To whom correspondence should be addressed.