BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus.

INTRODUCTION: Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. OBJECTIVES: This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. METHODS: We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. RESULTS: The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. CONCLUSION: Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

[Clinical significance of erythrocyte phosphoenolpyruvate in elderly and senior patients with iron deficiency anemia].

The article presents a part of the study dealing with erythrocyte glycolysis observed in 29 elderly and senior patients with iron deficiency anemia. The accumulation of phosphoenolpyruvate which is high-energy product of glycolysis testifies that erythrocyte glycolytic reactions are further enhanced. The change in phosphoenolpyruvate content may reflect the restoration and normalization of erythropoiesis and testify the appearance of new forms of erythrocytes, in particular, reticulocytes with natural type of glycolysis and energy metabolism. Clinical significance of obtained data is discussed in the article.

Transport of glucose by a phosphoenolpyruvate:mannose phosphotransferase system in Pasteurella multocida.

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.

Relationship between the concentrations of glycolytic intermediates and expression of the L-type pyruvate kinase gene in cultured hepatocytes.

Previous studies have suggested that some glycolytic intermediates are involved in the regulation of L-type pyruvate kinase gene expression by carbohydrates such as glucose and fructose. To find such intermediates, we examined the relationship between the levels of L-type pyruvate kinase mRNA and glycolytic metabolites in hepatocytes cultured under various conditions. Of the metabolites, the levels of 3-phosphoglycerate and phosphoenol-pyruvate only increased significantly under conditions under which the expression of the L-type pyruvate kinase gene was stimulated. The level of glucose 6-phosphate, which was reported to be involved in dietary stimulation of this gene expression, was not correlated with the mRNA level since marked accumulation of deoxyglucose 6-phosphate occurring on the addition of deoxyglucose, a nonmetabolizable glucose analog, was not accompanied by an increase in the L-type pyruvate kinase mRNA level. In addition, we found that fructose at a low concentration in the presence of glucose failed to increase the mRNA and metabolite levels in contrast to other reports that the promoter activity of the L-type pyruvate kinase gene is stimulated by this treatment. Thus we propose that 3-phosphoglycerate and/or phosphoenolpyruvate are involved in the carbohydrate regulation of L-type pyruvate kinase gene expression.

Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH4, enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. Therefore, to assay the enrichment of [2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [U-13C3]lactate or [U-13C3]glycerol. There is a 78% isotopic equilibration of 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.

Analysis of glycolysis metabolites by capillary zone electrophoresis with indirect UV detection.

The glycolysis metabolites glucose 6-phosphate (G6-P), fructose 6-phosphate (F6-P), fructose 1,6-bisphosphate (F1,6-BP), fructose 2,6-bisphosphate (F2,6-BP), glyceraldehyde phosphate (GAP), dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP), pyruvate, and lactate were analyzed by capillary zone electrophoresis (CZE) with indirect UV detection. The chromophores phthalic acid, sorbic acid, and 4-hydroxybenzoic acid were studied as background electrolytes. Both detection sensitivity and resolution were found to depend on the pH and the concentration of the carrier electrolyte. Optimum separation and detection of the phosphate compounds were accomplished upon reversal of electroendosmotic flow (EOF) with OFM Anion-BT (Waters Corp., Milford, MA) at a concentration of 4-6 mM 4-hydroxybenzoic acid, pH 11.6-12.0, with the detection wave-length set at 280 nm. The highly alkaline pH allowed the successful separation of the isomers F6-P and G6-P, as well as F1,6-BP and F2,6-BP, respectively. The effect of sample ionic strength on the detection limits of G6-P, F6-P, F1,6-BP, and F2,6-BP was also investigated: These limits ranged from 1 to 3 microM in both low- and high-ionic-strength samples. However, high Mg2+ concentrations in the sample led to a progressive loss of resolution between F1,6-BP and F2,6-BP, unless the inlet reservoir was replenished with fresh electrolyte after every injection. Linearity of detection was observed over one to two orders of magnitude.

[Marker histochemical demonstration of phosphoenolpyruvate at the light-optical and electron-microscopic levels]. / Markerno-gistokhimicheskoe vyiavlenie fosfoenolpiruvata na svetoopticheskom i élektronno-mikroskopicheskom urovniakh.

Results of staining section of rat liver using colloidal gold-labeled pyruvate kinase are described. On light microscopic level, centrolobular localized hepatocytes displayed the most intense specific staining. With electron microscopic observations, phosphoenolpyruvate residues were seen over granular zones in the nuclei, over the cisternae of the granular ER, mitochondria, and hepatocyte microvilli.

Metabolomics of supragingival plaque and oral bacteria.

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

Metabolic characterization of Escherichia coli strains adapted to growth on lactate.

In comparison with intensive studies of genetic mechanisms related to biological evolutionary systems, much less analysis has been conducted on metabolic network responses to adaptive evolution that are directly associated with evolved metabolic phenotypes. Metabolic mechanisms involved in laboratory evolution of Escherichia coli on gluconeogenic carbon sources, such as lactate, were studied based on intracellular flux states determined from 13C tracer experiments and 13C-constrained flux analysis. At the end point of laboratory evolution, strains exhibited a more than doubling of the average growth rate and a 50% increase in the average biomass yield. Despite different evolutionary trajectories among parallel evolved populations, most improvements were obtained within the first 250 generations of evolution and were generally characterized by a significant increase in pathway capacity. Partitioning between gluconeogenic and pyruvate catabolic flux at the pyruvate node remained almost unchanged, while flux distributions around the key metabolites phosphoenolpyruvate, oxaloacetate, and acetyl-coenzyme A were relatively flexible over the course of evolution on lactate to meet energetic and anabolic demands during rapid growth on this gluconeogenic carbon substrate. There were no clear qualitative correlations between most transcriptional expression and metabolic flux changes, suggesting complex regulatory mechanisms at multiple levels of genetics and molecular biology. Moreover, higher fitness gains for cell growth on both evolutionary and alternative carbon sources were found for strains that adaptively evolved on gluconeogenic carbon sources compared to those that evolved on glucose. These results provide a novel systematic view of the mechanisms underlying microbial adaptation to growth on a gluconeogenic substrate.

Determination of glycolytic intermediates in a flow injection system using immobilized enzymes.

Some glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, enolase and phosphoglyceromutase) were immobilized on a polyacrylamide-type bead polymer containing carboxylic functional groups activated by water-soluble carbodiimide. The immobilized enzymes were used for the determination of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric acid and 3-phosphoglyceric acid in a flow injection system. The immobilized lactate dehydrogenase column was repeatedly employed for the determination of pyruvic acid in clinical samples. The results of the flow injection method accorded well in accuracy, sensitivity and reproducibility with those of soluble enzyme analysis.

Isocratic high-performance liquid chromatographic determination of the concentration and specific radioactivity of phosphoenolpyruvate and uridine diphosphate glucose in tissue extracts.

A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of phosphoenolpyruvate and uridine diphosphate glucose (UDPG) has been developed. For each compound this method can measure tissue concentrations in the range 0.1-1000 nmol/g of tissue and determine specific radioactivity. All measurements can be performed in 200 mg of tissue. The recoveries of uridine diphosphate [6-3H]glucose and phosphoenol[1-14C]pyruvate from liver tissue homogenates were 97 and 99%, respectively. Following intra-arterial infusion of [6-3H]glucose and [U-14C]lactate in conscious rat, the concentration and specific radioactivity of phosphoenolpyruvate and UDPG were determined in rat liver. The method may be applied to experimentation in small animals using radiolabelled precursors in order to quantitate in vivo the glycogenic and gluconeogenic fluxes.

Regulation of autotrophic metabolism in Pseudomonas oxalaticus OX1 wild-type and an isocitrate-lyase-deficient mutant.

In Pseudomonas oxalaticus the activity and synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are regulated by inactivation and endproduct repression, respectively. Phosphoenolpyruvate (PEP) has been suggested to function as a signal molecule for the latter control system. During growth of the organism in carbon-source-limited continuous cultures with various ratios of acetate and formate in the feed, the RuBisCO levels varied considerably, but no correlation was observed with the intracellular concentrations of PEP. To study whether the repression exerted by acetate utilization was dependent on the synthesis of glycolytic intermediates from this compound, an acetate-negative mutant defective in isocitrate lyase was isolated and characterized. Clear evidence was obtained that in this mutant acetate is as effective in repressing RuBisCO synthesis as in the wild-type. It therefore appears more likely that acetyl-CoA or a closely related metabolite functions as a signal molecule in the regulation of RuBisCO synthesis.

Determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate by an enzymatic assay coupled to firefly luciferase/luciferin luminescence.

A procedure for the determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate is described. These metabolites were utilized by the glycolytic enzymes phosphoglycerate mutase, enolase, and pyruvate kinase to generate ATP which was determined by firefly luciferase/luciferin luminescence. The phosphoglycerate mutase used was of the glycerate 2,3-bisphosphate-independent type and was prepared from wheat germ. Stoichiometric conversion of glycerate 3-P, ranging in amount from 9 to 275 pmol, occurred after 25 min preincubation and required a narrow range of added mutase. The application of the procedure for determining these metabolites in suspensions of plant protoplasts is described.