Studies on the entry of fructose-2,6-bisphosphate into chloroplasts

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P₂) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P₂ controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P₂ is present mainly in the cytosol. Here we present evidence that Fru-2,6-P₂ can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P₂ (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P₂ in the stroma. Elevated plastid Fru-2,6-P₂ levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P₂ enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P₂ on chloroplast enzymes.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate

The aim of this work was to examine the role of fructose 2,6-bisphosphate (Fru-2,6-P₂) in photosynthetic carbon partitioning. The amount of Fru-2,6-P₂ in leaves of tobacco (Nicotiana tabacum L. cv. Samsun) was reduced by introduction of a modified mammalian gene encoding a functional fructose-2,6-bisphosphatase (EC 3.1.3.46). Expression of this gene in transgenic plants reduced the Fru-2,6-P₂ content of darkened leaves to between 54% and 80% of that in untransformed plants. During the first 30 min of photosynthesis sucrose accumulated more rapidly in the transgenic lines than in the untransformed plants, whereas starch production was slower in the transgenic plants. On illumination, the proportion of ¹⁴CO₂ converted to sucrose was greater in leaf disks of transgenic lines possessing reduced amounts of Fru-2,6-P₂ than in those of the control plants, and there was a corresponding decrease in the proportion of carbon assimilated to starch in the transgenic lines. Furthermore, plants with smaller amounts of Fru-2,6-P₂ had lower rates of net CO₂ assimilation. In illuminated leaves, decreasing the amount of Fru-2,6-P₂ resulted in greater amounts of hexose phosphates, but smaller amounts of 3-phosphoglycerate and dihydroxyacetone phosphate. These differences are interpreted in terms of decreased inhibition of cytosolic fructose-1,6-bisphosphatase resulting from the lowered Fru-2,6-P₂ content. The data provide direct evidence for the importance of Fru-2,6-P₂ in co-ordinating chloroplastic and cytosolic carbohydrate metabolism in leaves in the light. Received: 8 February 2000 / Accepted: 25 April 2000     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.     

The effect of oxygen on fructose-2, 6-bisphosphate concentration and fructose-6-phosphate, 2-kinase activity in yeast cells

Fructose-2,6-bisphosphate concentration and fructose-6-phosphate,2-kinase activity were measured in yeast cells grown aerobically or anaerobically using glucose as a carbon source. A new improved analytical method using HPLC was employed to measure fructose-2,6-P₂ concentration. Anaerobically-grown yeast cells contain approximately 4-fold higher levels of fructose-2,6-P₂ as compared to aerobically-grown cells in the growth phase of culture. Similarly, fructose-6-P,2-kinase activity is approximately 7-fold higher in the anaerobically-grown cells. These results suggest that the presence of oxygen in the growth medium decreases the content of fructose-2,6-P₂ through inactivation of fructose-6-P,2-kinase.     

Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.     

Enzymatic preparation of high-specific-activity β-d-[6,6′-H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase

β-d-Fructose-2,6-bisphosphate (Fru-2,6-P₂) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P₂ levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-³²P]Fru-2,6-P₂ has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P₂. The preparation involves conversion of readily available, carrier-free d-[6,6′-³H]glucose to [6,6′-³H]Fru-2,6-P₂ using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6′-³H]Fru-2,6-P₂ having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6′-³H]Fru-2,6-P₂ serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10⁻¹⁴ to 10⁻¹⁵ mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.     

The tissue distribution of fructose-2,6-p2 and fructose-6-P,2-kinase in rats and the effect of starvation diabetes and hypoglycemia on hepatic fructose-2,6-P2 and fructose-6-P,2-kinase

The tissue distribution of fructose-2,6-P₂ and fructose-6-P,2-kinase in rats was determined. The highest concentration of fructose-2,6-P₂ was found in liver, followed by brain, heart muscle, kidney, testis and skeletal muscle in decreasing order. Similar results were obtained with fructose-6-P,2-kinase activities in these tissues. Starvation, streptozotocin-induced diabetes or hypoglycemia lowers the fructose-2,6-P₂ levels and fructose-6-P,2-kinase activity in the liver.     

Reciprocal changes in fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase activity in response to glucagon and epinephrine

The effect of hormones on the enzymes responsible for the synthesis (fructose-6-P,2-kinase) and degradation (fructose-2,6-Pase) of fructose-2,6-P₂ was examined in isolated rat hepatocytes. Glucagon (10^?11 M), epinephrine (10^?5 M), or calcium (2.4 mM) and A23187 (10^?5 M) administration to hepatocytes produced simultaneous activation of fructose-2,6-Pase and inactivation of fructose-6-P,2-kinase within 2 minutes. The effect of epinephrine on these two enzymes was dependent on the presence of Ca⁺⁺. These results suggest that the level of fructose-2,6-P₂ is controlled by recriprocal changes in fructose-2,6-Pase and fructose-6-P,2-kinase activities.     

Identification of two forms of PFK and a fructose-2,6-bisphosphate independent form of PFP in a green alga

Cell-free preparations from the green alga, Chlorella pyrenoidosa, contained two forms of phosphofructokinase (PFK), designated PFK I and PFK II. This represents the first evidence for a second form of PFK in green algae. A pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase (PFP) activity, that was unaffected by the regulatory metabolite, fructose-2,6-bisphosphate, co-purified with PFK II through several steps. The data suggest that Chlorella pyrenoidosa resembles higher plants in containing two forms of PFK, but differs in containing an atypical form of PFP.Abbreviations PFK phosphofructokinase - PFP pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase, Fru-2,6-P₂-fructose-2,6-bisphosphate - DEAE diethylaminoethyl-     

Role of fructose-1,6-bisphosphatase, fructose phosphotransferase, and phosphofructokinase in saccharide metabolism of four C3 grassland species under elevated CO2

We studied the effects of 15-months of elevated (700 μmol mol⁻¹) CO₂ concentration (EC) on the CO₂ assimilation rate, saccharide content, and the activity of key enzymes in the regulation of saccharide metabolism (glycolysis and gluconeogenesis) of four C₃ perennial temperate grassland species, the dicots Filipendula vulgaris and Salvia nemorosa and the monocots Festuca rupicola and Dactylis glomerata. The acclimation of photosynthesis to EC was downward in F. rupicola and D. glomerata whereas it was upward in F. vulgaris and S. nemorosa. At EC, F. rupicola and F. vulgaris leaves accumulated starch while soluble sugar contents were higher in F. vulgaris and D. glomerata. EC decreased pyrophosphate-D-fructose-6-phosphate l-phosphotransferase (PFP, EC 2.7.1.90) activity assayed with Fru-2,6-P₂ in F. vulgaris and D. glomerata and increased it in F. rupicola and S. nemorosa. Growth in EC decreased phosphofructokinase (PFK, EC 2.7.1.11) activity in all four species, the decrease being smallest in S. nemorosa and greatest in F. rupicola. With Fru-2,6-P2 in the assay medium, EC increased the PFP/PFK ratio, except in F. vulgaris. Cytosolic fructose-1,6-bisphosphatase (Fru-1,6-P₂ase, EC 3.1.3.11) was inhibited by EC, the effect being greatest in F. vulgaris and smallest in F. rupicola. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) activity was decreased by growth EC in the four species. Activity ratios of Fru-1,6-P₂ase to PFP and PFK suggest that EC may shift sugar metabolism towards glycolysis in the dicots.     

Coordinate control of sucrose formation in soybean leaves by sucrose-phosphate synthase and fructose-2,6-bisphosphate

Net photosynthesis (CER), assimilate-export rate, sucrose-phosphate-synthase (EC 2.4.1.14) activity, fructose-2,6-bisphosphate content, and 6-phosphofructo-2-kinase (EC 2.7.1.105) activity were monitored in leaves of soybean (Glycine max (L.) Merr.) plants during a 12:12 h day-night cycle, and in plants transferred, at regular intervals throughout the diurnal cycle, to an illuminated chamber for 3 h. In the control plants, assimilate-export rate decreased progressively during the day whereas in transferred plants, a strongly rhythmic fluctuation in both CER and export rate was observed over the 24-h test period. Two maxima during the 24-h period for both processes were observed: one when plants were transferred during the middle of the normal light period, and a second when plants were transferred during the middle of the normal dark period. Overall, the results indicated that export rate was correlated positively with photosynthetic rate and sucrose-phosphate-synthase activity, and correlated negatively with fructose-2,6-bisphosphate levels, and that coarse control and fine control of the sucrose-formation pathway are coordinated during the diurnal cycle. Diurnal changes in sucrose-phosphate-synthase activity were not associated with changes in regulatory properties (phosphate inhibition) or substrate affinities. The biochemical basis for the diurnal rhythm in sucrose-phosphate-synthase activity in the soybean leaf thus appears to involve changes in the amount of the enzyme or a post-translational modification that affects only the maximum velocity.Abbreviations FBPase fructose-1,6-bisphosphatase - SPS sucrose-phosphate synthase - F26BPase fructose-2,6-bisphosphatase - PGI glucose-6-phosphate isomerase - F6P fructose-6-phosphate - F26BP fructose-2,6-bisphosphate - G6P glucose-6-phosphate - CER net carbon exchange rate - Pi inorganic phosphate - DHAP dihydroxyacetone phosphate - PGA glycerate 3-phosphate - F6P,2-kinase 6-phosphofructo-2-kinase Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10503 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.     

Regulation of fructose 2,6-P2 concentration in isolated hepatocytes

The effect of hormones on fructose-2,6-P₂ level and fructose-6-P,2-kinase activity was examined using rat hepatocytes. The dose response curve shows the half-maximal effect of glucagon on fructose-2,6-P₂ occurs at 3 X 10^?13 M glucagon, whereas the half-maximal effect on cyclic AMP occurs at 3 × 10^?0 M. The decrease in fructose-2,6-P₂ parallels the decrease in fructose-6-P,2-kinase activity. Incubation of cells with dibutryl cyclic AMP and cyclic AMP results in a 2- to 3-fold decrease in fructose-2,6-P₂. Epinephrine (10^?5 M) mediates a 2-fold decrease in fructose-2,6-P₂; isoproterenol has no effect. These results suggest that regulation of fructose-6-P,2-kinase is complex, involving cyclic AMP-dependent and -independent mechanisms.     

Pi和PGA对玉米叶片PFK-2/FBPase-2的作用

Fructose-6-phosphate 2-kinase and fructose 2,6-bisphosphatase have been partially purified from maize leaves by PEG fractionadon and by chromatography on TSK-DEAE ion exchanger and Blue-Sepharose 4B. Fructose-6-phosphate 2-kinase was activated by phosphate and inhibited by 3-pnosphoglycerate. Furctose 2,6-bisphosphatase was inhibited by inorganic phosphate and fructose-6-phosphate.The observed pattern of reguladon suggests that systhesis and degradation of Fru-2,6-P_2 respond to changes in the concentration of effectors. An increase in the level of glycerate-3-phosphate or dihydroxyacetonephosphate will result in a decrease in the level of Fru-2,6-P_2. Conversely a rise in Fru-6-P concentration will lead to an increase in the Fru-2, 6-P_2 concentration.