Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase     

Pyrophosphate:fructose-6-phosphate 1-phosphotransferase from barley seedlings. Isolation, subunit composition and kinetic Characterization

Pyrophosphate:fructose-6-phosphate I-phosphotransferase (PFP: EC 2.7.1.90) was purified 260-fold from leaves of etiolated barley seedlings. The purified enzyme consisted of two subunits, with apparent molecular masses of 65 (α) and 60 (β) kDa. Polyclonal antibodies were raised against the denatured PFP protein eluted from an SDS-polyacrylamide gel. The antibodies recognized both denatured and native PFP. Western blots of crude extracts showed that the activity of PFP in barley leaves is correlated to the amount of PFP protein, and that both the α- and the β-subunits are present in near stoichiometric amounts in all investigated tissues. The apparent molecular mass of the boloenzyme. as determined by gel filtration chromatography, was dependent on the presence of pyrophosphate. In absence of pyrophosphate. barley PFP elutes as a heterotetramer whereas it elutes as a heterooctamer in the presence of 20 m M pyrophosphate. Pure PFP obtained by gel filtration chromatography in the presence of 20 m M pyropnosphaie reached a specific activity of 28 U mg⁻¹. Barley PFP was characterized with respect 10 kinetic properties in the forward direction (use of PP₁) and in the reverse direction (formation of PP₁). The affinity for the activator Fru-2.6-P₂: was very high, with an estimated K₃ of 2.8 n M when PFP activity was assayed in the forward direction.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

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     

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.     

Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via inhibition by phosphoenolpyruvate

The two enzymatic activities of the highly conserved catalytic core of 6PF2K/Fru-2,6-P₂ase are thought to be reciprocally regulated by the amino- and carboxy-terminal regions unique to each isoform. In this study, we describe the recombinant expression, purification, and kinetic characterization of two human brain 6PF2K/Fru-2,6-P₂ase splice variants, HBP1 and HBP2. Interestingly, both lack an arginine which is highly conserved among other tissue isoforms, and which is understood to be critical to the fructose-2,6-bisphosphatase mechanism. As a result, the phosphatase activity of both HBP isoforms is negligible, but we found that it could be recovered by restoration of the arginine by site directed mutagenesis. We also found that AMP activated protein kinase and protein kinases A, B, and C catalyzed the phosphorylation of Ser-460 of HBP1, and that in addition both isoforms are phosphorylated at a second, as yet undetermined site by protein kinase C. However, none of the phosphorylations had any effect on the intrinsic kinetic characteristics of either enzymatic activity, and neither did point mutation (mimicking phosphorylation), deletion, and alternative-splice modification of the HBP1 carboxy-terminal region. Instead, these phosphorylations and mutations decreased the sensitivity of the 6PF2K to a potent allosteric inhibitor, phosphoenolpyruvate, which appears to be the major regulatory mechanism.     

Changes in the protein composition of cytoplasmic ribsomes during the greening of etiolated barley leaves

We examined changes in the protein composition of cytoplasmic ribosomes in etiolated barley leaves following illumination. Cytoplasmic ribosomes were isolated from greening barley leaves by sucrose density gradient centrifugation, and were analyzed by radical-free highly reducing polyacrylamide gel electrophoresis (RFHR-PAGE). Eighty-nine proteins were resolved from the ribosomal fraction; among them, 8 proteins changed their copy numbers depending on the stage of greening. We designated these as phase dependent ribosomal proteins (PD1–PD8). Two of the proteins (PD1 and 5) present in the ribosomes of etiolated leaves showed a decrease in level during greening. In contrast, the levels of 6 ribosomal proteins (PD2, 3, 4, 6, 7 and 8) increased as greening proceeded. N-terminal amino acid sequence of PD8 showed high homology to rat ribosomal protein L34. The ribosomal proteins that appeared after illumination were not found in any fraction of the etiolated leaves, suggesting that they were synthesized after the onset of illumination. Copy numbers of other ribosomal proteins did not change during greening.     

Preparation of monoclonal antibodies against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and their effects on enzyme activities

The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2,5-P2asc as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2,6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme and     

Changes in fructose-2,6-bisphosphate levels after glucose loading of starved rats

Fructose-2,6-bisphosphate levels in freeze-clamped livers of starved rats were 0.5 nmol/g liver. Oral administration of 1 g glucose per kg body weight to starved rats increased glycogen levels from 4 mg/g liver to 13.5 mg/g in 2 hr but did not significantly alter fructose-2,6-bisphosphate levels. The low level of this effector is consistent with an active gluconeogenic process and the results support the hypothesis that carbon atoms for glycogen synthesis can be derived from 3-carbon precursors via this pathway, even in the presence of glucose.     

Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress

Changes in the content of fructose-2,6-bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non-oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK-2/FBPase-2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose-2,6-bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Photoinhibition and recovery of photosynthesis in intact barley leaves at 5 and 20°C

Photoinhibition of photosynthesis and its recovery were studied in intact barley ( Hordeum vuigare L. cv. Gunilla) leaves grown in a controlled environment by exposing them to two temperatures, 5 and 20°C, and a range of photon flux densities in excess of that during growth. Additionally, photoinhibtion was examined in the presence of chloramphenicol (CAP, an inhibitor of chloroplast protein synthesis) and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Susceptibility to photoinhibition was much higher at 5 than at 20°C. Furthermore, at 20°C. CAP exacerbated photoinhibition strongly, whereas CAP had little additional effect (10%) at 5°C. These results support the model that net photoinhibition is the difference between the inactivation and repair of photosystem II (PSII); i.e. the degradation and synthesis of the reaction centre protein, Dl. Furthermore, the steady-state extent of photoinhibition was strongly dependent on temperature and the results indicated this was manifested through the effects of temperature on the repair process of PSII. We propose that the continuous repair of PS II at 20°C conferred at least some protection from photoinhibition. At 5°C the repair process was largely inhibited, with increased photoinhibition as a consequence. However, we suggest where repair is inhibited by low temperature, some protection is alternatively conferred by the photoinhibited reaction centres. Providing they are not degraded, such centres could still dissipate excitation energy non-radiatively, thereby conferring protection of remaining photochemically active centres under steady-state conditions.
A fraction of PS II centres were capable of resisting photoinhibition when the repair process was inhibited by CAP. This is discussed in relation to PS II heterogeneity. Furthermore, the repair process was not apparently activated within 3 h when barley leaves were transferred to photoinhibitory light conditions at 20°C.     

Effects of combined NaCl and CaCl2 salinity on photosynthetic parameters of barley grown in nutrient solution

The changes caused by NaCl− and CaCl₂-induced salinity on several leaf parameters have been measured in two cultivars of barley ( Hordeum vulgare L.) growing in a growth chamber in nutrient solution. Salinity was induced by adding to the nutrient solution equal weights of NaCl and CaCl₂, to obtain conductivities of 2, 6, 12, 19 and 26 dS m⁻¹. Salinity induced decreases in the leaf water potential and in the osmotic potential. Salinity did not induce significant changes in the relative photosynthetic pigment composition of barley leaves, the photosynthetic pigment stoichiometry for neoxanthin:violaxanthin cycle pigments:lutein:β-carotene:Chl b :Chl a being close to 3:6:14:12:25:100 (mol:mol). Salinity per se did not induce interconversions in the carotenoids within the violaxanthin cycle in most barley leaves. The PSII photochemistry of most barley leaves was unchanged by salinity. However, some apparently healthy leaves growing in high salinity exhibited sudden decreases in PSII photochemistry and increases in zeaxanthin (at the expense of violaxanthin), that preceded rapid leaf drying. Salinity induced significant changes in the slow part of the chlorophyll fluorescence induction curve from barley leaves.