Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Kinetic evidence for the presence of two forms of M2-type pyruvate kinase in rat small intestine

Some kinetic properties of pyruvate kinase from rat small intestine have been investigated. The relative insensitivity of the enzyme to ATP inhibition and the amino acid inhibition pattern allows the conclusion that intestinal pyruvate kinase belongs to the M₂-type. The pyruvate kinase activity as a function of the phosphoenol pyruvate concentration is characterized by two different n values. The activity correlating with the low n value is stimulated by Fru-1,6-P₂, whereas the activity at higher phosphoenol pyruvate concentrations is not influenced by this glycolytic intermediate. These results, together with the partial relief of the amino acid inhibition by Fru-1,6-P₂, show that two forms of the enzyme are present with different kinetic properties. The metabolic implication of the kinetic properties of pyruvate kinase for rat small intestine is discussed.     

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.     

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD⁺-dependent l-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d-fructose-1,6-bisphosphate (Fru-1,6-P₂). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V _max for the reduction of pyruvate being approximately tenfold that of the value for l-lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P₂, while a sigmoidal response (indicative of positive cooperativity) was seen towards l-lactate without Fru-1,6-P₂. Strikingly, when introduced into an lldD ⁻ strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on l-lactate as the sole carbon source.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

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)     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

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.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

Summary A quantative cytochemical assay for PP_i-PFK activity in the presence of Fru-2,6-P₂ is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PP_i-PFK activity, has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PP_i-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.Abbreviations AMP adenosine monophosphate - ATP adenosine triphosphate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Fru-2,6-P₂ fructose 2,6-bisphosphate - Fru-6-P fructose 6-phosphate - FB Pase 2 fructose 2,6-bisphosphatase (EC 3.1.3.46) - Gl-3-PD glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT nitroblue tetrazolium - PEP phosphoenolpyruvate - PFK 6-phosphofructokinase (EC 2.7.1.11) - PFK₂ 6-phosphofructo-2-kinase (EC 2.7.1.105) - PP_i pyrophosphate - PP_i-PFK pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) - PVA polyvinyl alcohol (G04/140 Wacke Chemical Company)     

Multifunctional Properties of Beef Liver Phosphoglucomutase

Liver phosphoglucomutase was found to catalyze also the reaction of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P_z or Glc-1-P and glycerate-1,3-P₂. The specific activity of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P₂ was 1/9200 of that of the mutase activity. The activity of Glc-1,6-P₂ formation from Glc-1-P and glycerate-1,3-P₂ was 1/122,000 of the mutase activity. From the results of the kinetics and the thermal inactivation experiments, the reaction of the mutase and Glc-1,6-P₂ synthesis were strongly suggested to occur at the same active site of liver phosphoglucomutase.

Liver phosphoglucomutase exhibited the Glc-1,6-P₂ phosphatase activity only in the presence of xylose 1-phosphate. The specific activity of phosphatase was only 1/154,000 of that of the mutase activity.     

Glucose- and mannose-1,6-P2 as activators of phosphofructokinase in red blood cells

Glucose-1,6-P₂ and mannose-1,6-P₂ are concluded to be important activators from “reconstruction” experiments showing that the other known effectors of phosphofructokinase poise it at ～ 0.1% of its V_max, compared with the $\text{in vivo}$ rate of ～ 1%. These activators may explain the relative insensitivity of red cell glycolysis to fructose-1,6-P₂. Glucose-1,6-P₂ is elevated more than two-fold in pyruvate kinase deficient cells but not in cells from patients with alkalosis although both have increased levels of the inhibitor, glycerate-2,3-P₂.     

Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.     

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.     

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     

Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

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.     

Effect of thiamin on the content of fructose 2,6-bisphosphate in Euglena

• 1.1. Thiamin deprived Euglena cells contained twice as high a concentration of Fru-2,6-P₂ in sufficient cells and the rate of the uptake of glucose from the medium was twice as great as that found in sufficient cells, indicating that Fru-2,6-P₂ is responsible for the glycolysis.
• 2.2. Moreover, incubation of thiamin deprived cells with increasing thiamin concentrations caused a decrease of Fru-2,6-P₂. The activity of Fru-6-P, 2-kinase was affected by thiamin and the activity of PFK-2 in thiamin deprived cells was 2.3 times greater than that observed in sufficient cells.
• 3.3. Incubating thiamin-deficient cells in a thiamin-containing medium, the activity of Fru-6-P, 2-kinase decreased rapidly and became the same activity as that found in thiamin sufficient cells.
• 4.4. In contrast, the two thiamin dependent 2-OGDC and PNOR activities showed a reverse relationship, being higher in thiamin-sufficient cells and lower in thiamin deficient cells.
• 5.5. We concluded that the carbohydrate metabolism of Euglena is regulated by Fru-2,6-P₂ through an intracellular concentration of thiamin based on the present evidence.

     

On the biochemical basis of hereditary fructose intolerance.

In hereditary fructose intolerance it was found that in addition to an increased K_m value for Fru-1-P, the K_m of aldolase for Fru-1,6-P₂ was also increased. Furthermore, human phosphorylase $\text{a}$ was found to be inhibited by Fru-1-P in a non-competitive way.