Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

PFP的研究进展

焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP)可催化果糖-6-磷酸与果糖-1,6-二磷酸间的可逆转变.该酶广泛存在于各种高等植物及一些微生物体内.文章综述了90年代以来有关PFP的一些研究进展.包括:PFP的种类与亚基构成、活性中心、底物特异性、酶活性的调节及功能等.     

玉米果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶基因的结构与表达分析

通过RT-PCR,结合RACE技术,得到了玉米(Zea mays L.)果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶的全长cDNA克隆,命名为mF2KP.氨基酸序列同源性比较发现,mF2KP蛋白可以分为两个部分:C端包含高度保守的催化功能区,N端为植物中特有的多肽.将mF2KP基因中一段包含完整催化功能区的片段在大肠杆菌(Escherichia coli)中表达,融合蛋白具有果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶活性.Northern杂交证明在种子活力不同的幼苗中,mF2KP的转录水平存在明显差异.种子活力越高,幼苗中mF2KP的转录水平越低.     

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.     

玉米果糖—6—磷酸，2—激酶／果糖—2，6—二磷酸酶基因的结构与表达分析

通过RT-PCR,结果RACE技术,得到了玉米（Zea maysL.)果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶的全长cDNA克隆。命名为mF2KP,氨基酸序列同源性比较发现,mF2KP蛋白可以分为两个部分;C端包含高度保守的催化功能区。N端为植物中特有的多肽,将mF2KP基因中一段包含完整催化功能区的片段在大肠杆菌（Escherichia coli)中表达,融合蛋白具有果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶活性,Northern杂交证明在种子活力不同的幼苗中,mF2KP的转录水平存在明显差异。种子活力越高,幼苗中mF2KP的转录水平越低。     

Modification of cys-128 of pig kidney fructose 1,6-bisphosphatase with different thiol reagents: Size dependent effect on the substrate and fructose-2,6-bisphosphate interaction

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem. 262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.     

Oxygen Radical Injury and Loss of High-Energy Compounds in Anoxic and Reperfused Rat Heart: Prevention By Exogenous Fructose-1, 6-Bisphosphate

Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1, 6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and the TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1, 6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1, 6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release oflactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1, 6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed.

On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1, 6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.     

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.     

光/暗对玉米叶片果糖-6-磷酸激酶-2和果糖-2,6-二磷酸酯酶活力的影响

比较了照光和黑暗条件下玉米叶片果糖—6—磷酸激酶—2(PFK-2)和果糖—2,6—二磷酸酯酶(FBPase-2)的活力变化。当玉米植株从暗中转入光下后,其叶片PFK—2的活力随光照时间的延长而逐渐降低,而FBPase-2活力变化不明显;从光下转入暗后叶片PFK-2活力明显上升,FBPase-2活力仍无明显变化;其PFK-2/FBPase-2比值在光处理时下降,暗处理时上升。同时叶片中果糖—2,6—二磷酸的含量与PFK-2/FBPase-2活力比值的变化趋势一致。连续光照 20 h,PFK-2活力持续下降,表明PFK-2的光钝化现象与玉米植株的昼夜节律变化无关。     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Natural products as sources of new fungicides (IV): Synthesis and biological evaluation of isobutyrophenone analogs as potential inhibitors of class-II fructose-1,6-bisphosphate aldolase

Several recently identified antifungal compounds share the backbone structure of acetophenones. The aim of the present study was to develop new isobutyrophenone analogs as new antifungal agents. A series of new 2,4-dihydroxy-5-methyl isobutyrophenone derivatives were prepared and characterized by ¹H, ¹³C NMR and MS spectroscopic data. These products were evaluated for in vitro antifungal activities against seven plant fungal pathogens by the mycelial growth inhibitory rate assay. Compounds 3, 4a, 5a, 5b, 5e, 5f and 5g showed a broad-spectrum high antifungal activity. On the other hand, for the first time, these compounds were also assayed as potential inhibitors against Class II fructose-1,6-bisphosphate aldolase (Fba) from the rice blast fungus, Magnaporthe grisea. Compounds 5e and 5g were found to exhibit the inhibition constants (Ki) for 15.12 and 14.27?μM, respectively, as the strongest competitive inhibitors against Fba activity. The possible binding-modes of compounds 5e and 5g were further analyzed by molecular docking algorithms. The results strongly suggested that compound 5g could be a promising lead for the discovery of new fungicides via targeting Class II Fba.     

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.     

Identification of a nerve ending-enriched 29-kDa protein,labeled with [3-32P]1,3-bisphosphoglycerate,as monophosphoglycerate mutase: inhibition by fructose-2,6-bisphosphate via enhancement of dephosphorylation

Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3-32P]1,3-bisphosphoglycerate and [32P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3-32P]1,3-bisphosphoglycerate or [32P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P2) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P2 potently stimulated release of [32P]phosphate from the 32P-labeled PGAM (EC50 = 1 microM), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.     

Phototransformation of protochlorophyll(ide) by a proplastid fraction from Euglena

The phototransformation of protochlorophyll(ide) (Pchl(ide)) to chlorophyll(ide) (Chl(ide)) can be demonstrated in a proplastid fraction from Euglena gracilis Klebs var. bacillaris Cori if appropriate conditions are employed. Pigments were measured fluorometrically in acetone extracts of cell or organelles. Pchl(ide) and the phototransformation to Chl(ide) are at their highest levels in cells grown in darkness on normal or low vitamin B₁₂-containing medium (pH 3.5) to the late exponential phase (1.2–1.4 × 10⁶ cells ml^?1). Late exponential cells on low B¹² medium yield a proplastid fraction that contains Pchl(ide) which is phototransformed to Chl(ide) when illuminated with red light (5.6 W m^?2 for 4 min) in the presence of 10 mM Hepes, 20 mM TES, 0.5 mM potassium phosphate (pH 7.4), 70 mM sorbitol, 5 mM DTT, 5 mM ATP, 5 mM fructose-1, 6-bisphosphate, 10 mM malate and 2 mM MgCl²; intact organelles appear to be involved since deletion of osmoticum gives a lower activity, and addition of NAD(P)H is without effect. Phototransformation of Pchl(ide) to Chl(ide) in red light shows Bunsen-Roscoe reciprocity between fluence rate and duration of illumination. Although mitochondria are present, they do not appear to be involved since inhibitors of respiration and uncouplers of oxidative phosphorylation fail to block the phototransformation. The percentage phototransformation of Pchl(ide) to Chl(ide) in late exponential normal B¹² cells is 61 ± 10, and is 52 ± 3 in low B¹² cells. About 67% of the activity in low B¹² cells is recovered in the proplastid fraction incubated with the complete incubation mixture in saturating light. In both types of cells and in the proplastid fraction, the stoichiometry of conversion of Pchl(ide) to Chl(ide) is about 1:1 (mol/mol).     

Antibody inhibition of external aldolase activity in spinach chloroplast preparations

Abstract Antibodies (rabbit) have been prepared against total stroma from isolated spinach (Spinacia oleracea L. cv. Viking II) chloroplasts. These antibodies inhibited most of the aldolase activity present outside the chloroplasts in preparations of intact (80–95%) chloroplasts. They also reduced the amount of labelled fructose-1,6-bisphosphate found in the medium after ¹⁴CO₂ fixation with such preparations. Both intact and broken chloroplasts were strongly agglutinated by the antibodies. The results indicate that the external fructose-1,6-bisphosphate was formed from excreted dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase present outside the chloroplasts. The contamination of organelle preparations with free enzymes or enzymes adsorbed on the outer surface of the organelles is probably a general phenomenon. It is suggested that antibodies can be used as a tool to detect and selectively inhibit such contaminating enzyme activities.