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.     

Quantitative Aspects of the in Vivo Regulation of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase by Fructose-2,6-Bisphosphate

Pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was quantified in developing barley (Hordeum vulgare) leaves by immunostaining on western blots using a purified preparation of barley leaf PFP as standard. Fructose-2,6-bisphosphate (Fru-2,6-bisP) was quantified in the same tissues. Depending on age and tissue development, the concentration of PFP varied between 11 and 80 [mu]g PFP protein g-1 fresh weight, which corresponds to 0.09 to 0.65 nmol g-1 fresh weight of each of the [alpha] and [beta] PFP subunits. The level depends primarily on the maturity of the tissue. In the same tissues the concentration of Fru-2,6-bisP varied between 0.07 and 0.46 nmol g-1 fresh weight. Thus, the concentrations of PFP subunits and Fru-2,6-bisP were of the same order of magnitude. In young leaf tissues the concentration of PFP subunits may exceed the concentration of Fru-2,6-bisP. This means that the amount of Fru-2,6-bisP present will be too low to occupy all the allosteric binding sites on PFP even though the concentration of Fru-2,6-bisP exceeds the Ka(Fru-2,6-bisP) by several orders of magnitude. These results are discussed in relation to Fru-2,6-bisP as a regulator of enzyme activities under in vivo conditions.     

Fructose-2,6-bisphosphate Activates Pyrophosphate: d-Fructose 6-Phosphate 1-Phosphotransferase from Euglena gracilis

When hexavalent chromium (Cr⁶⁺) tolerant Pseudomonas ambigua G-l was cultivated in nutrient broth containing 150 ppm Cr^{6 +}, the Cr⁶⁺ content of the broth rapidly decreased. The Cr⁶⁺ reducing enzyme found in a cell-free extract of P. ambigua G-l required NADH but not NADPH as a hydrogen donor for the reduction of Cr^{6 +}. The specific activities of cell-free extracts of several Cr⁶⁺ sensitive mutants derived from P. ambigua G-l showed decreases to one fourth to one tenth of that of P. ambigua G-l. Glucose protected the Cr⁶⁺ reducing enzyme against inac-tivation on dialysis.     

Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant,Pineapple

Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.     

Pyrophosphate Fructose-6-P 1-Phosphotransferase from Tomato Fruit : Evidence for Change during Ripening

Three forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) were purified from both green and red tomato (Lycopersicon esculentum) fruit: (a) a classical form (designated Q₂) containing α- (66 kilodalton) and β- (60 kilodalton) subunits; (b) a form (Q₁) containing a β-doublet subunit; and (c) a form (Q₀) that appeared to contain a β-singlet subunit. Several lines of evidence suggested that the different forms occur under physiological conditions. Q₂ was purified to apparent electrophoretic homogeneity; Q₁ and Q₀ were highly purified, but not to homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q₂, 29% (90%); Q₁, 47% (6%); and Q₀, 24% (4%). The major difference distinguishing the red from the green tomato enzymes was the fructose-2,6-bisphosphate (Fru-2,6-P₂)-induced change in K_m for fructose-6-phosphate (Fru-6-P), the `green forms' showing markedly enhanced affinity on activation (K<sub>m</sub> decrease of 7-9-fold) and the `red forms' showing either little change (Q₀, Q₁) or a relatively small (2.5-fold) affinity increase (Q₂). The results extend our earlier findings with carrot root to another tissue and indicate that forms of PFP showing low or no affinity increase for Fru 6-P on activation by Fru-2,6-P₂ (here Q₁ and Q₀) are associated with sugar storage, whereas the classical form (Q₂), which shows a pronounced affinity increase, is more important for starch storage.     

Immunological Characterization of the Pyrophosphate Dependent Fructose-6-Phosphate Phosphotransferase

The pyrophosphate dependent phosphofructokinase (PFP, EC 2.7.1.90 [EC] )was purified from potato tubers, bean seeds and cucumber seeds.The PFP of all three species appears to contain two subunitswith a molecular weight of approximately 60,000 and 66,000 dalton.The purified proteins were used as the antigens to produce polyclonalantibodies in rabbits. Two of the obtained sera (anti-potatoPFP and anti-cucumber PFP) proved to be monospecific for thePFP polypeptides on protein blots. The antipotato serum crossreacts with the PFP from all the tested higher plant specieson protein blots, but no cross reaction with the PFP of Propionibacteriumsharmanii was found. This shows that the PFP subunits from thehigher plant species have similar antigenic determinants inthe primary structure but differes largely from that of thePropionibacterium. The differences observed in the efficiencyof the sera to inactivate the PFP from the different species,however, indicate that the surface antigenic determinants onthe native PFP enzymes differ between the higher plant speciesand even within the Cucurbitaceae. (Received June 15, 1987; Accepted November 20, 1987)     

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.     

Ischemia and Reperfusion: Effect of Fructose-1,6-Bisphosphate

Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the “oxygen paradox” in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.     

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.     

Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds

The distribution of pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles·min·milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K_m values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P₂. The activation is caused by lowering the K_m for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P₂ and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K_m of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K_m for fructose 6-P. The ATP content appears to be at saturating levels for PFK.     

Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus

After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA₃) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control.     

Molecular Properties of the ATP:D-Fructose-6-Phosphate 1-Phosphotransferase Isoenzymes from Cucumis sativus

The ATP:D-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.11 [EC] )isoenzymes from cucumber seeds were separated and purified.The calculated molecular weights of the two isoenzymes (approximately180,000) are similar and the isoenzymes are probably hetro-tetramers.The purified isoenzymes contained three polypeptides of 53.3,41.5 and 39.0 kDa for the plastid and 47.2, 42.4 and 40.4 forthe cytosolic isoenzyme, respectively. The purified phosphofructokinaseisoenzymes were used as the antigen for the production of polyclonalantibodies in rabbits. The obtained antisera clearly indicatedthat there is no immunological similarity between the two isoenzymes.The results also show that the phosphofructokinase isoenzymesin cucumber are not merely different stages of association ofthe same protein. (Received June 29, 1987; Accepted October 21, 1987)     

番茄果实中焦磷酸：果糖－6－磷酸1－磷酸转移酶的两种亚基特性的探讨

番茄果实由绿转红的过程中,焦磷酸：果糖－６－磷酸１－磷酸转移酶（ＰＦＰ）的酸型发生转化。在体外通过胰蛋白酶处理部分纯化的番茄绿果实中ＰＦＰ来探讨酶型转化的原因。蛋白免疫印渍结果证实ＰＦＰ的α－亚基比β－亚基更容易受到胰蛋白酶的降解,这也是ＰＦＰ经胰蛋白酶处理后酵解与生糖活性下降的原因。然而ＰＦＰ的亚基经尿素解离后,以胰蛋白酶处理的蛋白免疫印渍分析却表明ＰＦＰ的两种亚基均被胰蛋白酶更加有效地降解,显然α－亚基在ＰＦＰ的高级结构中有更多的酶切位点外露,而β－亚基上的酶切酶点可能位于分子的内部受到有效的保护。     

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

胡杨果糖-1,6-二磷酸醛羧酶基因PeALD的克隆及耐盐性分析

胡杨(Populus euphratica Oliv)是优良的抗逆树种,有很强的耐盐碱性.前期胡杨转录组研究结果显示盐胁迫可诱导果糖-1,6-二磷酸醛羧酶基因(PeALD)转录上调,提示该基因可能对胡杨耐盐性方面有某种贡献.为分析PeALD基因在胡杨耐盐性中的作用,本研究以胡杨为材料,利用RT-PCR方法克隆了胡杨果糖-1,6-二磷酸醛羧酶基因的全长cDNA,通过基因转化法尝试在烟草中过量表达该基因,并对转基因株系的耐盐性进行初步分析.研究结果显示,克隆的cDNA编码果糖-1,6-二磷酸醛羧酶,ORF为1 177 bp,是由247个氨基酸编码的疏水性蛋白,理论等电点为6.84,分子量为26.79 kD.PCR与RT-PCR检测结果表明,外源的PeALD基因通过转化已经整合到烟草的染色体中,并在4个株系中得到较强的表达.转化株系表型筛选实验表明,在200 mmol/L NaCl的MS培养基中培养15 d后,野生型烟草植株无明显高生长,叶片全部黄化并萎缩;而转基因烟草高生长基本没有受到抑制,植株生长良好.说明在烟草中过表达PeALD使转化植株的耐盐性显著提高.光合数据结果显示,ALD基因能够降低盐胁迫对烟草叶片净光合速率的影响,可能是PeALD过表达加速了卡尔文循环的运转,提高了CO2的固定速率,进而促进了光合活性,通过光合作用促进碳的积累,利于呼吸作用和氧化磷酸化产生ATP,为维持跨膜质子浓度梯度提供能量,从而有可能促进质膜上的Na+/H+逆向转运,利于细胞质膜保持拒盐性.这些结果初步验证了PeALD基因的耐盐性功能,为进一步深入研究该基因在耐盐机制中的作用奠定了良好基础.     

Fructose-1-phosphate-6-sulfate as an alternative substrate for aldolase and fructose-1,6-diphosphatase.

Fructose-1-phosphate-6-sulfate was prepared by direct sulfurylation of fructose, and selective phosphorylation of the 6-sulfuryl isomer by phosphofructokinase. The ketose derivative was used as a substrate for aldolase and fructose-1,6-diphosphatase. Kinetic studies with aldolase showed that the alternative substrate binds one third as well as fructose-1,6-P₂ yet 900 fold greater than fructose-1-P. The V_m was intermediate between the two ketose phosphates. From kinetic studies with skeletal muscle fructose-1,6-diphosphatase at pH 7.5 a K_m of 8 μM and a V_m approximately 6% that for fructose-1,6-P₂ was obtained.     

Characterization of ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferase Isozymes from Leaf and Endosperm Tissues of Ricinus communis

Plastid and cytosolic isozymes of ATP:fructose 6-phosphate 1-phosphotransferase (PFK_p and PFK_c, respectively) have been isolated from leaves and developing endosperm tissues of the castor oil plant (Ricinus communis L). Endosperm PFK_p has been purified to apparent homogeneity. Polyclonal antibodies raised against one of the four polypeptides associated with potato tuber PFK (molecular mass, 46 kilodaltons) immunoprecipitated developing endosperm and leaf PFK_p, but not PFK_c isozymes. Western blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and analytical gel filtration show that PFK_p from developing endosperm is a 220 kilodalton homotetramer composed of 57 kilodalton subunits. Kinetic studies of leaf PFK_p and PFK_c isozymes reveal both similarities and differences to the characteristics of the respective endosperm isozymes studied previously (WJ Garland, DT Dennis [1980] Arch Biochem Biophys 204: 302-317). The immunological and kinetic data suggest that leaf and developing endosperm PFK_p are different but structurally related proteins.     

Effects of Fructose-1,6-Bisphosphate on Glutamate Release and ATP Loss from Rat Brain Slices During Hypoxia

Abstract: Fructose-1,6-bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase)-catalyzed conversion of glutamate to α-ketoglutarate) during hypoxia (Po₂ 15 mm Hg) or hypoxia plus 100 µ M cyanide. FBP (3.5 m M , with glucose 20 m M ) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% ( p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose-free conditions with 20 but not 3.5 m M FBP, and fell to <20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 m M FBP without glucose decreased hypoxia-evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia-evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.