果糖-2,6-二磷酸对香蕉成熟过程中焦磷酸果糖-6-磷酸转移酶活性的调节作用

从成熟香蕉果实中部分纯化了焦磷酸:果糖—6—磷酸磷酸转移酶(PFP)。研究了酶的果糖—2,6—二磷酸的活化动力学特性.果糖—2,6—二磷酸通过降低酶的K_m(F6P)值和增进最大反应速度(V_(max))促进酶的果糖—6—磷酸磷酸化活性。底物(F6P)浓度和温度影响果糖—2,6—二磷酸对酶的活化作用。 本工作中还观察了香蕉成熟过程中PFP和依赖ATP的磷酸果糖激酶(PFK)活性的变化,并对PFP在果实成熟中的生理意义和调节特性进行了讨论。     

番茄果实中焦磷酸:D-果糖-6-磷酸 1-磷酸转移酶的两种酶型的分离纯化及其特性

用快速蛋白液相层析仪(FPLC)Mono Q柱(HR5/5)分离纯化成熟绿番茄果实中PFP的两种分子酶型及其特性。一种酶型为Q_1,是含两个β-亚基(60kD)的二聚体,比活为5μmol min~(-1) mg~(-1);另一种为Q_2,由四个α-亚基(66kD)和四个β-亚基(60kD)组成八聚体,比活为70.5μmol/min~(-1)·mg~(-1)。Q_1的分子量是120kD,Q_2的分子量介于500kD和530kD之间。用纯化的Q_2制备的抗血清专一地与Q_2起沉淀反应。PFP酶液贮存后,其Q_1/Q_2蛋白量比值增加明显,表明部分Q_2转化为Q_1。Q_1具有催化活力表明PFP的活性中心位于β-亚基。α-亚基可能借增强PFP酶对F2,6P_2的亲和力以提高酶的比活而起调节功能,但是Q_1的活力依赖于F2,6P_2的激活,表明β-亚基处也可能存在F_2,_6P_2的调节位点。Q_2含紧密结合的F2,6P_2分子,并表现出对F2,6P2_的不敏感性,基于此种现象,有必要重新认识PFP对F2,6P_2敏感性的内在实质。     

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.     

鸡肝果糖-2,6-二磷酸酯酶结构域的初步晶体学研究

从鸡肝6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酯酶分离的果糖-2,6-二磷酸酯酶结构域(残基245～468)已在E.coli中获得高效表达,并经分离得到纯化,使用悬滴气相扩散法成功地培养出该果糖-2,6-二磷酸酯酶结构域单晶.该酶晶体属于四方晶系,空间群为P4₁2₁2或P4₃2₁2,晶胞参数为:a=b=10.02nm,c=13.98nm,α=β=γ=90°.晶胞内每个结晶学不对称单位含有2个果糖-2,6-二磷酸酯酶分子.利用日本 Photon Factory同步辐射光源收集了分辨率为 0.32 nm的母体衍射据.     

玉米叶片依赖焦磷酸的磷酸果糖激酶两种分子型动力学特性的研究

从玉米叶片中部分纯化了依赖焦磷酸的磷酸果糖激酶(PPi-PFK),对果糖2,6-二磷酸具有很高的敏感性(K_a≈15nmol/L)。纯化过程中酶的生糖方向活性对酵解方向活性的比值逐渐增加。F2,6-P_2的参与使这一比值下降,并且解除高浓度PPi对酶FBP形成活性的抑制。 用胰蛋白酶限制酶解,90min使80％酶的酵解方向活性丧失而仍然保持几乎全部的酶的生糖方向活性。胰蛋白酶修饰的酶的动力学结果表明F6P饱和曲线呈明显S型而且V_(max)大大下降。在F2,6-P_2存在下修饰酶的K_m(F6P)值比天然酶约大4倍。 酶的生糖方向活性动力学特性的比较说明天然酶和胰蛋白酶修饰酶几乎具有相同的催化能力和底物(F6P)亲合力。 实验支持植物PPi-PFK存在两种可以相互转化的酶分子型的调节酶的活性和作用方向的模型。     

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.     

鸡肝6-磷酸果糖-2-激酶/果糖-2-,6-二磷酸酯酶单克隆抗体制备及对酶活力影响

利用鸡肝-6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酯酶(6PF-2-K/Fru-2,6-P₂ase)的单克隆抗体对其结构和功能进行了初步研究.用鸡肝6PF-2-K/Fru-2,6-P₂ase为抗原免疫Balb/C小鼠,最后获得7株单克隆抗体.其中6株抗体的抗原决定簇位于6PF-2-K/Fru-2,6-P₂ase的酯酶结构域部分,而另一株H₂的抗原决定簇则位于其激酶结构域部分.7株单克隆抗体都能引起鸡肝6PF-2-K/Fru-2,6-P₂ase的激酶活力提高2倍左右,而对酯酶活力的影响大致相同.它们激活该酶的酯酶活力至4倍左右,但却不影响分离的鸡肝果糖-2,6-二磷酸酯酶结构域的酯酶活力.以上结果再次提示6PF-2-K/Fru-2,6-P₂ase双功能酶和分离的Fru-2,6-P₂ase结构域的酯酶处于2种不同的构象和活性状态.     

番木瓜果糖-2,6-二磷酸酶CpF2KP基因克隆和蛋白表达纯化

番木瓜是岭南四大名果之一,在我国东南部地区广泛种植,因其具有食用和药用双重价值,因此深受人们的青睐。果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酯酶(fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase,F2KP)是一个独特的双功能酶,具有激酶功能域和酯酶功能域,能催化生物体内糖代谢的重要调节物果糖-2,6-二磷酸(Fru-2,6-P_(2))的合成和降解。为了研究番木瓜中编码该酶的基因CpF2KP的功能,得到目的蛋白尤为重要。本研究从番木瓜基因组中提取到CpF2KP基因的编码序列(coding sequence,CDS)序列,该基因CDS全长2274 bp。将该基因CDS全长扩增之后选用pGEX-4T-1载体进行原核表达。对载体pGEX-4T-1用EcoRⅠ和BamHⅠ进行双酶切,利用基因重组的方式将扩增序列构建到原核表达载体上。经过诱导条件探索,SDS-PAGE结果显示GST-CpF2KP重组蛋白的大小约为110 kDa,诱导CpF2KP蛋白表达的最适条件为:异丙基β-D-硫代半乳糖苷(isopropyl beta-D-thiogalactopyranoside,IPTG)浓度为0.5 mmol/L,温度28℃。对诱导后的CpF2KP蛋白进行纯化,得到了纯化的单一目的蛋白。此外,检测了该基因的组织表达特性发现该基因在种子中表达量最高,在果肉中表达量最低。该研究为进一步深入揭示番木瓜CpF2KP蛋白的功能及研究该基因参与的生物学过程提供了重要基础。     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

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。     

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.

The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).     

Fructose-1,6-Bisphosphate Is an Allosteric Activator of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase

The activity of highly purified pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) from barley (Hordeum vulgare) leaves was studied under conditions where the catalyzed reaction was allowed to approach equilibrium. The activity of PFP was monitored by determining the changes in the levels of fructose-6-phosphate, orthophosphate, and fructose-1,6-bisphosphate (Fru-1,6-bisP). Under these conditions PFP activity was not dependent on activation by fructose-2,6-bisphosphate (Fru-2,6-bisP). Inclusion of aldolase in the reaction mixture temporarily restored the dependence of PFP on Fru-2,6-bisP. Alternatively, PFP was activated by Fru-1,6-bisP in the presence of aldolase. It is concluded that Fru-1,6-bisP is an allosteric activator of barley PFP, which can substitute for Fru-2,6-bisP as an activator. A significant activation was observed at a concentration of 5 to 25 [mu]M Fru-1,6-bisP, which demonstrates that the allosteric site of barley PFP has a very high affinity for Fru-1,6-bisP. The high affinity for Fru-1,6-bisP at the allosteric site suggests that the observed activation of PFP by Fru-1,6-bisP constitutes a previously unrecognized in vivo regulation mechanism.     

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.     

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.     

Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase

Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.     

Pi和PGA对玉米叶片PFK-2/FBPase-2的作用

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

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     

Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.     

Purification and characterization of pyrophosphate-dependent phosphofructokinase from phosphate-starved Brassica nigra suspension cells.

Previously, we reported that inorganic phosphate (Pi) deprivation of Brassica nigra suspension cells or seedlings leads to a progressive increase in the alpha: beta-subunit ratio of the inorganic pyrophosphate (PPi)-dependent phosphofructokinase (PFP) and that this coincides with a marked enhancement in the enzyme's activity and sensitivity to its allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2). To further investigate the effect of Pi nutrition on B. nigra PFP, the enzyme was purified and characterized from Pi-starved B. nigra suspension cell cultures. Polyacrylamide gel electrophoresis, immunoblot, and gel-filtration analyses of the final preparation indicated that this enzyme exists as a heterooctamer of approximately 500 kD and is composed of a 1:1 ratio of immunologically distinct alpha (66 kD) and beta (60 kD) subunits. The enzyme's alpha subunit was susceptible to partial proteolysis during purification, but this was prevented by the presence of chymostatin and leupeptin. In the presence and absence of 5 microM Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and 7.6, respectively. Maximal activation of the forward and reverse reactions by Fru-2,6-P2 occurred at pH 6.8. The potent inhibition of the forward activity by Pi (concentration of inhibitor producing 50% inhibition of enzyme activity [I50] = 1.3 mM) was attributed to a marked Pi-dependent reduction in Fru-2,6-P2 binding. The reverse reaction was substrate-inhibited by Pi (I50 = 13 mM) and product-inhibited by PPi (I50 = 0.9 mM). The kinetic data are consistent with the hypothesis that PFP may function to bypass the ATP-dependent PFP in Pi-starved B. nigra. The importance of the Pi nutritional status to the regulation and predicted physiological function of PFP is emphasized.     

Oscillation in fructose 2,6-bisphosphate levels and in the phosphorylation states of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in ischemic rat liver.

In order to determine the role of fructose (Fru) 2,6-P2 in stimulation of phosphofructokinase in ischemic liver, tissue contents of Fru-2,6-P2, hexose-Ps, adenine nucleotides, and Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated during the first few minutes of ischemia. The Fru-2,6-P2 concentration in the liver changed in an oscillatory manner. Within 7 s after the initiation of ischemia, Fru-2,6-P2 increased from 6 to 21 nmol/g liver and decreased to 5 nmol/g liver within 30 s. Subsequently, it reached the maximum value at 50, 80, and 100 s and decreased to the basal concentration at 60, 90, and 120 s. Oscillatory patterns were also observed with Glc-6-P and Fru-6-P, but the ATP/ADP ratio decreased monotonically. Determination of Fru-6-P,2-kinase activity and the phosphorylation states of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase demonstrated that at 7 and 50 s, where Fru-2,6-P2 was the highest, the enzyme was activated and mostly in a dephosphorylated form. On the other hand, at 0, 30, and 300 s, the enzyme was predominantly in the phosphorylated form. The concentration of cAMP in the liver also changed in an oscillatory manner between 0.5 to 1.3 nmol/g with varying frequency of 10 to 40 s. These results indicated that: (a) Fru-2,6-P2 was important in rapid activation of phosphofructokinase in the first few seconds and up to 2-3 min, and (b) the oscillation of Fru-2,6-P2 concentration was the result of activation and inhibition of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, which was caused by changes in the phosphorylation state of the enzyme.