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.     

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)     

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.     

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.     

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.     

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.     

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.     

菠萝叶片依赖焦磷酸的磷酸果糖激酶的纯化及其特性

经硫酸铵分部,DEAE—纤维素、羟基磷灰石、Sephadex G—200及磷酸纤维素柱层析,从菠萝叶片分离得到电泳均一的依赖焦磷酸的磷酸果糖激酶(PFP)。SDS电泳图谱表明有一条分子量为62kD的主带和一条57 kD的弱带。Fru—2,6—P_2对酶的正反应活性有促进作用。动力学研究表明,Fru—2,6—P_2增加V_(max)及酶对底物Fru—6—P和Mg~(2+)的亲和性。     

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。     

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).     

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.     

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.     

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.     

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP_i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP_i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.     

Isolation and Characterization of Isolectins from Talisia esculenta Seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm × 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M_r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.     

Pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase activity patterns in relation to sucrose storage across sugarcane varieties

Pyrophosphate: d -fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) and ATP: d -fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activities were determined in sugarcane varieties differing in sucrose content. For this purpose, activities were measured in those internodes where the maximum rate of sucrose accumulation occurs. The specific activity of internodal PFP varied significantly between the sugarcane varieties and was inversely correlated with the sucrose content. There was also a highly significant inverse correlation between PFP and sucrose content in a segregating F1 population. PFK activity was comparable to, or lower than, PFP activity and no relationship was evident between PFK activity and sucrose content. In all tissues investigated, the fructose 2,6-bisphosphate levels were probably sufficient to ensure full activation of PFP. The levels of PFP activity appear to be controlled by the expression of the β -subunit of the protein. The molecular mass of the PFP _β subunit polypeptide(s) was approximately 63 kDa. There was an inverse correlation between sucrose content and the partitioning of radiolabel into respiration in internodal tissue slices labelled with [U-¹⁴C]glucose across 3 sugarcane lines. The estimated flux of carbon into respiration correlated directly with PFP activity.     

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) regulates carbon metabolism during grain filling in rice

Key message

Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and reproductive growth stages.

Abstract

Starch is a primary energy reserve for various metabolic processes in plant. Despite much advance has been achieved in pathways involved in starch biosynthesis, information was still lacked for precise regulation related to carbon metabolism during seed filling in rice (Oryza sativa). The objective of this study was to identify and characterize new gene associated with carbon metabolism during grain filling. By screening our chemical mutant pool, two allelic mutants exhibiting floury endosperm were isolated. No visible phenotypic defects were observed during both the vegetative and reproductive growth stages, except for the floury-like endosperm of grains with significantly reduced kernel thickness, 1000-grain weight and total starch content. Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) β subunit (PFP_β), which catalyzes reversible interconversion between fructose-6-phosphate and fructose-1, 6-bisphosphate. The identity of PFP _β was further confirmed by a genetic complementation test. Subcellular analysis demonstrated that PFPβ was localized in cytoplasm. Quantitative PCR and histochemical staining indicated PFP _β was ubiquitously expressed in various tissues. Furthermore, we found PFP _β could express in both the early and late phases of starch accumulation during grain filling and decreased activity of PFP _β in pfp mutants resulted in compromised carbon metabolism with increased soluble sugar contents and unfavorable starch biosynthesis. Our results highlight PFP_β functions in modulating carbon metabolism during grain filling stage.

     

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase is involved in the tolerance of Arabidopsis seedlings to salt and osmotic stresses

In plants, pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) is a regulatory enzyme that participates in glycolysis and gluconeogenesis. Arabidopsis contains two PFPα subunit genes (PFPα1 and PFPα2) and two PFPβ subunit genes (PFPβ1 and PFPβ2). The single-knockout mutants of the PFP subunit genes were isolated, and double and quadruple pfp mutants were generated by crossing the single mutants. To elucidate the role of PFP in stress tolerance, the responses of the double and quadruple pfp knockout mutants to stress conditions, including osmotic and salt stresses, were examined. The seedling growth of the pfpα1/α2 and pfpβ1/β2 double mutants and the pfpα1/α2/β1/β2 quadruple mutant was severely retarded under salt and osmotic stress conditions compared with that of the wild type. The expression of PFP subunit genes increased in response to salt and osmotic stresses. In contrast, the vegetative growth of the wild type and pfp mutants after the seedling stage was similarly affected by salt and osmotic stresses. These findings suggest that PFP plays a role in the adaptation of Arabidopsis seedlings to salt and osmotic stresses.     

丝瓜籽中蛋白质生物合成抑制蛋白的分离、纯化及性质研究

丝瓜（Ｌｕｆｆａｃｙｌｉｎｄｒｉｃａ）种籽,经捣碎、抽提、硫酸铵分级沉淀,ＣＭ－５２离子交换层析,Ｓｅｐｈａｃｒｙ１Ｓ－１００分子筛,阳离子交换ＦＰＬＣ等步骤,分离到两种单链蛋白质生物合成抑制蛋白：Ｌｕｆｆｉｎ－Ａ和Ｌｕｆｆｉｎ－Ｂ。它们都是等电点接近１０的碱性蛋白,ＳＤＳ－ＰＡＧＥ测定分子量分别约为２７ｋｄ和２８ｋｄ,氨基酸组成分析表明两者具很大同源性,但免疫双扩散及ＥＬＩＳＡ检测证明两者的免疫原性有差异。Ｌｕｆｆｉｎｓ对兔网织红细胞裂解液的蛋白质生物合成有强烈的抑制作用,ＩＣ５０分别为１．４×１０－１１ｍｏｌ／Ｌ和２．０×１０－１１ｍｏｌ／Ｌ,比ＴＣＳ的２．９×１０－１０ｍｏｌ／Ｌ低得多。因而,Ｌｕｆｆｉｎｓ很有可能成为肿瘤导向药物的高效＂弹头＂。