Measurement of phosphate esters in extract of fetal rat heart by automated high pressure liquid chromatography

Creatine phosphate, nucleotides and glycolytic phosphate esters were estimated in extract of beating, in situ freeze clamped, $\text{131}\text{2}$ to $\text{191}\text{2}$ day fetal rat hearts by automated phosphate ester chromatography. Creatine phosphate increased more than 4-fold to almost 9 n moles per mg. protein at $\text{191}\text{2}$ days, while ATP remained relatively constant at about 19 to 21 n moles per mg. protein. Most other nucleotides decreased as gestation advanced. ATP rather than creatine phosphate appears to be the major energy source of fetal rat heart. Except for glucose-6-phosphate, which increased, the glycolytic phosphate esters decreased only very slightly with advancing gestational age, suggesting a relatively stable basal glycolytic activity. Methodology includes correction for phosphate esters of whole blood trapped in extracts of in situ freeze clamped tissues.     

Changes in fructose-2,6-bisphosphate levels after glucose loading of starved rats

Fructose-2,6-bisphosphate levels in freeze-clamped livers of starved rats were 0.5 nmol/g liver. Oral administration of 1 g glucose per kg body weight to starved rats increased glycogen levels from 4 mg/g liver to 13.5 mg/g in 2 hr but did not significantly alter fructose-2,6-bisphosphate levels. The low level of this effector is consistent with an active gluconeogenic process and the results support the hypothesis that carbon atoms for glycogen synthesis can be derived from 3-carbon precursors via this pathway, even in the presence of glucose.     

Structural analysis of human glutamine:fructose-6-phosphate amidotransferase, a key regulator in type 2 diabetes

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosynthetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose-6-phosphate and linear glucosamine-6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.     

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.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

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。     

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。     

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.     

Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular ‘hunter’ attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.     

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.     

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PPi:fructose 6-phosphate phosphotransferase

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PP_i:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PP_i:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.     

Glycogen phosphorylase from Dictyostelium: a kinetic analysis by computer simulation.

A model of carbohydrate metabolism during differentiation in Dictyostelium discoideum has been used to investigate which enzyme kinetic mechanism(s) might be operative for glycogen phosphorylase in vivo. The model, which has been described previously, is capable of simulating experimentally observed changes in metabolite concentrations and fluxes during differentiation under both the standard starvation condition and in the presence of glucose (25 mM). The concentrations of saccharide end products of differentiation under these 2 conditions differ substantially.Glycogen phosphorylase is described in the model by a rapid equilibrium random bi bi mechanism and the effect of substituting 4 other kinetic mechanisms was examined. Each of these mechanisms in the model allows simulations compatible with the saccharide accumulation patterns found during differentiation in the absence of glucose. However, in the presence of glucose, only a reversible mechanism (random or ordered) is compatible with the experimental data. It is concluded that glycogen degradation in vivo is controlled by an enzyme catalyzing a reversible reaction, the rate of which is inversely related to the glucose-1-P concentration.     

Some antitumor derivatives of ellipticine deprived of mutagenic properties

Seven derivatives of the antitumor alkaloid ellipticine were assayed for activity against murine leukaemia L1210 and for mutagenicity in Ames' Salmonella-microsomes test. Not only did the results show a complete lack of correlation between these two properties, but it was possible to choose a highly efficient analog which was completely devoid of mutagenic and hence, probably, carcinogenic effect. The lack of interaction of this product (2-methyl-9-hydroxyellipticinium acetate) with Cytochrome P-450 of hepatic monooxygenases prevents the formation of reactive intermediates and their subsequent binding to DNA. These data are discussed in view of the currently admitted mode of action of ellipticines i.e., intercalation in DNA and their therapeutic use.     

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.     

Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1, of which the high-M_r nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two amidases differing in substrate specificity, judging from the effects of various amides on amidase activity in this strain. These amidases seemed to be inducible enzymes depending on amide compounds.     

人谷氨酰胺:6-磷酸果糖酰胺转移酶的体外表达、纯化及活性检测

目的:制备重组谷氨酰胺∶6-磷酸果糖酰胺转移酶(GFAT),检测其活性。方法:利用RT-PCR扩增人肝脏cDNA中GFAT1基因全长片段,克隆到表达载体pET32b中;在大肠杆菌Origami(DE3)中诱导表达,用镍离子螯合柱(Ni-NTA)纯化重组GFAT1;用体外酶学的方法检测GFAT的活性。结果:构建了pET32b-GFAT1质粒,经诱导表达及纯化,得到具有一定生物活性的GFAT。结论:利用原核表达系统可得到具有良好生物学活性的重组人GFAT1。     

Pyrophosphate as an Energy Donor in the Cytosol of Plant Cells: an Enigmatic Alternative to ATP

Pyrophosphate serves as an alternative energy donor to ATP for sucrose mobilisation via sucrose synthase, for glycolysis via pyrophosphate: fructose-6-phosphate phosphotransferase, and for tonoplast energisation via the tonoplast proton-pumping pyrophosphatase. This review considers the possible roles of these pyrophosphate-driven reactions. Correlative evidence based on expression patterns, the distribution of proteins and activities in various tissues, and comparisons of the in vitro properties of the enzymes with the in vivo metabolite levels indicates an important role in young growing tissues and in stress conditions including anaerobiosis, but interpretation is complicated by the reversibility of the pyrophosphate-driven reactions and by their duplication by ATP-dependent reactions. The review then considers the evidence emerging from experiments using reversed genetics to alter expression of sucrose synthase, the pyrophosphate: fructose-6-phosphate phosphotransferase, and the tonoplast proton-pumping pyrophosphatase. This approach has revealed that sucrose synthase plays an essential role in sucrose breakdown in potato tubers, and that pyrophosphate: fructose-6-phosphate phosphotransferase catalyses a near-equilibrium reaction with a net flux in the direction of glycolysis. However, it does not support a special role of the latter enzymes in stress responses. Interpretation is complicated by compensation, which can include expression of other members of a gene family, use of alternative pathways, and relaxation of the feed back regulation in response to decreased expression of the enzyme. In an alternative approach, ectopic overexpression of soluble pyrophosphatase from E. coli has been used as a tool to decrease the levels of pyrophosphate in the cytosol. Constitutive overexpression leads to dramatic changes in sucrose and starch synthesis, sink-source relations and plant growth, phloem-specific overexpression of soluble pyrophosphatase leads to an inhibition of phloem transport, leaf mesophyll-specific overexpression leads to a small stimulation of sucrose synthesis, and potato tuber-specific overexpression leads to an inhibition of starch accumulation.     

Methods for the determination of intracellular levels of ribose phosphates

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.