Kinetic pathways of formation and dissociation of the glycerol-3-phosphate dehydrogenase-fructose-1,6-bisphosphate aldolase complex.

Quantitative analysis of the time courses of fluorescence anisotropy changes due to the binding of fructose-1,6-bisphosphate aldolase to the dissociable cytoplasmic glycerol-3-phosphate dehydrogenase covalently labelled with fluorescent dye was carried out. The behaviour of the aldolase-dehydrogenase system seems to be consistent with a cyclic reversible model characterized by the formation and dissociation of complexes of both the monomeric and the dimeric forms of dehydrogenase with aldolase, and rapid equilibrium between the free monomeric and dimeric forms of dehydrogenase. The half-life time of the formation of dimeric dehydrogenase-aldolase complex at the concentration of the enzymes expected to exist in the cell (i.e. in the micromolar range) is some minutes, and the time needed for equilibration between the aldolase-bound dimeric and monomeric forms of dehydrogenase is a few minutes as well. Consequently, one may expect that both the formation and the dissociation of this heterologous enzyme complex have physiological relevance.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.     

Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies

The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6-bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose-1,6-bisphosphate concentration, with a second-order rate constant of 2.0 X 10(4) M-1 s-1, but were independent of enzyme concentration. Binding of fructose 1,6-bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second-order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction. The results suggest that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.     

Aldolase-localization in cultured cells: cell-type and substrate-specific regulation of cytoskeletal associations.

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20-2000 microM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20-60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin-aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.     

Dynamic interactions of enzymes involved in triosephosphate metabolism

A steady-state kinetic analysis of the coupled reactions catalysed by the three-enzyme system, aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end-product formation calculated for noninteracting enzymes were compared with those measured in the two-enzyme and three-enzyme systems. Changes in the fluorescence anisotropy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured. Glyceraldehyde-3-phosphate oxidation catalysed by glyceraldehyde-3-phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first-order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase-dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes. The steady-state velocity of 3-phosphoglycerate formation from fructose 1, 6-bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6-bisphosphate/glyceraldehyde 3-phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady-state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase-isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase. The decrease in the steady-state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentrations in the three-enzyme system. All of these observations fit the assumption that the amount of aldolase-dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.     

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.     

The influence of fructose-1:6-bisphosphate on the release of glycolytic enzymes from cellular structure

In order to provide information on the relative binding characteristics of glycolytic enzymes, the effect of fructose-1,6-bisphosphate (FBP) on the release of glycolytic enzymes from cultured pig kidney cells treated with digitonin has been studied. In the absence of FBP, a differential release of these enzymes was observed, with the order of retention being aldolase greater than glyceraldehyde-3-phosphate dehydrogenase greater than glucosephosphate isomerase, triosephosphate isomerase, phosphoglycerokinase, phosphoglucomutase, lactate dehydrogenase, enolase, pyruvate kinase and phosphofructokinase. In the presence of fructose-1,6-bisphosphate, the release of aldolase was considerably enhanced, whereas the release of phosphofructokinase and pyruvate kinase was decreased by this metabolite. No significant alterations in the rate of release of the other enzymes was caused by FBP. These data have been discussed in relation to their contribution to the knowledge of the degree of association and order of binding between glycolytic enzymes and the cytoplasmic matrix.     

Structure-based Reevaluation of the Mechanism of Class I Fructose-1,6-bisphosphate Aldolase

The enzymatic reaction carried out by class I fructose-1,6-bisphosphate aldolase is known in great detail in terms of reaction intermediates, but the precise role of individual amino acids in the active site is poorly understood. Therefore, on the basis of the crystallographic structure of the complex between aldolase and dihydroxyacetone phosphate a molecular modelling study was undertaken to predict the Michaelis complex with fructose-1,6-bisphosphate and several covalent enzymatic reaction intermediates. This model reveals the unknown 6-phosphate binding site and assigns distinct roles to crucial residues. Asp33 is responsible for aligning the 2-keto function of the substrate correctly for nucleophilic attack by Lys229, and plays a role in carbinolamine formation. Lys146 assists in carbinolamine dehydration and is essential for stabilising the developing negative charge on O4 of fructose-1,6-bisphosphate during hydroxyl proton abstraction by Glu187. Subsequently, Glu187 is also responsible for protonating C1 of the dihydroxyacetone phosphate enamine. In addition, the absolute configuration of the fructose-1,6-bisphosphate carbinol intermediate is shown to be (2S), in agreement with the crystal structure, but opposite from the interpretation in the literature of the stereospecific reduction of the aldolase fructose-1,6-bisphosphate complex with sodium borohydride. It is demonstrated that the outcome of the latter type of experiment critically depends on conformational changes triggered by Schiff base formation. Electronic Supplementary Material available.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Interaction of enzymes involved in triosephosphate metabolism. Comparison of yeast and rabbit muscle cytoplasmic systems

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution. A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Quantitative determination of triosephosphates during enzymatic reaction by high performance liquid chromatography: effect of isomerase on aldolase activity

Fructose-1,6-bisphosphate and triosephosphates have been separated by high performance liquid chromatography utilizing a SynChropack AX anion exchange column with 50-200 mM KH2PO4, pH 2.5-4.6 as mobile phase. The best resolution for each compound was reached in a system of 150 mM KH2PO4, pH 2.5. If radioactive fructose-1,6-bisphosphate as initial substrate was enzymatically converted in triosephosphates, the recoveries of metabolites after the precipitation and chromatographic procedures were higher than 95%. The concentration of radioactive 3-phosphoglycerate measured by liquid scintillation shows a good correlation (correlation coefficient: 0.997) with the spectrophotometrically determined concentration of NADH, which is formed from [U-14C]fructose-1,6-bisphosphate in equimolar concentration with 3-phosphoglycerate in aldolase and glyceraldehyde-3-phosphate dehydrogenase system. The method developed was applied to detect the inhibitory effect of triosephosphate isomerase on aldolase activity which takes place due to the heterologous complex formation.     

Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy

The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.     

An exploration of the binding site of aldolase using alkanediol monoglycolate bisphosphoric esters

Alkanediol monoglycolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The binding constants estimated from difference spectra correlate well with Ki values for the substrate analogues. Propanediol monoglycolate bisphosphoric ester protected aldolase from inactivation by 1,2-cyclohexanedione, which preferentially attacks arginine-55. However, propanol phosphate had little protective effect. The synthesized phosphate compounds protected the enzyme against inactivation by trypsin, and also against spontaneous denaturation. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site, which tends to keep the distance constant between the two phosphate-binding sites for the open-chain form of fructose 1,6-bisphosphate, and stabilize the natural conformation of the enzyme. Both arginine-55 and lysine-146 are shown to participate in the phosphate-binding site for the C-1-phosphate of fructose 1,6-bisphosphate.     

Inositol polyphosphate-mediated repartitioning of aldolase in skeletal muscle triads and myofibrils

The effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been hypothesized to be a chemical transmitter in excitation-contraction coupling in skeletal muscle, on aldolase bound to isolated triad junctions were investigated. Fructose-1,6-bisphosphate aldolase was identified as the major specific binding protein for the Ins(1,4,5)P3 analogue glycolaldehyde (2)-1-phospho-D-myo-inositol 4,5-bisphosphate which can form covalent bonds with protein amino groups by reduction of the Schiff's base intermediate with [3H]NaCNBH3. This analogue, Ins(1,4,5) P3, and the inositol polyphosphates inositol 1,3,4,5-tetrakisphosphate and inositol 1,4-bisphosphate were nearly equipotent in selectively releasing membrane bound aldolase with a K0.5 of about 3 microM. The rank order of the K0.5 values was identical to the KI values for inhibition of aldolase. Aldolase was also released by its substrate fructose 1,6-bisphosphate and by 2,3-bisphosphoglycerate. Ins(1,4,5)P3-induced aldolase release did not disrupt the triad junction; glyceraldehyde-3-phosphate dehydrogenase, a known junctional constituent, was displaced only at much higher Ins(1,4,5)P3 concentrations. Ins(1,4,5)P3 was as effective as fructose 1,6-bisphosphate in releasing aldolase from myofibrils. A finite number of binding sites for aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase/mg of triad protein, KD = 23 nM). The junctional foot protein was implicated as an aldolase binding site by affinity chromatography with the junctional foot protein immobilized on Sepharose 4B. The potential consequences of aldolase being bound in the gap between the terminal cisternae and the transverse tubule to inositol polyphosphate and glycolytic metabolism in that local region are discussed.     

亚适温弱光对黄瓜幼苗光合酶活性和基因表达的影响

以‘津优3号’为试材,研究亚适温弱光(18℃/12℃,100 μmol·m-2·s-1)下黄瓜幼苗叶片核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)、果糖-1,6-二磷酸酶(FBPase)、甘油醛-3-磷酸脱氢酶(GAPDH)、果糖-1,6-二磷酸醛缩酶(FBA)、转酮醇酶(TK) mRNA表达量及活性的变化.结果表明:亚适温弱光处理的单株叶面积和干物质量均明显减小.处理初期,Rubisco大亚基(rbcL)、小亚基(rbcS)、FBPase、GAPDH、FBA及TK的基因表达量大幅度下降,多数酶活性明显减弱(TK变化不明显),光合速率(Pn)快速降低;处理3d后,亚适温弱光处理的rbcL、rbcS基因表达量和Rubisco初始活性持续下降,但下降幅度明显减小,Rubisco总活性及FBPase、GAPDH、FBA和TK基因表达与活性均呈上升趋势,Pn同步回升;处理时间超过6d时,Rubisco和FBPase基因表达与活性趋于平稳,其他酶和Pn呈下降趋势.可见,亚适温弱光下黄瓜光合酶基因表达量和活性的降低是Pn降低的重要原因,光合机构对亚适温弱光的适应与光合酶的活化机制有关.     

Change of Carbon Metabolic Activity of Rhizobium Under Carbon Starvation

One fast growing strain of Rhizobium sp (Vigna mungo) VBS 1 was tested for its metabolic activities under carbon starvation. Specific activities of the catabolic enzymes like phosphofructokinase, fructose-1,6-bisphosphate aldolase, iso-citrate dehydrogenase and malate dehydrogenase decreased remarkably whereas, induction of two anapleurotic enzymes like fructose-1,6-bisphosphatase and iso-citrate lyase took place in the cell-free extract of the strain. Almost unchanged specific activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase indicated its key role in maintaining a balance between catabolic and anabolic activities under carbon starvation.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.