Properties of two high-molecular-mass forms of glyceraldehyde-3-phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity.

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-Mr glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G.R., Ortwerth, B.J. & Koeppe, O.J. (1970) J. Biol. Chem. 245, 4193-4198]. The other, a glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-Mr glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach. Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-Mr glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2). Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.     

Activation of Glyceraldehyde-Phosphate Dehydrogenase (NADP) and Phosphoribulokinase in Phaseolus vulgaris Leaf Extracts Involves the Dissociation of Oligomers

Phosphoribulokinase (EC 2.7.1.19, ATP: d-ribulose-5-phosphate-1-phosphotransferase) resembles the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, d-glyceraldehyde-3-phosphate: NADPH(+) oxidoreductase [phosphorylating]) of chloroplasts in that the activation of both of these enzymes involves the dissociation of oligomers (apparently tetrameric forms) with low catalytic activity to give protomers which possess higher catalytic activity. Gel filtration on Sepharose 6B has shown that the molecular weights of the oligomer and active protomer of phosphoribulokinase are, respectively, about 6.8 x 10(5) and 1.7 x 10(5), whereas the corresponding values for glyceraldehyde-3-phosphate dehydrogenase are 8.2 x 10(5) and 2.2 x 10(5). Activation of both enzymes occurs in response to either ATP, dithiothreitol, or cholate while the glyceraldehyde-3-phosphate dehydrogenase is also activated by NADPH. Activation/dissociation of these enzymes may involve conformational changes resulting from nucleotide binding, the reduction of sulfur bridges, and the cholate induced loosening of hydrophobic interactions.     

Characterisation of two forms of phosphoribulokinase isolated from the green alga, Scenedesmus obliquus

Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42,000-Mr subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of Mr 470,000 and apparent subunit composition X8Y4. The subunits X and Y are of Mr 39,000 and 42,000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD. Depolymerisation of the latent phosphoribulokinase to give the low-Mr form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol. The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-Mr latent form of the enzyme.     

Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus. Effects of dithiothreitol and nucleotide on coenzyme specificity.

NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.--) of the photosynthetic alga Scenedesmus obliquus is converted to an NADPH specific form by incubation with dithiothreitol. The change in nucleotide specificity is accompanied by a reduction in the molecular weight of the enzyme from 550 000 to 140 000. Prolonged incubation with dithiothreitol results in the further dissociation of the enzyme to an inactive 70 000 dalton species. The 140 000 dalton, NADPH-specific enzyme is stabilized against dissociation and inactivation by the presence of NAD(H) or NADP(H). Optimum stimulation of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity is achieved on incubation of the NADH-specific enzyme with dithiothreitol and NADPH, or dithiothreitol and a 1,3-diphosphoglycerate generating system. The relevance of these observations to in vivo light-induced changes in the nucleotide specificity of the enzyme is discussed.     

Glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus: Promotion of NADPH-dependent activity and antagonisation by NAD

The glyceraldehyde 3-phosphate dehydrogenase activity of extracts from heterotrophic Scenedesmus obliquus was linked predominantly to NADH. However, on DEAE-cellulose chromatography the enzyme was eluted by a gradient of phosphate in a form characterized by high NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity. This interconversion of enzyme forms could be prevented by the presence of NAD during DEAE-cellulose chromatography.High concentrations of phosphate stimulated the NADPH-dependent activity of the purified enzyme at the expense of activity linked to NADH and these changes were associated with depolymerization of a hexadecamer to a tetramer. The effect of phosphate on the rates of increase in NADPH-dependent activity and of a decrease in activity linked to NADH was cooperative with a Hill coefficient of 3.2. The inversely related changes in coenzyme specificity were inhibited to the same extent by NAD and the response to this ligand was anticooperative. These findings imply a strictly inverse proportional relationship between the rates of change of NADH and NADPH-linked activity. In the presence of dithiothreitol, low concentrations of phosphate promoted NADPH-dependent activity by stabilising the unstable tetrameric form produced from the hexadecamer by the thiol.These phenomena are discussed in relation to a general mechanism for the in vivo promotion of NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity.     

Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases.

1. NAD(P)+-induced changes in the aggregational state of prepurified NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativaum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55--70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70--95%. It contains a sigle subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38,000/40,000, 38,000/42,000 and 36,000/39,000 respectively. 2. The NAD-specific glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL-6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39,000. 3. The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde-3-phosphate dehydrogenases from higher plants.     

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.     

Thermodynamic analysis of the emergence of new regulatory properties in a phosphoribulokinase-glyceraldehyde 3-phosphate dehydrogenase complex

Glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase exist as stable enzymes and as part of a complex in Chlamydomonas reinhardtii. We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 +/- 0.5 and 1.3 +/- 0.3 kJ.mol(-1). Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase within the complex are regulated by NADP(H) but not by NAD(H). The activities of the metastable phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase released from the complex preincubated with NADP(H) are different from those of the metastable enzymes released from the untreated complex. NADP(H) increases phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase activities with a (~)K(0.5 (NADP)) of 0.68 +/- 0.16 mm and a (~)K(0.5 (NADPH)) of 2.93 +/- 0.87 mm and decreases NADH-dependent activity. 1 mm NADP increases the energy barrier of the NADH-glyceraldehyde 3-phosphate dehydrogenase-dependent reaction by 1.8 +/- 0.2 kJ.mol(-1) and decreases that of the reactions catalyzed by phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase by 3 +/- 0.2 and 1.2 +/- 0.3 kJ.mol(-1), respectively. These cofactors have no effect on the independent stable enzymes. Therefore, protein-protein interactions may give rise to new regulatory properties.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Changing Activities During Senescence and Sites of Synthesis of Photosynthetic Enzymes in Leaves of the Labiate, Perilla frutescens (L.) Britt.

The changing activities of several regulatory enzymes of thephotosynthetic carbon reduction cycle accompanying ageing ofthe third leaf pair of Perilla frutescens fall into two distinctcategories: firstly, enzymes which reach maximum activity priorto the completion of leaf expansion followed by a rapid decline(phosphoribulokinase, ribulose-l,5-diphosphate carboxylase,and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase);secondly, enzymes which maintain high activity beyond completionof leaf expansion and decline only at a late stage in senescence(phosphoglycerate kinase, NADH-linked glyceraldehyde-3-phosphatedehydrogenase, alkaline fructose-1,6-diphosphatase, and ribose-5-phosphateisomerase). The introduction of the ribosomal inhibitors D-threochloramphenicol, lincomycin, D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and cycloheximide to illuminated, detached shootsystems of 2-d-darkened Perilla plants has suggested that synthesisof ribulose diphosphate carboxylase, NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase, and possibly phosphoribulokinase, is mediatedby 70 S-based chloroplastic ribosomes. A chloroplastic siteof synthesis of these three photosynthetic enzymes is consistentwith their early deterioration during leaf ageing.     

Algal glyceraldehyde-3-phosphate dehydrogenases. Conversion of the NADH-linked enzyme of Scenedesmus obliquus into a form which preferentially uses NADPH as coenzyme.

Scenedesmus obliquus contains two glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.-) one of which uses NADH as its preferred coenzyme (D-enzyme) and the other NADPH (T-enzyme). On incubation of the D-enzyme with cysteine and a 1,3-diphosphoglycerate-generating system the specific activity with NADH as coenzyme decreased whilst that with NADPH increased by a factor of 10. The components of the generating system had no effect on the D-enzyme individually and it is concluded that 1,3-diphosphoglycerate was probably responsible for the change in nucleotide specificity. The coenzyme specificity of the T-enzyme was not affected by such treatment. A similar type of activation occurred to a lesser extent on incubation of the D-enzyme with 2,3-diphosphoglycerate. The NADPH-dependent activity of the D-enzyme could also be promoted by incubation with NADPH. However, in this case the activation was less than that seen with either 1,3- or 2,3-diphosphoglycerate. The change in coenzyme specificity of the D-enzyme occurred in parallel with changes in sedimentation behaviour. Initially, a single boundary of S20,w equals 14.5 S was present, but on conversion to NADPH-dependent activity by incubation with the 1,3-diphosphoglycerate-generating system, new boundaries of 7.5 S and 5.5 S appeared. The first of these corresponds in sedimentation coefficient to the native T-enzyme. On removal of 1,3-diphosphoglycerate the 7.5 S boundary disappeared accompanied by an increase in that of 14.5 S, whilst the 5.5 S boundary persisted. These changes are consistent with the reversible conversion of the D-enzyme into a form similar to the native T-enzyme in response to cysteine and 1,3-diphosphoglycerate. These effects may be explained if acylation of the active site of the D-enzyme by 1,3-diphosphoglycerate results in displacement of the bound nucleotide, thus promoting nucleotide exchange. These findings are consistent with the kinetic mechanism established for other glyceraldehyde-3-phosphate dehydrogenases. Similar activation was seen in extracts of other species of the Chlorophyta but not in other photosynthetic organisms. The significance of this type of activation of enzyme activity to the metabolism of these species of algae is discussed.     

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction (“binding fraction”) which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP⁺ causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.

Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.

The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD⁺ and NADP⁺ can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).

The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of “surplus” nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Glyceraldehyde-3-phosphate dehydrogenase activity studied under physiological conditions with a linear assay.

An assay method for glyceraldehyde-3-phosphate dehydrogenase in which none of the primary products accumulate and which gives linear kinetics under physiological conditions has been developed. It is based on the use of the 1,3-diphosphoglycerate produced by the enzyme for the formation of NADPH, while the NADH produced is recycled with an auxiliary system. Revised Km values at pH 7.4 for the muscle (rabbit and rat) enzyme are: glyceraldehyde-3-P, 50 μM; NAD, 100 μM; Pi, 10 mM. The rat erythrocyte enzyme gave similar values except for glyceraldehyde-3-P which was 300 μM. Cooperativity for NAD⁺ tends to be positive but is a variable parameter.     

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.     

Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans.

We report the sequencing of a 2,019-bp region of the Streptococcus mutans NG5 genome which contains a 1,428-bp open reading frame (ORF) whose putative translation product had 50% identity to the amino acid sequences of the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenases (GAPN) from maize and pea. This ORF is located approximately 200 bp downstream of the ptsI gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system. Mutant BCH150, in which the putative gapN gene had been inactivated, lacked GAPN activity that was present in the wild-type strain, thus positively identifying the ORF as the S. mutans gapN gene. Another strain of S. mutans, DC10, which contains an insertionally inactivated ptsI gene, still possessed GAPN activity, as did S. salivarius ATCC 25975, which contains an insertion element between the ptsI and gapN genes. Since the wild-type S. mutans NG5 lacks both glucose-6-phosphate dehydrogenase and NADH:NADP oxidoreductase activities, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is important as a means of generating NADPH for biosynthetic reactions.     

Striking conformational change suspected within the phosphoribulokinase dimer induced by interaction with GAPDH.

A multitechnique approach was used to study the [glyceraldehyde-3-phosphate dehydrogenase](2 x 4)-[phosphoribulokinase](2 x 2) multienzymatic complex of the alga Chlamydomonas reinhardtii. On the one hand, each component of the complex was compared with known atomic structures of related enzymes or of similar enzymes originating from different organisms. On the other hand, the overall low resolution architecture of the whole complex was studied using cryoelectron microscopy and image processing techniques. The dimers of phosphoribulokinase are suspected to undergo a dramatic change in activity during a cycle of binding and detaching from tetramers of glyceraldehyde-3-phosphate dehydrogenase. This is likely supported by strong structural differences between the modeled phosphoribulokinase dimers and the counterpart in the three-dimensional reconstruction volume of the whole complex obtained from cryoelectron microscope images.     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

Covalent linkage of lipoprotein to peptidoglycan is not essential for outer membrane stability inProteus mirabilis

Incubation of hexadecameric glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus with dithiothreitol caused a transient increase in NADPH-dependent activity. When ATP was also included in the incubation, the induced NADPH-dependent activity was stabilised. The rate of induction of NADPH-dependent activity increased hyperbolically with respect to ATP concentration. The effect of binding of ATP to the enzyme was two-fold; not only did it stimulate the dithiothreitol-promoted formation of an enzyme species with high NADPH-dependent activity it also prevented the inactivation of this species. The high concentration of ATP required for the stimulation of the NADPH-dependent activity compared with those for NADPH suggest that activation by ATP is unlikely to be of physiological significance.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.