Localization of glycerate kinase and some enzymes for sucrose synthesis in c(3) and c(4) plants

The localization of some key enzymes leading to sucrose synthesis in photosynthetic tissue of C₃ and C₄ species was investigated. These included UDP-glucose (UDPG) pyrophosphorylase, sucrose phosphate synthetase, and glycerate kinase. Whether glycerate kinase is localized exclusively in the chloroplast or partly outside the chloroplast could influence the fate of carbon flow to sucrose through the glycolate pathway.     

CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.     

Rapid Increase in UDP-Glucose during Early Wheat Embryo Germination

The cellular content of UDP-glucose in isolated wheat (Triticum aestivum L.) embryo increases 8-fold during the first 40 minutes of imbibition. An additional 3-fold increase in the amount of UDP-glucose was observed in the next 5 hours of germination. This communication also describes a unique, quantitative method to achieve a high sensitivity in a direct determination of UDP-glucose with Na [³²P]pyrophosphate and UDP-glucose pyrophosphorylase. The sensitivity of the assay for UDP-glucose is 10 picomoles.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Occurrence of sucrose and sucrose metabolizing enzymes in achlorophyllous algae

The presence of sucrose and the enzymes related to sucrose metabolism, i.e. sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13), sucrose phosphate synthase (SPS) (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was demonstrated in Prototheca zopfii, a colorless alga. The levels of enzyme activities were lower than those obtained in Chlorella vulgaris, which is generally considered the photosynthetic counterpart of P. zopfii. Whem enzyme activities were measured in bleached cells of C. vulgaris, the levels were of the same order than those found in P. zopfii. These results would indicate that the sucrose metabolizing enzymes are not related to the algae ability to carry on photosynthesis.     

Synthesis of osmoprotectants by halophilic and alkaliphilic methanotrophs

The¹H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5–1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium ofMethylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-contaihing osmoprotectants, ectoine and 5-oxo-1-proline.M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH ₄ ⁺ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

An improved radiochemical assay for uridine diphosphoglucose pyrophosphorylase

UDP glucose is an important intermediate in numerous metabolic pathways (1). It is therefore not surprising that the enzyme which catalyses its formation, UDP-glucose pyrophosphorylase is ubiquitous (see (2) for references). The reaction catalysed by UDP-glucose pyrophosphorylase is:

$\text{glucose-1}\text{-P +}\text{UTP ? UDP glucose + PPi}$

and the enzyme has been assayed either in the direction of pyrophosphorolysis of the nucleoside diphosphate sugar or in the direction of UDP-glucose formation.Spectrophotometric assays of UDP-glucose pyrophosphorylase in the direction of pyrophosphorolysis are often nonspecific by virtue of the nature of the coupling enzymes (3), whereas similar assays in the direction of UDPG formation may lack the expected stoichiometry of reaction (3,4). Radioisotopic techniques for the assay of UDP-glucose pyrophosphorylase (5,6) are to be preferred to spectrophotometric assays both for their increased sensitivity and specificity. However, these methods depend upon the specific isolation of the radioactive UDP glucose formed, either by a somewhat tedious adsorption to and elution from charcoal (5) or a hazardous precipitation using mercuric acetate. For routine assay of a large number of samples it would be advantageous to replace these techniques with one involving a safer, more rapid method of radioactive UDP-glucose isolation. The radiochemical assay described in this note utilises the binding of UDP glucose to commercially available, anion-exchange filter-paper discs for this purpose. Although the technique was designed to assay UDP-glucose pyrophosphorylase in cell extracts of the cellular slime mould, Dictyostelium discoideum, it should be applicable to most sources of the enzyme.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.     

Sucrose metabolism in Sorghum vulgare at ripening

Enzymes associated with sucrose metabolism in root, stem, leaf and grain of Sorghum vulgare Pers. (cv. JS 263) were studied at the ripening stage. Sucrose phosphate synthetase was dominating in the leaf and sucrose synthetase in the grain. Invertases were more active in leaf, root and stem tissues than in grains. The maximum activities of ADPG pyrophosphorylase and UDPG pyrophosphorylase were found in grains and leaves, respectively. Sucrose synthetase from grains catalyses both synthesis and cleavage of sucrose but the two activities differed in their responses to the effect of temperature, pH and type of buffer. The Km values of the enzyme for UDPG, ADPG, GDPG, TDPG and CDPG were 8.5, 5.3, 16.8 2.2 and 10.7 mM, and for UDP and ADP they were 17.2 and 55.0 mM respectively.     

Oligomerization,Membrane Association,and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H₂O₂ strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Spectral changes arising from the action of spinach chloroplast ribosephosphate isomerase on ribose 5-phosphate

Following addition of spinach chloroplast ribosephosphate isomerase (RPI) to ribose 5-phosphate (R5P) buffered with neutral phosphate, the sequential appearance of compounds absorbing at 280, 308.5, and 285 nm was observed. The 280-nm absorbing compound has previously been identified as ribulose 5-phosphate (Ru5P). The 308.5-nm absorbing compound has a high molar extinction coefficient and was a transient species, appearing only after production of Ru5P and preceding the accumulation of the 285-nm absorbing compound. The 285-nm absorbing compound has been identified as 4-hydroxy-5-methyl-3(2H)-furanone (I). Following addition of rabbit skeleton muscle RPI to R5P buffered with phosphate only the 280-nm absorbing compound, Ru5P, could be observed. Preparations of RPI from photosynthetic tissues have invariably led to the rapid accumulation of I whereas preparations of RPI from nonphotosynthetic tissues produce only Ru5P. In solutions of R5P buffered with citrate, the transient band at 308.5 nm was not observed although I accumulates in solution in the presence of spinach chloroplast RPI. Prior to formation of I a compound with a low molar extinction coefficient and wavelength of maximum absorption at approximately 310 nm can be detected. These results suggest that purified preparations of RPI from spinach chloroplasts and other photosynthetic tissues possess an enzymatic activity not present in crude extracts obtained from non-photosynthetic tissues. This activity is in addition to the aldol-ketol activity. The product of this heretofore unrecognized enzymic activity is postulated to be 3,4-epoxy-Ru5P and could be derived from Ru5P by removing the elements of water from the hydroxyl groups at C-3 and C-4.     

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate

Background

Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes.

Results

Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification.

Conclusions

Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides.

General significance

The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.