UDP-glucose Pyrophosphorylase Activity Research Articles

Properties of uridine diphosphate glucose pyrophosphorylase from Golgi apparatus of liver

Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:α- d-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.

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.

The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach wheat and barley

Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [ 14C]sucrose from 14CO 2 at rates comparable with those of the parent tissue. CO 2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO 2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO 2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.

Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin.

When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells.

Changes in activity of enzymes associated with prespore differentiation in a suspension culture of Dictyostelium discoideum

Abstract It has been shown (Okamoto, K. (1981) J. Gen. Microbiol. in the press) that Dictyostelim discoideum cells dissociated at early aggregation can differentiate into prespore cells in a suspension containing glucose, albumin, EDTA and cyclic AMP. Strict requirement of cyclic AMP in this process has also been demonstrated. In the present paper, changes in activity of eight developmentally regulated enzymes were examined in this culture system and compared to those occuring in the normal course of development on the solid substratum. The results show that (a) formation in this medium is not accompanied by increases in activity of UDPglucose pyrophosphorylase and trehalose phosphate synthetase, unlike the case of the normal development, (b) among the enzymes examined, only UDPgalactose: polysaccharide galactosyl transferase can be regarded as a specific marker of the prespore formation, and (c) development in this system does not proceed beyond the slug stage of the normal development, in the case of a wild-type strain NC4.

Localization of Glycerate Kinase and Some Enzymes for Sucrose Synthesis in C3 and C4 Plants

The localization of some key enzymes leading to sucrose synthesis in photosynthetic tissue of C(3) and C(4) 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.In the C(3) species wheat, intact chloroplasts were isolated from protoplasts. Following separation of the chloroplasts by differential centrifugation and by sucrose density gradient centrifugation, UDPG pyrophosphorylase, sucrose phosphate synthetase, and sucrose synthetase were found outside the chloroplast while glycerate kinase was localized in the chloroplast.In the C(4) species (maize, NADP-malic enzyme-type; Panicum miliaceum, NAD-malic enzyme type and Brachiaria erucaeformis, phosphoenolpyruvate carboxykinase type) the distribution of UDPG pyrophosphorylase and glycerate kinase between mesophyll and bundle sheath cells and their intracellular localization in mesophyll protoplasts was determined. Substantial activity of UDPG pyrophosphorylase was found in both mesophyll and bundle sheath cells while glycerate kinase was localized only in mesophyll cells. From C(4) mesophyll protoplasts, UDPG pyrophosphorylase was found to be cytoplasmic while glycerate kinase appears exclusively localized in the chloroplasts as determined by differential centrifugation and sucrose density gradients.It seems that in both C(3) and C(4) plants, the terminal steps of sucrose synthesis occur exclusively in the cytoplasm while carbon flow in the glycolate pathway to sucrose must occur through glycerate kinase in the chloroplasts. The localization of glycerate kinase in mesophyll cells of C(4) plants may have further implications for intercellular flow of carbon during metabolism in the glycolate pathway in these species.

The role of NDP-glucose pyrophosphorylases in growing mung bean seedlings in relation to cell wall biosynthesis

NDP-glucose pyrophosphorylase activities were measured in different parts of growing mung bean seedlings. Amongst the different enzymic activities only UDP-glucose pyrophosphorylase proved to be active, showing highest activity in the lower, no-longer elongating, sections of the hypocotyl. In contrast UDP-glucose : glucan synthetase activity was highest in the upper, still-elongating sections of the hypocotyl. Using hypocotyls of 2–6-day-old seedlings it could be demonstrated that UDP-glucose pyrophosphorylase activity was increasing as elongation ceased. At the same time UDP-glucose : glucan synthetase activity was decreasing.

Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.

Changes in rat carbohydrate metabolism after acute and chronic treatment with dichlorvos

Administration to rats of a single dose of dichlorvos (DDVP) equal to 50% of the LD50 caused hyperglycemia which could be prevented, and the effect of the pesticide completely abolished, by administration of increasing doses of insulin. The glucose tolerance curves after po administration of glucose showed impaired regulation of carbohydrate metabolism and changes of the diabetic type. A statistically significant increase in glycogen phosphorylase activity was observed after administration of a single dose (50% LD50) of DDVP. UDPglucose pyrophosphorylase activity was decreased both after acute treatment and after administration of DDVP (5% of the LD50 daily) for 3 or 7 days, but the enzyme activity became adapted to the pesticide after 14 days of treatment with low doses of DDVP. In experiments in vitro, dichlorvos (at concentration of 10 −4, 10 −3, and 10 −2 m) stimulated the glycogen phosphorylase activity in liver extract only after 30-min preincubation. Dichlorvos inhibited synthesis of UDPglucose from UTP and G-1-P but only at concentrations as high as 10 −3 and 10 −2 m. Changes in glycogen synthetase activity were observed only at a very high dichlorvos concentration (10 −2 m).

Accumulation of UDP-glucose pyrophosphorylase by axenically grown amoebae of Dictyostelium discoideum

Amoebae of the slime mould Dictyostelium discoideum AX2 possess only low UDP-glucose pyrophosphorylase activity when grown on autoclaved Klebsiella aerogenes (approx. 30 units/mg of protein), but accumulate the enzyme to approx. 150-200 units/mg of protein during vegetative growth in axenic medium. The vegetative accumulation of UDP-glucose pyrophosphorylase by axenically grown cells is prevented if autoclaved K. aerogenes are included in the axenic medium, suggesting the absence of a specific inducer. Affinity chromatography using anti-(UDP-glucose pyrophosphorylase) antibody and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicate that the enzyme accumulated during axenic growth and that normally accumulated during development are immunologically cross-reactive and that both are composed of two subunits with mol.wts. 55,600 and 57,500 present in approximately equal amounts in the active enzyme.

Presence of a single enzyme catalyzing the pyrophosphorolysis of UDP-glucose and UDP-galactose in Bifidobacterium bifidum

The enzyme preparation catalyzing the pyrophosphorolyses of UDP-glucose and UDPgalactose almost at the same rate was purified about 900-fold from Bifidobacterium bifidum grown on glucose medium. The two activities were always associated with each other, and their activity ratio was always constant throughout the purification steps. The final preparation was revealed homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. There was no significant difference in thermal stabilities of the two activities. From these results it was concluded that the UDPglucose and UDPgalactose pyrophosphorylase activities in B. bifidum are catalyzed by a single enzyme protein.

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 beta-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only beta-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only beta-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.

Hyaluronic acid synthesis in rooster comb. Effect of testosterone on nucleotide sugar metabolism.

Enzymes involved in the synthesis of UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc), the nucleotide sugar precursors of hyaluronic acid, were assayed in extracts of the mucoid layer of combs of normal roosters, castrated roosters (capons) and capons treated with testosterone. UDP-glucose (UDP-G) dehydrogenase and UDP-GlcNAc pyrophosphorylase activities were found to be present in extracts of both normal and capon comb. Although UDP-G pyrophosphorylase activity could be demonstrated in extracts of normal combs, it was impossible, initially, to demonstrate this activity in capon extracts. A deficiency of this enzyme would result in less UDP-GlcUA available for the synthesis of hyaluronic acid. A protein inhibitor of this enzyme was found in capon extracts which inhibited the activity of the enzyme from normal comb. Glucose 1-phosphate (G1P) was found to overcome this inhibition. By increasing the G1P or UTP concentrations in assays of UDP-G pyrophosphorylase in capon comb extracts,...

Grain size, sucrose level and starch accumulation in developing rice grain

Five rices ( Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K + to the medium did not improve dry matter accumulation in the developing grain.

Entamoeba histolytica: Localization and characterization of phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, and glycogen synthase

Localization and characterization of phosphoglucomutase, (EC 2.7.5.1) uridine diphosphate glucose (UDPglucose) pyrophosphorylase (EC 2.7.7.9), and glycogen synthase (EC 2.4.1.11) were investigated in axenized Entamoeba histolytica. After differential centrifugation of homogenized amoebae, phosphoglucomutase activity was concentrated in the 110,000g supernatant (“soluble”) fraction. UDPglucose pyrophosphorylase was detected in all subcellular fractions; in the absence of exogenous Mg 2+, the highest specific activity was in the soluble fraction. All fractions exhibited a low but readily detectable level of glycogen synthase activity. The specific activity of UDPglucose pyrophosphorylase was enhanced by Mg 2+ in all particulate but not soluble fractions. The amoebal enzyme thus differs from that of mammalian liver and muscle in its intracellular distribution, being primarily particulate. The 15,000g pellet, which exhibited the highest specific activity, was treated with hydrolytic enzymes and sodium deoxycholate to elucidate the nature of enzyme binding. None of these treatments affected UDPglucose pyrophosphorylase, but trypsin abolished phosphoglucomutase activity. The detailed localization of both enzymes was investigated using fractions obtained by sucrose density centrifugation of the 15,000g pellet. Seven fractions and a pellet were isolated. Phosphoglucomutase was detected only in the lightest fraction which, under the electron microscope, appeared to consist largely of membrane fragments. UDPglucose pyrophosphorylase was detected in all of the density gradient fractions.

The effect of epsilon-aminocaproic acid on biochemical changes in the development of the cellular slime mould Dictyostelium discoideum.

epsilon-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain AX2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of N-acetylglucosaminidase, alpha-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells.

Carbohydrate and Enzymic Characterization of a High Sucrose Sugary Inbred Line of Sweet Corn

Reserve carbohydrates were determined on developing endosperm of a new line of sugary maize (Zea mays L.). Other entries, included for comparative purposes, were Midway (sugary), Funks G4646 (starchy), and Illini X-tra Sweet (shrunken-2). Sucrose in the new line, Illinois 677a, was more than twice that of Midway at most stages of development, and reached a maximum of 40% of dry weight at 18 days after pollination. Appreciable phytoglycogen accumulated in Illinois 677a, reaching 30% or more of dry weight as endosperm tissue matured. Thus, Illinois 677a is a typical sugary maize concerning phytoglycogen content, but it resembles shrunken-2 concerning the extent of sucrose accumulation.Enhanced sucrose accumulation by Illinois 677a was not accounted for by altered in vitro activities of invertase, sucrose synthase, UDP-glucose pyrophosphorylase, or ADP-glucose pyrophosphorylase. Its normal level of ADP-glucose pyrophosphorylase set Illinois 677a apart from shrunken-2 in which the enzyme was drastically reduced.

ADP-Glucose Pyrophosphorylase and UDP-Glucose Pyrophosphorylase Activity in Callus Tissue During Shoot Initiation and Callus Growth

Summary The level of activity of UDP-glucose pyrophosphorylase increased in callus tissue cultured under shoot-forming or nonshoot-forming conditions. Under the former conditions enzyme activity peaked between day 8 and day 10 whereas non-shoot-forming callus showed highest activity at day 14. A similar pattern of activity is shown for ADP-glucose pyrephosphorylase. Gibberellic acid stimulated UDP-glucose-pyrophosphorylase activity under both conditions while ADP-glucose pyrophosphorylase activity was reduced. Reduction in activity was more pronounced in non-shoot-forming callus. The level of UDPG in the soluble nucleotide pool was not significantly different in both tissues but ADPG was not detected. It is concluded that starch accumulation during shoot formation may not be regulated at the level of sugar nucleotide synthesis.

Pyrophosphorylases in Solanum tuberosum

Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.Evidence that UDP-glucose and ADP-glucose pyrophosphorylases are separate protein entities was indicated by their (a) activity ratio variations during tuber development and storage, (b) extraction stabilities, (c) morphological localization, (d) separation with ammonium sulfate, (e) pH optima, and (f) differential activation with 3-P-glycerate.

Glycogen synthesis by a strain of ehrlich ascites tumor

Summary A strain of Ehrlich Ascites cells, H, contains glycogen and synthesizes glycogen in vitro from added glucose or fructose. Only a fraction of the cells contain glycogen, and it is distributed in cytoplasm and nucleus. The metabolism of glucose and activities of enzymes of glycogen metabolism in the H cells, was compared to a strain (HL) which does not contain or form glycogen. The activities of glycogen phosphorylase, glycogen synthase, phosphoglucomutase and UDP glucose pyrophosphorylase were of similar magnitude in both strains. Glucogon, epinephrine, and dibutyryl CAMP had no effect on the metabolism of glucose and its incorporation into glycogen in H cells, and were also without effect on its catabolism.