Gad Avigad

Sugar nucleotide transformations in plants

Publisher Summary This chapter discusses sugar nucleotide transformations in plant. The study of nucleotide sugars and of their central role in metabolism started with the discovery of UDP-Glc. UDP-Glc and other sugar nucleotides are ubiquitous in living organisms. The amount of nucleotide sugars is usually found to be 10–25 % of the pool of soluble nucleotides extracted from plant tissues. Many enzymes directly associated with the metabolism of the nucleotide sugars are known. The chapter also reviews the enzymes responsible for the formation of compounds and for inters conversions of the sugar skeleton in the nucleotide sugar molecules. The major mechanism by which nucleotide sugars are produced de novo is by the action of a pyrophosphorylase that catalyzes the reaction. The pyrophosphorylase reactions are actually nucleotidyl transfer reactions. The requirement for nucleotide sugars varies according to the stage of plant growth, and different control mechanisms may contribute to regulating the supply of nucleotide sugar precursors at the site of utilization.

Accumulation of trehalose and sucrose in relation to the metabolism of α-glucosides in yeasts of defined genotype

Abstract 1. 1. Glucose-grown haploid yeast hybrids lacking the ability to ferment sucrose and affording an extract devoid of sucrose activity failed to accumulate [14C]maltose or methyl-α-glucoside but accumulated [14C]sucrose up to 3% of cell dry weight. The accumulated [14C]sucrose could be removed by incubation with [12C]glucose or fermentable α-glucosides. Its accumulation was inhibited by 10−3M NaN3 or 10−3M 2,4-dinitrophenol. 2. 2. Although the cells when grown on maltose or methyl-α-glucoside contained an α-glucosidase which can hydrolyse sucrose, they failed to ferment or hydrolyse sucrose. They accumulated sucrose as did glucose grown cells. The possibility that a fragile intracellular barrier separating sucrose from the α-glucosidase exists in these cells is considered. 3. 3. The bulk of radioactivity fixed by cells incubated with 14C fermentable sugars (glucose, maltose, methyl-α-glucoside) was shown to reside in trehalose. In washed cells the amount of the accumulated trehalose is fairly stable but a rapid turnover and a small accretion of trehalose occurs in the presence of the fermentable sugar.

[59] UDP-glucose: Fructose transglucosylase from sugar beet roots☆

Publisher Summary This chapter discusses the synthesis of uridine diphosphate (UDP)-glucose from sugar beet roots. The UDP-dependent liberation of fructose from sucrose is determined colorimetrically. Several alternative procedures for assay of the reaction in both directions are described in the chapter. The most useful procedures are spectrophotometric determination of fructose liberated from sucrose in presence of UDP; spectrophotometric measurement of UDPG and UDP; colorimetric determination of the sucrose formed after treatment by alkali or borohydride or after hydrolysis by invertase. The degree of contamination of enzyme solutions obtained from different plant material by endogenous substrates, invertase, and phosphatases, determines the method of choice to be employed. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter. The paper chromatographic procedures for nucleotides and for sugars are employed for the detection and isolation of products, particularly in analyses based on the use of radioactive substrates. Identification of components is achieved by thin layer chromatography. The maximum rate of sucrose cleavage occurs between pH 6.0 and 6.5, where the enzyme is relatively unstable.

An enzymic synthesis of a sucrose analog: α-d-xylopyranosyl-β-d-fructofuranoside☆

Abstract Levansucrase transferred the β-fructofuranosyl group of raffinose to the anomeric carbon of xylose. The disaccharide “xylsucrose” formed by this reaction has been isolated and is shown to be α- d -xylopyranosyl-β- d -fructofuranoside. Xylsucrose was hydrolyzed by ordinary yeast invertase and was utilized both as donor and acceptor by levansucrase. Some other sucrases (dextransucrase, amylosucrase, and a special yeast sucrase) were inert towards this sucrose analog. The substrate specificity of levansucrase and the possible role of sucrose analogs in biological polymer synthesis are discussed.

Partition of sugars by thin-layer chromatography at low temperatures.

Abstract Several monosaccharides give two components when analysed by thin-layer chromatography on cellulose at −18°C. For D -fructose, the two spots corresponded to the furanoid and pyranoid forms. For d -mannose, the two spots could be correlated with the α and β anomers of the pyranoid form, and for d -galactose, the minor component is probably the hexofuranose. Other sugars, including d -glucose, gave a single spot under these conditions. It is suggested that chromatography at low temperatures is of potential vlaue as a useful analytical tool for the identification of minute amounts of certain reducing sugars.

[4] Colorimetric ultramicro assay for reducing sugars

Publisher Summary This chapter describes the colorimetric ultramicro assay for reducing sugars. Ferrocyanide produced as the result of the reduction of ferricyanide by a reducing sugar is assayed indirectly by the formation of ferrous ions. These subsequently interact with an iron chelate to yield a product with high color intensity. The reagent for iron employed in the following procedure is 2,4,6-tripyridyl- s -triazine. Reactions should be shielded from direct light. Aliquots (1.0 ml) of solutions containing 2–100 nmoles of reducing sugar are placed in test tubes. Reagent A (1.0 ml) is added, and the tubes are kept for 10 min in a boiling-water bath. Reagent B (1.0 ml) and reagent C (2.0 ml) are then added immediately, without cooling. The solutions are stirred vigorously with a Vibromixer, after 5 min in the dark, the absorbance at 595 nm of the violet Fe (TPTZ) 2+ 2 is against a reagent blank. It has been found out that 3-(2-pyridyl)-5,6-diphenyl-l,2,4-triazine can substitute for TPTZ as the reagent for ferrous ions used for the determination of reducing sugars. A procedure for the determination of ferrocyanide by direct UV-spectrophotometry in an assay for reducing sugars has been described. This method may be employed only when no components other than ferrocyanide that adsorb significantly in the far ultraviolet range are present in the solution analyzed.

[5] Colorimetric assays for hexuronic acids and some keto sugars

Publisher Summary This chapter discusses the colorimetric assays for hexuronic acids and some keto sugars. In the reaction of the reducing sugars with an acid-copper reagent, hexuronic acids and some keto sugars rapidly reduce dilute Cu + solutions (15.6 m M ) at acid pH in the presence of high salt concentrations to suppress Cu + reoxidation. The Cu + produced is measured colorimetrically with the arsenomolybdate reagent. The method is useful for the determination of hexuronic acids and certain ketoses in the presence of other sugars, aldoses in particular. It may also be employed for a specific measurement of hexuronic acids at the reducing ends of oligosaccharides, such as those appearing in enzymic digests of polysaccharides. In the reaction with m -hydroxydiphenyl, hexuronic acids, free or as glycosides, produce a distinctive chromogen when treated with tetraborate in concentrated sulfuric acid and subsequently allowed to react with m-hydroxydiphenyl. Interference by aldohexoses and aldopentoses that often creates a serious problem with the other colorimetric procedures, is significantly reduced in the reaction with m -hydroxydiphenyl.

Synthesis of D-galactose-6-t and D-galactosides-6-t

The aldehyde obtained by oxidation of o-nitrophenyl β-D-galactopyranoside with D-galactose oxidase was reduced with tritiated sodium borohydride. The labelled glycoside was subsequently hydrolyzed with β-D-galactosidase to yield D-galactose-6-t. Raffinose-t, labelled in the galactose residue, was prepared by a similar procedure. It is suggested that this method may be of general application to galactosides which can serve as substrates for galactose oxidase.

Purification of levansucrase by precipitation with levan

Summary Levansucrase (β-2,6-fructan: D -glucose 1-fructosyltransferase, EC 2.4.1.10) forms a complex with levan, which is the product of the enzymic reaction. The specificity of this interaction and the fact that the enzyme-polysaccharide complex precipitates from solution at 100 000 × g , was used as the basis for an efficient procedure for an extensive enzyme purification.

NADP dependent oxidation of TDP-glucose by an enzyme system from sugar beets.

Abstract Analysis of the free nucleotide pool in the mature sugar beet root demonstrated the presence of TDP-D-glucose (TDPG) and TDP-galacturonic acid (TDPGa1A) among other sugar nucleotides ( Katan and Avigad, 1966 ). TDP-glucose was also found to be formed in the ‘sucrose synthetase’ reaction by transglucosylation from sucrose to TDP ( Milner and Avigad, 1965 ). Further experiments have now shown that extracts prepared from sugar beet roots catalyzed the reduction of NADP when TDPG was added as a substrate. A preliminary analysis of this enzymic system is described in this communication.