Diphosphate Sugars Research Articles

Yeast Cell-Wall Glucans

Publisher Summary This chapter describes the present state of knowledge on the structure and metabolism of the related glucan components of yeast cell walls. Many of the earlier structural studies involved commercial samples of pressed baker's yeast and, although these enabled the main structural features to be identified, they were of limited value from a metabolic point of view. With recent improvements in polysaccharide fractionation and in the methods of structural analysis, it is now possible to use smaller samples of yeast from laboratory-scale cultures as the experimental material, and hence, to relate the changes in cell–wall structure with cell growth. The structural analysis of glucan preparations isolated from various species of yeast is also described, where all of the constituent monosaccharide residues are assumed to have the D-configuration. The chapter discusses the synthesis of glucan from nucleoside diphosphate sugar compounds by a variety of yeast enzyme preparations, and lastly it also deals with the physiological control of the glucan content of yeast cell walls during various conditions of growth.

The synthesis of uridine diphosphate N-acetylhexosamines and uridine 5′-(2-acetamido-2-deoxy-α-D-mannopyranosyluronic acid diphosphate)

2-Methyl-(2-acetamido-3,4,6-tri-O-acetyl-l,2-dideoxy-β-D-mannopyrano)-[2,1-d]-2-oxazoline was efficiently converted into 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-mannopyranosyl phosphate, by treatment with dibenzyl phosphate, followed by catalytic hydrogenolysis of the benzyl groups. Similarly, 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl phosphate and -galactopyranosyl phosphate were synthesized from the respective peracetyl oxazolines. In each case, the procedures for preparing the oxazoline, and conversion into the glycosyl phosphate, were modified to give high yields of pure products. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-mannopyranosyl phosphate was coupled with 2′,3′-di-O-acetyluridine 5′-monophosphate by a modification of the mixed anhydride procedure, to give 2',3'-di-O-acetyluridine 5′-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-mannopyranosyl diphosphate), which was readily purified by preparative tic and O-deacetylated to give "uridine diphosphate N-acetylmannosamine" in high yield. Similarly, uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl- and -galactopyranosyl diphosphates) were synthesized by rapid, efficient procedures, not involving ion-exchange chromatography. Uridine 5′-(2-acetamido-2-deoxy-α-D-mannopyranosyl diphosphate) was converted into uridine 5′-(2-acetamido-2-deoxy-α-D-mannopyranosyluronic acid diphosphate), required for biosynthetic studies, without the preparation of a special platinum catalyst. All the synthetic uridine diphosphate sugars were characterized by optical rotation, 1H nmr spectrum, and elemental analysis.

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase.

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.

3-Ribosyluric acid and nucleotide content of erythroid cells in phenylhydrazine-induced anemia in cattle

1. 1. Steers injected with phenylhydrazine became anemic accompanied by an increase in the number of reticulocytes. 2. 2. The concentration of 3-ribosyluric acid in the red cells decreased until at day 5 it was about 20% that of the initial concentration and remained at this level until day 12 when it began to increase and reached the original'level by day 19. 3. 3. The concentration of the nucleoside di- and triphosphates and the uridine diphosphate sugars was markedly elevated in the blood samples with high reticulocyte counts, the concentration of these intermediates returned to their initial level by day 12. 4. 4. Bovine reticulocytes appear to have higher concentrations of nucleoside di- and triphosphates and a lower concentration of 3-ribosyluric acid compared to bovine erythrocytes.

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase d-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisophosphatase resembles other regulatory enzymes involved in photosynthetic CO 2 assimilation in its activation by chloroplast metabolites.

On the stability of nucleotide and nucleoside diphosphate sugar concentrations in isolated hepatocytes [proceedings

On the stability of nucleotide and nucleoside diphosphate sugar concentrations in isolated hepatocytes [proceedings

CO2‐Fixierung und Stofftransport in benthischen marinen Algen

When discs punched out of the median part of the phylloid of Laminaria saccharina Lamour. were exposed to H14CO3- in the light for periods of 10 sec to 10 min, 14C was rapidly incorporated into various photosynthetic products. As compared with dark fixation, 14C-photosynthesis increased exponentially during the first 60 sec of incubation in H14CO3-. Fixation rates were found to be 76 μmol CO2·dm-2·h-1 or 100 μmol CO2·mg-1 chlorophyll a·h-1. Eighty-five per cent of the total 14C assimilated after 10 sec was fixed in phosphoglycerate and in the sugar monophosphates, 2% in the sugar diphosphates, and only 3.5% in malate and aspartate. While the radioactivity of malate and aspartate only rose to a constant level, the percentage of the total 14C in phosphoglycerate and-to a lower extent-that in the sugar monophosphates rapidly decreased with the duration of light exposure. Simultaneously, mannitol and glycine+serine became labelled with 43% and 32% respectively of the total 14C after 10 min light fixation. In the dark, the percentage of the total 14C in malate decreased with the time of H14CO2--incubation, while there was a remarkable increase in radioactivity of aspartate and glutamate. Within 60 min darkness no labelling of mannitol was found.

Deoxythymidine sugars are not direct precursors of DNA-thymine

A theoretical model for the kinetics of uptake of a putative precursor molecule into nucleotide pools and into replicating DNA has been developed. The relationship between the accumulation of radioactively labeled precursors in the pool and the appearance of radioactivity in DNA is then derived. Experiments have been carried out in bacteria to compare the uptake of radioactive thymine into deoxythymidine triphosphate, deoxythymidine diphosphate sugars, and DNA to test the suitability of either compound as the direct precursor of thymine in DNA. New one-dimensional, thin-layer chromatographic procedures were used to determine the specific activity of deoxythymidine triphosphate and deoxythymidine triphosphate and deoxythymidine diphosphate sugars in growing cultures of 32PO4-labeled Escherichia coli during pulse labeling with [3H]-thymine. A comparison of the experimental data with our theoretical model supports the hypothesis that deoxythymidine triphosphate, but not deoxythymidine sugar, is the direct precursor of thymine in normally replicating DNA in vivo.

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars: I. Determination of the degradation rate during the glycosyltransferase assays

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phophatases; 3, chemical decomposition of UDP- D-[ 14C]Gal; UDP- D-[ 14C]Glc and UDP- D-[ 14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn 2+, Mg 2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP- D-Gal. The results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.

Nucleoside Diphosphate Sugar Hydrolase Gene of Salmonella typhimurium : Chromosomal Location Determined by Intergeneric Crosses

The gene specifying a membrane-bound nucleoside diphosphate sugar hydrolase of Salmonella typhimurium was mapped near the metA locus by using intergeneric crosses between this bacterium and Escherichia coli.

.alpha.- and .beta.-Glycopyranosyl phosphates and 1,2-phosphates. Assignments of conformations in solution by carbon-13 and proton NMR

The 1H and 13C NMR parameters of the anomeric pairs of aldopyranosyl phosphates and their rigid 1,2-phosphate derivatives are reported.The derivatives of D-glucose, D-galactose, and D-mannose exist in the 4C1 conformation while the L-fuco derivatives are in the C4 conformation. As judged by 31P--1H and 31P--13C coupling constants, all of the alpha anomers of the aldopyranosyl phosphates have the phosphate moiety predominantly trans to C(2) while in the beta anomers other rotamers make significant contributions. This relationship remains the same for the biologically important nucleoside diphosphate sugars (UDPGlc, UDPGal, GDPMan, and GDPFuc). From the pH dependence of 13C chemical shifts, observed in 0.5 M solutions, the pK'a2 of the alpha anomers is 6.1 while the pK'a2 of the beta anomers is 0.6--0.8 pH unit lower. In the 1.2-phosphates, the chair conformation of the parent aldose is retained while an envelope conformation is formed by the cyclic phosphate. In the alpha anomers, the plane is formed between C(2), C(1), O(1), and P while O(2) is above the plane. In the beta anomers, O(1) is out of the plane formed by the other atoms. The beta anomers have phosphorus coupled to C(3) with coupling constants of 10.8--11.7 Hz, approximately 2 Hz greater than the maximum reported for trans coupling (Lapper, R. D., & Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2880).

Polyprenyl phosphates as coenzymes in protein and oligosaccharide glycosylation

Partly saturated polyprenols of the dolichol type occur in all branches of the plant kingdom although in green plants they are quantitatively minor compared with the shorter polyprenols of the ficaprenol and betulaprenol type. Nevertheless, in all members of the plant kingdom so far studied it is the dolichyl phosphates that function in glycosyl transfer. In yeasts, dolichyl phosphate mannose is primarily an intermediate in the O-mannosylation of some membrane-bound proteins. Further mannosylation of the mannoprotein does not involve dolichyl phosphate. Yeast membrane preparations also catalyse the sequential transfer from the appropriate nucleoside diphosphate sugar of N -acetylglucosaminyl phosphate, N -acetylglucosamine, several mannose residues and several glucose residues to dolichyl monophosphate to form a dolichyl diphosphate oligosaccharide. The oligosaccharide is then transferred en bloc to protein to form an N-glycosidically linked glycoprotein. The transfer of mannose and glucose to the dolichyl diphosphate oligosaccharide is probably via dolichyl monophosphate sugar derivatives. Tunicamycin inhibits specifically the transfer of JV-acetylglucosamine phosphate which in turn blocks protein N-glycosylation. Evidence for corresponding processes in O- and N-glycosylation of proteins of hyphal fungi is also available but much less well established. The N-glycosylation of proteins by membrane preparations of green plants appears to occur by a process very similar to that found in yeast. In addition, the biosynthesis of β1-3 and β1-4 linked oligoglucans and also of an algal cellulose primer (a glucoprotein) via dolichyl phosphate glucose has been reported. Possible consequences of these phenomena are discussed.

ACID‐SOLUBLE NUCLEOTIDES OF ACHOLEPLASMA (MYCOPLASMA) LAIDLAWII A

A study was made of the acid-soluble nucleotides present in Acholeplasma (Mycoplasma) laidlawii A during growth. The nucleotide pool of this organism is meagre, both qualitatively and quantitatively. The main nucleotides are uridine mono- and diphosphates, and none of the more complex nucleotide compounds, such as uridine diphosphate (UDP) sugars or sugar peptides, required for cell wall formation, were detected. The nucleotide composition of A. laidlawii A is compared with that of the Streptococcus L-form.

Comparison of polyprenyl derivatives in yeast glycosyl transfer reactions

Seven homogeneous, semi-synthetic polyprenyl phosphates ranging in chain length from 20 to 95 carbon atoms and differing in the presence of saturated isoprene residues, were tested in several glycosyl transfer reactions catalyzed by yeast membranes. They were compared not only as acceptors of sugar phosphate from nucleoside diphosphate sugars but also as donors of the carbohydrate units to endogenous protein. All polyprenyl phosphates tested were effective substrates for the synthesis of mannosyl-lipid and differed by no more than a factor of two in activity. In contrast, a 5–8-fold preference for derivatives containing a saturated rather than an unsaturated α-isoprene residue was observed for utilization of the mannosyl-lipids as substrates for mannosylation of protein. The enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryl-polyprenol showed the highest specificity and only polyprenyl phosphates resembling natural yeast dolichyl phosphate in nearly all respects were effective substrates. Mono- N-acetylglucosaminyl-lipids were glycosyl donors to protein only after conversion to more polar lipids containing additional residues of N-acetylglucosamine and mannose. In all reactions tested, changes in the chain length of the polyprenyl moiety had little or no effect on activity.

The measurement of the rate of synthesis of uridine diphosphate sugars in animal tissues by [ 14C]orotate incorporation

1. 1. [ 14C]orotate incorporation into UDP-sugars was used for in-vivo studies of their synthesis. An equation which takes into account the relative molar specific activity (that of UMP = 100) and concentration of the compound newly formed was proposed to calculate the rate of UDP-sugar formation. 2. 2. Approximately a 2-fold and 5-fold lower rate of UDP-glucose synthesis was estimated in guinea-pig and chicken liver, respectively, than in rat liver. The same order in the rate of UDP- N-acetylglucosamine formation was observed. 3. 3. Approximately 50% of UDP-glucose was estimated to enter the UDP-glucuronic acid pathway in guinea-pig liver, while in rat and chicken liver this percentage was considerably lower (12 and 8, respectively). 4. 4. Compared to rat liver, a 20-fold lower rate of UDP-glucose synthesis was calculated in rat small intestine and pancreas. 5. 5. In contrast with rat liver, an equal rate in the synthesis of UDP-glucose and UDP-N-acetylglucosamine was found in rat small intestine and pancreas.

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.

Specificity of polyprenyl phosphates in the in vitro formation of lipid-linked sugars

Various polyprenyl phosphates were prepared by chemical phosphorylation of native and partially hydrogenated polyprenols. They were tested as lipid acceptors of sugars from nucleoside diphosphate sugars using a microsomal preparation from rat liver and membrane preparations from B. stearothermophilus, S. typhimurium, and Sh. flexneri. With the microsomal glycosyl transferase system, a demand for saturation of the α-isoprene residue of polyprenyl phosphate was observed; the chain length and cis/trans configuration of polyprenyl radical were less important. With bacterial glycosyl transferases, a demand for the unsaturated α-isoprene residue was observed. In B. stearothermophilus, the rate of synthesis of polyprenyl monophosphate glucose did not depend on the chain length of fully unsaturated polyprenyl phosphate. In S. typhimurium, C 55-polyprenyl phosphate was the most effective precursor of polyprenyl diphosphate galactose.

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir

A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar

During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.

Possible control sites of polysaccharide synthesis during cell growth and wall expansion of pea seedlings (Pisum sativum L.)

The activities of the enzymes of uridine diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) were measured by using enzymic preparations (protein precipitated between 40-65% (NH4)2SO4 saturation) isolated from segments at different stages of elongation of the third internode of pea seedlings. All enzymic activities increased from dividing and non-elongated cells to fully elongated cells. At all stages of growth, the specific activity or the activity per cell of UDP-D-glucose dehydrogenase was much lower than that of UDP-D-glucuronate decarboxylase and this may represent a controlling step in the formation of UDP-D-xylose. During elongation, changes were also found in the activities of the epimerases. These could be correlated with the corresponding variations which occur in the chemical structure and physical properties of pectins during cell wall extension. However, the high levels of the epimerases present in cells which have completed elongation growth suggest that pectin synthesis is mainly controlled at the sites of the synthetase reactions.