Uridine sugar nucleotides modified in their nucleoside moieties and their effect on lipid-linked saccharide formationin vitro

Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol [GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50% at 230 micron periodate oxidised UDP-Glc and 70 micron periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol. The sugar nucleotide containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GlcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc.     

Ontogeny of Glucose and Lipid Metabolism in Dorsal Root Ganglia of Chickens.: Similarities and Contrasts with Sympathetic Ganglia

Dorsal root ganglia, excised from the lumbar roots of the sciatic nerve of white Leghorn chicken embryos 6-13 days of age, were incubated usually for 5 h, at 36 degrees C in 20 microliters of a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose. [U-14C]Glucose, [1-14C]glucose, [6-14C]glucose, or [5-3H]uridine was also added. Lipid synthesis and lactate output were measured by incorporation of 3H from [5-3H]uridine. Glucose uptake and labeled lactate output declined rapidly from 6 to 8-9 days of age, more slowly thereafter. Synthesis of lipids was relatively constant throughout the ages studied, without the increased rate at intermediate ages seen previously in sympathetic ganglia of the same species. RNA synthesis declined progressively throughout the ages studied. The output of C-6 of glucose to CO2 was about the same at all ages, whereas that of C-1 declined rapidly from 6 to 7 days of age and then more slowly, but always remained higher than that of C-6 and thus indicated that much glucose was metabolized via the hexosemonophosphate shunt.     

Glycosyl transferase activity in the brush border membrane of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta incorporates radiolabeled glucose when incubated in the presence of uridine diphospho(UDP)-D-(6-3H)glucose. The pH optimum for incorporation was 7.0 to 7.2 regardless of the buffer used. Transferase activity was maximal in 200 mM Tris buffer; 100 mM phosphate buffer inhibited significantly the incorporation of radiolabeled glucose, whereas 50 mM Tris-maleate and 100 mM PIPES resulted in moderate inhibition of activity. Incorporation of labeled glucose was not inhibited by low concentrations (0.01%) of Triton X-100, but activity was inhibited 50% by 0.25% Triton X-100. Addition of divalent cations to the brush border membrane preparation did not activate transferase activity, but addition of chelating agents (i.e., EDTA or EGTA) inhibited transferase activity nearly 90%. Incorporation of labeled glucose was inhibited by UDP, guanosine diphosphate (GDP), UDP- and GDP-activated monosaccharides, and monosaccharides, indicating that the transferase activity lacked substrate specificity.     

Biosynthesis of tunicamycin and metabolic origin of the 11-carbon dialdose sugar,tunicamine

Tunicamycin is a reversible inhibitor of polyprenol-phosphate: N-acetylhexosamine-1-phosphate translocases and is produced by several Streptomyces species. We have examined tunicamycin biosynthesis, an important but poorly characterized biosynthetic pathway. Biosynthetic precursors have been identified by incorporating radioactive and stable isotopes, and by determining the labeling pattern using electrospray ionization-collision induced dissociation-mass spectrometry (ESI-CID-MS), and proton, deuterium, and C-13 nuclear magnetic resonance (NMR) spectroscopy. Preparation and analysis of [uracil-5-(2)H]-labeled tunicamycin established the complete ESI-CID-MS fragmentation pathway for the major components of the tunicamycin complex. Competitive metabolic experiments indicate that 7 deuteriums incorporate into tunicamycin from [6,6'-(2)H,(2)H]-labeled D-glucose, 6 of which arise from D-GlcNAc and 1 from uridine and/or D-ribose. Inverse correlation NMR experiments (heteronuclear single-quantum coherence (HSQC)) of (13)C-labeled tunicamycin enriched from D-[1-(13)C]glucose suggest that the unique tunicamine 11-carbon dialdose sugar backbone arises from a 5-carbon furanose precursor derived from uridine and a 6-carbon N-acetylamino-pyranose precursor derived from UDP-D-N-acetylglucosamine. The equivalent incorporation of (13)C into both the alpha-1" and beta-11' anomeric carbons of tunicamycin supports a direct biosynthesis via 6-carbon metabolism. It also indicates that the tunicamine motif and the alpha-1"-linked GlcNAc residue are both derived from the same metabolic pool of UDP-GlcNAc, without significant differential metabolic processing. A biosynthetic pathway is therefore proposed for tunicamycin for the first time: an initial formation of the 11-carbon tunicamine sugar motif from uridine and UDP-GlcNAc via uridine-5'-aldehyde and UDP-4-keto-6-ene-N-acetylhexosamine, respectively, and subsequent formation of the anomeric-to-anomeric alpha, beta-1",11'-glycosidic bond.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.     

Influence of glycogen storage on vascular smooth muscle metabolism

The role of glycogen as an oxidative substrate for vascular smooth muscle (VSM) remains controversial. To elucidate the importance of glycogen as an oxidative substrate and the influence of glycogen flux on VSM substrate selection, we systematically altered glycogen levels and measured metabolism of glucose, acetate, and glycogen. Hog carotid arteries with glycogen contents ranging from 1 to 11 micromol/g were isometrically contracted in physiological salt solution containing 5 mM [1-(13)C]glucose and 1 mM [1, 2-(13)C]acetate at 37 degrees C for 6 h. [1-(13)C]glucose, [1, 2-(13)C]acetate, and glycogen oxidation were simultaneously measured with the use of a (13)C-labeled isotopomer analysis of glutamate. Although oxidation of glycogen increased with the glycogen content of the tissue, glycogen oxidation contributed only approximately 10% of the substrate oxidized by VSM. Whereas [1-(13)C]glucose flux, [3-(13)C]lactate production from [1-(13)C]glucose, and [1, 2-(13)C]acetate oxidation were not regulated by glycogen content, [1-(13)C]glucose oxidation was significantly affected by the glycogen content of VSM. However, [1-(13)C]glucose remained the primary ( approximately 40-50%) contributor to substrate oxidation. Therefore, we conclude that glucose is the predominate substrate oxidized by VSM, and glycogen oxidation contributes minimally to substrate oxidation.     

Starch synthesis by isolated amyloplasts from wheat endosperm

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of ¹⁴C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the ¹⁴C in the soluble fraction of the incubation mixture was recovered as [¹⁴C]glucose 1-phosphate. Thus it is unlikely that the added [¹⁴C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [¹³C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.Abbreviations PPase alkaline inorganic pyrophosphatase - UDPglucose uridine 5-diphosphoglucose     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose. The fractional enrichment pattern of teicoplanin produced in the medium containing [1-(13)C]glucose was obtained from a one-dimensional (13)C spectrum. The enrichment pattern showed characteristic peaks indicating that amino acids 3 and 7 are derived from acetate, whereas amino acids 1, 2, 4, 5, and 6 are derived from tyrosine. Multiplet structures in heteronuclear single quantum coherence spectra of teicoplanin produced in the medium containing [U-(13)C]glucose showed characteristic coupling patterns supporting these results. Fractional enrichment patterns and multiplet structures of the three sugars in teicoplanin showed that about 50% of the sugars have the same labeling pattern as the glucose substrate whereas the rest have a labeling pattern showing that they are reassembled, probably from precursors in the primary metabolism.     

Network flux analysis: impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures

The aim of this study was to test the assumption that (13)C-enrichment of respiratory substrate does not perturb metabolism. Cell suspension cultures of Arabidopsis thaliana were grown in MS medium containing unlabelled glucose (with (13)C at natural abundance), 100% [1-(13)C]glucose, 100% [U-(13)C(6)]glucose or 10% [U-(13)C(6)]glucose plus 90% unlabelled glucose. There was no significant difference in the metabolism of [U-(14)C]glucose between the cultures. Similarly, the pattern of (14)CO(2) release from specifically labelled [(14)C]-substrates was unaffected. Principal component analysis of (13)C-decoupled (1)H NMR metabolite fingerprints of cell extracts was unable to discriminate between the different culture conditions. It is concluded that (13)C-enrichment of the growth substrate has no effect on flux through the central pathways of carbon metabolism in higher plants. This conclusion supports the implicit assumption in metabolic flux analysis that steady-state (13)C-labelling does not perturb fluxes through the reactions of the metabolic network it seeks to quantify.     

Intermediatry steps in Acetobacter xylinum cellulose synthesis: studies with whole cells and cell-free preparations of the wild type and a celluloseless mutant.

Intermediatry steps in cellulose synthesis in Acetobacter xylinum were studied with resting cells and particulate-membranous preparations of the wild-type strain and of a celluloseless mutant. Exogenously supplied [1-14C]glucose was rapidly converted by resting cells of both types into glucose 6-phosphate, glucose 1-phosphate, and uridine glucose 5'-diphosphate (UDP)-glucose and incorporated into lipid-, water-, and alkali-soluble cellular fractions. The decrease in the level of labeled hexose-phosphates and UDP-glucose upon depletion of the exogenous substrate was accounted for by a continuous incorporation of [14C]glucose into cellulose in the wild type and into the above-mentioned cellular components in the mutant. [14C]glucose retained in the alkali- and water-soluble fractions of pulse-labeled wild-type cells was quantitatively chased into cellulose. Sonic extracts of both strains catalyzed the transfer of glucose from UDP-glucose into lipid-, water-, and alkali-soluble materials, as well as into an alkali-insoluble cellulosic beta-1,4-glucan. The results strongly support the sequence glucose leads to glucose 6-phosphate leads to glucose 1-phosphate leads to UDP-glucose leads to cellulose and indicate that lipid- and protein-linked cellodextrins may function as intermediates between UDP-glucose and cellulose in A. xylinum.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

Flexibility in the phosphorylase catalytic reaction. Glucosyltransfer from pyridoxal (5')-triphospho(1)-alpha-D-glucose to glycogen catalyzed by phosphorylase

When rabbit muscle phosphorylase reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose is incubated with glycogen, its glucosyl moiety is transferred to the nonreducing end of glycogen with the formation of a new alpha-1,4-glucosidic linkage. This finding provided the first evidence for the direct phosphate-phosphate interaction between the coenzyme pyridoxal 5'-phosphate and the substrate alpha-D-glucose 1-phosphate in the phosphorylase catalytic reaction (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3716-3719). We have examined whether pyridoxal(5')triphospho(1)-alpha-D-glucose can act in a similar manner to the diphospho compound or not. In the absence of glucan the enzyme-bound triphospho compound was stable for 1 day at pH 6-9. In the presence of glucan, however, its glucosidic linkage was cleaved, and the glucosyl moiety liberated was transferred to glycogen with the formation of a new alpha-1,4-glucosidic linkage. Allosteric activator AMP accelerated the reaction and allosteric inhibitor glucose 6-phosphate showed the reverse effect. The pH optimum of the reaction was pH 8.1-8.4. Mg2+ slightly but significantly accelerated the reaction, whereas Mn2+ and Ca2+ inhibited the reaction. These results indicate that the glucosyltransfer from the triphospho compound occurs in an identical manner to that from the diphospho compound. Based on the present and previous data, we discuss the catalytic mechanism of phosphorylase, especially in comparison with that of phosphoryltransferases.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

Non-enzymatic synthesis of the coenzymes,uridine diphosphate glucose and cytidine diphosphate choline,and other phosphorylated metabolic intermediates

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 °C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP. G1P and G6P were synthesized from glucose and inorganic phosphate at 70 °C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.     

Correction of glucose carbon recycling for the determination of 'true' hepatic glucose production rates by (1-13C1)glucose

Hepatic glucose production (HGP) and glucose carbon recycling are traditionally estimated by the combined use of hydrogen and carbon-labeled glucose tracers. A single-isotope method such as that of Reichard et al. for the determination of HGP and glucose carbon recycling requires the determination of activities in different glucose carbons by chemical degradation. Since the 13C content in the glucose carbon skeleton can be determined from mass fragmentography, the use of 13C-labeled glucose and mass fragmentography can provide a single-isotope method for the quantification of the recycled carbons. Correction for the recycling makes it possible to determine the true HGP. In this study, (1-13C1)glucose and mass fragmentography were used for the determination of HGP and glucose carbon recycling in six colon cancer patients. Molar enrichment of the molecular ion (m/z 328 cluster of glucose aldonitrile pentaacetate) was used to determine 'uncorrected' HGP, which was 1.93 +/- 0.11 mg kg-1 min-1 (mean +/- s.e.m.). The difference in molar enrichment of the molecular ion C1-C6 (m/z 328) and the ion corresponding to C1-C4 fragment (m/z 242) was used to determine the contribution of recycled label carbon. After this correction, the 'corrected' HGP was 2.04 +/- 0.12 mg kg-1 min-1, which is not significantly different from the 'true' HGP rate of 2.05 +/- 0.15 mg kg-1 min-1 determined by using (6-3H)glucose. HGP determined from the enrichment of the molecular ion C1-C6 underestimates true HGP, as expected. The corrected HGPs correlate well with those from 6-3H method (r = 0.86, y = 1.06x - 0.12; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of beta-(1-->3)-Glucan from Extracellular Uridine Diphosphate Glucose as a Wound Response in Suspension-cultured Soybean Cells

Soybean (Glycine max) suspension-cultured cells were incubated with 600 micromolar uridine diphosphate [¹⁴C]glucose, and the incorporation into alkali-insoluble material was studied. When the cells were kept in suspension by shaking on a linear shaker, the incorporation was very low. The incorporation was stimulated 30-fold when the cells were continually resuspended by stirring with a narrow glass rod. The stirring procedure was shown to damage some of the cells, and the incorporation appeared to be a wound response. The alkali-insoluble material formed was a β-(1→3)-glucan, and it was synthesized from uridine diphosphate glucose which did not penetrate through the plasma membrane of intact cells. The synthetase activity was probably induced by the stirring procedure. No evidence for cellulose synthesis from extracellular uridine diphosphate glucose was obtained.