Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.     

Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate.

Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates. The protein moiety is a large inner membrane molecule of about 319 kDa. In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively. Inner membranes of R. meliloti 102F34 and A. tumefaciens A348 were first incubated with UDP-[14C]Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions. A radioactive band corresponding to the 319-kDa protein was detected in both bacteria. Triton-solubilized inner membranes of A. tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-[14C]Glc. A [14C]glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa [14C]glucan protein intermediate was detected. In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc. A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-[14C]Glc. A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A. tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan. These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ. Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase). It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis.     

The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1----2)-glucan

The ndvB locus of Rhizobium meliloti was sequenced and found to encode a 319-kDa protein involved in the production of beta-(1----2)-glucan. Transposon Tn5 mutagenesis revealed that a large portion of the downstream half of this gene is not essential for symbiosis but is required for optimal production of beta-(1----2)-glucan. A high molecular weight inner membrane protein, believed to be the ndvB gene product, was absent from two different upstream ndvB::Tn5 mutants. This protein could be labeled in vitro with UDP-[U-14C]glucose in the wild type but not in the symbiotically defective mutants. Inner membrane preparations from the symbiotically competent downstream mutants labeled less well than did those from wild type with UDP-[U-14C] glucose and did not show distinct bands after polyacrylamide gel electrophoresis and fluorography, suggesting that C-terminal truncations of NdvB might affect the stability of this molecule. These downstream mutants had reduced amounts of periplasmic beta-(1----2)-glucan and exhibited several vegetative defects seen also in the upstream mutants. These included alterations in phage and antibiotic sensitivity, in motility, and in growth in low osmolarity media. Bacteroids produced by two of the downstream mutants were morphologically abnormal, indicating that ndvB is involved not only in invasion but also in bacteroid development.     

Biosynthesis of glycogen in Neurospora crassa. Existence of a glucoproteic intermediate in the initiation process.

A soluble enzyme preparation (20,000 X g supernatant fraction), prepared from the mycelia of wild-type Neurospora crassa, was capable of transferring [14C]glucose from UDP-[14C]glucose into both trichloroacetic acid (TCA)-soluble and TCA-insoluble macromolecule products in the absence of added primer. These reactions did not require either high concentrations of salts or any other chemical reagents. Two labeled products were formed; one was a glycogen-like polysaccharide and the other was a glycoprotein with glucosyl chains bound to protein through an acid-labile bond. After mild treatment of the glucoprotein with acid, the product liberated from the protein behaved as a mixture of malto-oligosaccharides and alpha-1,4-glucan with branches. The carbohydrate moiety of the glucoprotein seemed to be released upon prolonged incubation with the enzyme preparation. The glucan thus liberated from the glucoprotein may serve as a primer for the glycogen synthase. The results obtained are therefore suggestive of the existence of a glucoproteic intermediate in the initiation of glycogen biosynthesis.     

ISOLATION AND CHARACTERIZATION OF PARAMYLON SYNTHASE FROM EUGLENA GRACILIS (EUGLENOPHYCEAE)1,

The aim of this study was to isolate and characterize the paramylon synthesizing enzyme from Euglena gracilis Klebs. A method for enzyme solubilization with high synthase activity using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate is presented. Fractionated purification showed that the main enzyme activity was associated with the paramylon granula fraction, isolated from heterotrophically grown cells of E. gracilis. Further purification by sucrose density centrifugation resulted in a large enzyme complex with an apparent molar mass of 670 kDa (native). The complex remained active throughout the isolation procedures and produced beta-1,3-glucan in vitro. Two polypeptides of 37 and 54 kDa could be identified by photoaffinity labeling with [³²P]-UDP-glucose as substrate after SDS-PAGE.     

Synthesis of cyclic beta-(1,2)-glucans by Rhizobium leguminosarum biovar trifolii TA-1: factors influencing excretion.

The synthesis of cyclic beta-(1,2)-glucans from UDP-[14C]glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum bv. trifolii TA-1 was investigated. The crude membrane system needed Mn2+, ATP, and NAD+ for optimal activity. Hardly any difference in biosynthetic activity between membrane fractions of TA-1 cells grown in the presence (200 mM) or absence of NaCl was observed. Whole TA-1 cells grown in the presence of NaCl excreted labeled, neutral cyclic beta-(1,2)-glucan during incubation with added UDP-[14C]glucose. With NaCl-free cultured TA-1 cells, no excretion was observed; however, after these cells were alternately frozen and thawed eight times, they excreted glucans. Glucan formation in vitro and glucan excretion by whole cells were strongly inhibited in the presence of 50 mg of cyclic glucan per ml (about 15 mM), indicating that biosynthesis of cyclic beta-(1,2)-glucans in strain TA-1 is controlled by end-product inhibition. These observations indicate that TA-1 cells become more permeable to cyclic glucans at high NaCl concentrations. The constant loss of glucans from cells grown in the presence of 200 mM NaCl prevented end-product inhibition and resulted in glucan accumulation of up to 1,600 mg/liter in the medium.     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

The incorporation of glucose into alpha-1,4-glucan proteins of bovine retina membranes.

—Incubation of bovine retina membranes with UDP-[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous α-1, 4-glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m -sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein-I (GP-I), insoluble in TCA, and glucan protein-II (GP-II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP-I and GP-II were precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA). A third fraction, glucan protein-III (GP-III) was found when incubations were carried out with UDP-[¹⁴C]glucose at millimolar instead of micromolar concentrations. GP-III was soluble in TCA and in TCA-PTA and precipitable by ethanol from the TCA soluble fraction. GP-II was excluded from a Sephadex G-200 column and showed a greater size than GP-I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G-25 column and were of a greater size than the labelled residues released with salivary α-amylase. Only radioactive maltose was found after a-amylase treatment. When membranes containing labelled GP-I and GP-II were incubated with unlabelled UDP-glucose at millimolar concentrations, GP-I was converted into GP-II and GP-III was formed.     

The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

The fractionation of histones isolated from Euglena gracilis.

1. The histones of Euglena gracilis were separated by gel filtration into five fractions. 2. Each fraction was characterized in terms of its electrophoretic, solubility and compositional properties. 3. Euglena gracilis clearly contains histones corresponding to vertebrate H1, H2B, H3 and H4 fractions, although they all differ in containing more lysine. 4. The remaining Euglena histone is considered to be homologous to vertebrate histone H2A, but it differs in having a much higher ratio of lysine to arginine. 5. The Euglena histone H1 appears to be lacking in aspartic acid. 6. Electrophoresis in the presence of sodium dodecyl sulphate indicates that the molecular weights of the Euglena histones are close to those of the homologous vertebrate histones.     

Synthesis of Polysaccharides in Phragmoplasts Isolated from Tobacco BY-2 Cells

Autoradiographic experiments using preparations of isolatedphragmoplast obtained from tobacco cultured cells revealed thatthe radioactivity incorporated into insoluble material fromUDP-[³H]glucose was exclusively present at the cell plate ofisolated phragmoplasts. Most of the radioactivity incorporatedinto isolated phragmoplasts from UDP-[¹⁴C]glucose was solubilizedby 1,3-ß-glucanase and the solubilized radioactivitywas associated only with glucose, indicating that most of theradioactivity was incorporated into 1,3-ß-glucan.In the presence of high concentrations of unlabeled UDP-glucose,isolated phragmoplasts incorporated radioactivity from UDP-[³H]xylose.Most of the radioactivity incorporated into insoluble materialwas present at several sites distributed around the nuclei,while only little was found at the cell plate. (Received October 2, 1991; Accepted February 24, 1992)     

alpha-D-galactosyltransferase activity in calf thymus. A high-resolution 1H-NMR study

In calf thymus an alpha-D-galactosyltransferase activity has been detected that transfers galactosyl residues from UDP-galactose to suitable acceptors having galactose at the non-reducing terminus. For example, incubation of UDP-[14C]galactose and Gal beta(1 leads to 4) GlcNAc (N-acetyllactosamine) in the presence of a calf thymus cell-free suspension containing this galactosyltransferase activity resulted in the enzymic synthesis of a 14C-labelled trisaccharide. 500-MHz 1H-NMR spectroscopic analysis revealed the structure of the trisaccharide to be: Gal alpha (1 leads to 3) Gal beta (1 leads to 4) GlcNAc. This study illustrates the suitability of the 1H-NMR method for the analysis of enzymic conversions of carbohydrate chains.     

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.     

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.     

Participation of a trisaccharide-lipid in glycosylation of oviduct membrane glycoproteins.

Preincubation of a hen oviduct membrane preparation with UDP-Nactyl[14C]glucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (2/1)-extractable glycolipid. Treatment of the lipid with mild acid results in the release of a trisaccharide shown to have the structure beta-mannosyl-N-acetylglucosamineyl-N-acetylglucosamine. Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000...     

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.     

Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-beta-D-glucosyltransferase and 6-alpha-D-xylosyltransferase

A particulate enzyme fraction that catalyzes the transfer of glucose from UDP-[14C]glucose and of xylose from UDP-[14C]xylose into a xyloglucan has been isolated from suspension-cultured soybean cells. The incorporation of radioactivity from [14C]xylose into the polysaccharide was dependent on the presence of UDP-glucose in the incubation mixture, and that from [14C]glucose was dependent on the concentration of UDP-xylose in the mixture. Mn2+ was required for the incorporation of xylose and the optimum concentration of Mn2+ was about 10 mM. This reaction showed a pH optimum at 6.5 to 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and was inhibited by phosphate buffer and Tris buffer. On hydrolysis with Trichoderma endoglucanase, the polysaccharide synthesized in vitro gave a pentasaccharide, a hepatasaccharide, and a small amount of non-asaccharide. Based on the results from fragmentation and methylation analyses, the following structures were proposed for the penta- and the heptasaccharides from the xyloglucan synthesized in vitro: (formula, see text).     

Biosynthesis of pentosyl lipids by pea membranes.

Pea membranes were incubated with UDP-[14C]xylose or UDP-[14C]arabinose and sequentially extracted with chloroform/methanol/water (10:10:3, by vol.) and sodium dodecyl sulphate (2%, w/v). An active epimerase in the membranes rapidly interconverted the two pentosyl nucleotides. Chromatographic analysis of the lipid extract revealed that both substrates gave rise to xylose- and arabinose-containing neutral lipids, xylolipid with properties similar to a polyisoprenol monophosphoryl derivative, and highly charged lipid-linked arabinosyl oligosaccharide. When UDP-[14C]pentose or the extracted lipid-linked [14C]arabinosyl oligosaccharide were used as substrates, their 14C was also incorporating into sodium dodecyl sulphate-soluble and -insoluble fractions as major end products. Polyacrylamide-gel electrophoresis of sodium dodecyl sulphate-soluble products indicated the formation of mobile components with Mr values between 40 000 and 200 000 (Sepharose CL-6B). The lipid-linked [14C]arabinosyl oligosaccharide possessed properties comparable with those of unsaturated polyisoprenyl pyrophosphoryl derivatives. It was hydrolysed by dilute acid to a charged product (apparent Mr 2300) that could be fractionated in alkali. It was degraded to shorter labelled oligosaccharides by slightly more concentrated acid and eventually to [14C]arabinose as the only labelled component. Susceptibility to acid hydrolysis, and methylation analysis, indicated that the oligosaccharide contained approximately seven sequential alpha-1,5-linked arabinofuranosyl units at the non-reducing end. Several acidic residues appear to be interposed between the terminal arabinosyl units and the charged lipid.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.