Incorporation of glucose into lipid-linked saccharides in aorta and its inhibition by amphomycin

The particulate enzyme from pig aorta catalyzed the transfer of glucose from UDP-glucose into glucosyl-phosphoryl-dolichol, into lipid-linked oligosaccharides, and into glycoprotein. Radioactive lipid-linked oligosaccharides were prepared by incubating the extracts with GDP-[¹⁴C]mannose and UDP-[³H]glucose. When the labeled oligosaccharides were run on Bio-Gel P-4, the two different labels did not exactly coincide; the ³H peak eluted slightly earlier indicating that it was of higher molecular weight than the ¹⁴C material, but there was considerable overlap. The purified oligosaccharide(s) contained glucose, mannose, and N-acetylglucosamine but the ratios of these sugars varied from one enzyme preparation to another, probably depending on the endogenous oligosaccaride-lipids present in the microsomal preparation. Treatment of the [³H]glucose-labeled oligosaccharide with α-mannosidase gave rise to a ³H-labeled oligosaccharide which moved somewhat faster on Bio-Gel P-4 than the original oligosaccharide, suggesting it had lost one or two sugar residues. These data indicate that mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibited the transfer of glucose from UDP-glucose into the lipid-linked saccharides. However the synthesis of glucosyl-phosphoryl-dolichol was much more sensitive then was the synthesis of lipid-linked oligosaccharides. The glucose-labeled oligosaccharide produced in the absence of amphomycin was of high molecular weight based on paper chromatography. But in the presence of partially inhibitory concentrations of antibiotic, the oligosaccharide migrated more rapidly on paper chromatograms. However, amphomycin had no effect on the synthesis of glucosyl-ceramide by the aorta extracts. In fact, the antibiotic may stimulate glucosyl-ceramide by making more of the substrate, UDP-glucose, available for synthesis of this lipid.     

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Biosynthetic studies on N-acetylmannosaminuronic acid containing teichuronic acid in Bacillus megaterium

The particulate enzymes obtained from four strains of Bacillus megaterium AHU 1240, AHU 1373, AHU 1375, and T catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid, UDP-N-acetylglucosamine, and UDP-glucose. Chemical studies involving Smith degradation, acid hydrolysis, and N-acetylation revealed that the polysaccharide product has a backbone made up of trisaccharide repeating units comprising glucose, N-acetylmannosaminuronic acid, and N-acetylglucosamine and that the main oligosaccharide moieties of the glycolipids were identical with N-acetylmannosaminuronosyl-N-acetylglucosamine and glucosyl-N-acetylmannosaminuronosyl-N-acetylglucosamine. Incubation of the disaccharide-linked lipid with each particulate enzyme in the presence of UDP-glucose produced the trisaccharide-linked lipid and a polysaccharide. It is therefore suggested that in this polysaccharide-synthesizing system the repeating unit is formed on a carrier lipid from appropriate nucleotide derivatives first and the polymerization of the units then occurs to synthesize the backbone while the growing chain remains in pyrophosphate linkage to the carrier lipid presumed to be undecaprenol.     

Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland.

1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

Substrate Promiscuity: AglB,the Archaeal Oligosaccharyltransferase,Can Process a Variety of Lipid-Linked Glycans

Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform.     

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

Endo-β-N-acetylglucosaminidase H (endo H) is an enzyme which acts on asparagine- and lipid-linked oligosaccharides containing five or more mannose residues. Complex oligosaccharides and glycopeptides are completely resistant to the action of the enzyme. We have carried out pulse-chase experiments with ³⁵S-methionine and ³H-mannose in uninfected cells and in cells infected with Sindbis virus and vesicular stomatitis virus (VSV). In each case, the labeled materials were analyzed for sensitivity to endo H by polyacrylamide gel electrophoresis and gel filtration. We find that endo H releases all the labeled mannose from pulse-labeled proteins. Initially, the released material is nearly identical in size to the endo H cleavage product derived from lipid-linked oligosaccharides present in the same cells. During chase periods, ³⁵S-methionine and ³H-mannose protein becomes increasingly resistant to the enzyme. Moreover, the ³H-mannose-labeled material released from the protein during chase periods is smaller in size than the oligosaccharide from the lipid.On the basis of these results and results from other laboratories, we propose that during glycosylation of asparagine residues, a common oligosaccharide is transferred from the lipid carrier to protein and is subsequently processed to yield the so-called “high mannose” and “complex” oligosaccharides. Since, on the basis of present evidence, the lipid-linked oligosaccharide contains two N-acetylglucosamine, 8–12 mannose and 1–2 glucose molecules, it seems probable that the carbohydrate-processing systems remove half or more of the mannose and all of the glucose residues at sites destined to become complex glycopeptides. Removal of mannose and glucose residues may also occur at sites destined to become mature high mannose glycopeptides.     

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl₂. The K_m for UDP-glucose was determined as 27 micromolar, and the K_m for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [³²P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.     

Synthesis of mannosyl cellobiose diphosphate prenol in Acetobacter xylinum

The enzymatic synthesis of a β-mannosyl (1 → 3) β-glucosyl (1 → 4) α-glucose-1-pyrophosphate-prenol (allylic) by Acetobacter xylinum preparations is described. Glucose pyrophosphate lipid, already known to be formed from UDP-glucose and endogenous phosphate lipid, is demonstrated to accept another glucose from UDP-glucose to give a cellobiose pyrophosphate lipid. The latter in turn accepts mannose from GDP-mannose to form a mannosyl cellobiose pyrophosphate lipid. The structure of the trisaccharide and the way it is linked to the lipid moiety were established by enzymatic and chemical methods such as mild alkaline and acid hydrolysis, phenol treatment, partial acid hydrolysis and acetolysis, periodate oxidation, borohydride reduction, and treatments with glycosidases. The α-unsaturated, polyprenolic nature of the lipid was inferred from and confirmed by the reaction between UDP-glucose and ficaprenol monophosphate to give glucose pyrophosphate ficaprenol, which had the same properties as the glucose pyrophosphate lipid formed from the endogenous acceptor. The allylic structure proposed for the endogenous acceptor is suggested by the lability to phenol treatment and catalytic reduction of its glycosylated derivatives. The enzyme preparation also synthesizes a β-mannose phosphate prenol (allylic), which does not seem to participate in the trisaccharide synthesis. The possible role of these sugar prenols in the synthesis of exopolysaccharides is considered.     

The formation of oligoglucans linked to lipid during synthesis of beta-glucan by characterized membrane fractions isolated from peas.

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble beta-glucans, which like the oligosaccharides contained beta(1leads to 3) and beta-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in beta-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.     

Topography of glycosylation reactions in the rough endoplasmic reticulum membrane

The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.     

The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein.

The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.     

Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.     

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

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifoliiexoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.     

An improved radiochemical assay for uridine diphosphoglucose pyrophosphorylase

UDP glucose is an important intermediate in numerous metabolic pathways (1). It is therefore not surprising that the enzyme which catalyses its formation, UDP-glucose pyrophosphorylase is ubiquitous (see (2) for references). The reaction catalysed by UDP-glucose pyrophosphorylase is:

$\text{glucose-1}\text{-P +}\text{UTP ? UDP glucose + PPi}$

and the enzyme has been assayed either in the direction of pyrophosphorolysis of the nucleoside diphosphate sugar or in the direction of UDP-glucose formation.Spectrophotometric assays of UDP-glucose pyrophosphorylase in the direction of pyrophosphorolysis are often nonspecific by virtue of the nature of the coupling enzymes (3), whereas similar assays in the direction of UDPG formation may lack the expected stoichiometry of reaction (3,4). Radioisotopic techniques for the assay of UDP-glucose pyrophosphorylase (5,6) are to be preferred to spectrophotometric assays both for their increased sensitivity and specificity. However, these methods depend upon the specific isolation of the radioactive UDP glucose formed, either by a somewhat tedious adsorption to and elution from charcoal (5) or a hazardous precipitation using mercuric acetate. For routine assay of a large number of samples it would be advantageous to replace these techniques with one involving a safer, more rapid method of radioactive UDP-glucose isolation. The radiochemical assay described in this note utilises the binding of UDP glucose to commercially available, anion-exchange filter-paper discs for this purpose. Although the technique was designed to assay UDP-glucose pyrophosphorylase in cell extracts of the cellular slime mould, Dictyostelium discoideum, it should be applicable to most sources of the enzyme.