Cell Wall Ingrowths in Nematode Induced Syncytia Require UGD2 and UGD3

The cyst nematode Heterodera schachtii infects roots of Arabidopsis plants and establishes feeding sites called syncytia, which are the only nutrient source for nematodes. Development of syncytia is accompanied by changes in cell wall structures including the development of cell wall ingrowths. UDP-glucuronic acid is a precursor of several cell wall polysaccharides and can be produced by UDP-glucose dehydrogenase through oxidation of UDP-glucose. Four genes in Arabidopsis encode this enzyme. Promoter::GUS analysis revealed that UGD2 and UGD3 were expressed in syncytia as early as 1 dpi while expression of UGD1 and UGD4 could only be detected starting at 2 dpi. Infection assays showed no differences between Δugd1 and Δugd4 single mutants and wild type plants concerning numbers of males and females and the size of syncytia and cysts. On single mutants of Δugd2 and Δugd3, however, less and smaller females, and smaller syncytia formed compared to wild type plants. The double mutant ΔΔugd23 had a stronger effect than the single mutants. These data indicate that UGD2 and UGD3 but not UGD1 and UGD4 are important for syncytium development. We therefore studied the ultrastructure of syncytia in the ΔΔugd23 double mutant. Syncytia contained an electron translucent cytoplasm with degenerated cellular organelles and numerous small vacuoles instead of the dense cytoplasm as in syncytia developing in wild type roots. Typical cell wall ingrowths were missing in the ΔΔugd23 double mutant. Therefore we conclude that UGD2 and UGD3 are needed for the production of cell wall ingrowths in syncytia and that their lack leads to a reduced host suitability for H. schachtii resulting in smaller syncytia, lower number of developing nematodes, and smaller females.     

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.     

Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD(+) ( approximately 40 microM) but variable affinities for the substrate UDP-glucose (120-335 microM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.     

Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis

Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.     

UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.     

Changes in enzyme activities involved in formation and interconversion of UDP-sugars during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in the activities of enzymes involved in UDP-sugar formation [UDP-glucose pyrophosphorylase (EC 2.7.7.9), sucrose synthase (EC 2.4.1.13) and UDP-glucuronic acid pyrophosphorylase (EC 2.7.7.44)], and interconversion [UDP-glucuse 4-epimerase (EC 5.1.3.2), UDP-glucose dehydrogenase (EC 1.1.1.22), UDP-glucuronic acid decarboxylase (EC 4.1.1.35) and UDP-xylose 4-epimerase (EC 5.1.3.5)] were investigated during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. The specific activities of UDP-glucose pyrophosphorylase and UDP-glucose 4-epimerase increased in the G2 phase before the first cell division, and those of sucrose synthase, UDP-glucose dehydrogenase and UDP-glucuronic acid pyrophosphorylase increased in the G1 phase after the first cell division. However, during the cell cycle, UDP-glucuronic acid decarboxylase and UDP-xylose 4-epimerase did not change significantly in their specific activities. Changes in enzyme activities are discussed in relation to those reported previously for cell wall composition (S. Amino et al. 1984. Physiologia Plantarum 60: 326–332).     

基于双酶偶联法合成尿苷二磷酸葡萄糖醛酸的研究

尿苷二磷酸（uridine diphosphate,UDP）-葡萄糖醛酸是细胞内重要的糖基供体,参与多种代谢途径,也是体外进行糖基化反应的重要糖基供体,但其价格昂贵、工艺复杂,限制了其大量使用,无法满足生产需求。基于此,利用双酶偶联法氧化UDP-葡萄糖生成UDP-葡萄糖醛酸,并研究反应产物的合成情况。以UDP-葡萄糖为底物、烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD+）为辅因子,利用化脓性链球菌Streptococcus pyogenes源的尿苷二磷酸葡萄糖脱氢酶（UDP-glucose dehydrogenase,UGD）、猪源的乳酸脱氢酶（lactate dehydrogenase,LDH）,双酶偶联催化合成UDP-葡萄糖醛酸,并通过高效液相色谱、质谱及核磁共振氢谱对反应产物进行检测,确定产物的结构及产物的生成量。结果表明,利用双酶偶联法氧化UDP-葡萄糖所得到的产物为UDP-葡萄糖醛酸。在UGD的作用下,氧化UDP-葡萄糖生成UDP-葡萄糖醛酸,同时辅因子NAD+在LDH的作用下实现循环再生,减少高能产物辅酶还原型烟酰胺腺嘌呤二核苷酸（reduced nicotinamide adenine dinucleotide,NADH）对反应的反馈抑制作用,产物的生成率约为60.17%。研究提高了产物UDP-葡萄糖醛酸产物生成量,为后续工业化制备提供了新思路。     

Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the K(m) values for UDP-glucose and NAD(+) were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.     

Characterisation and immunolocation of an 87 kDa polypeptide associated with UDP-glucuronic acid decarboxylase activity from differentiating tobacco cells (Nicotiana tabacum L.)

UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown-Agarose. The native enzyme had an apparent M(r) of 220,000 which yielded a single subunit of 87,000 when analysed on SDS-PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around M(r) 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.     

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens.

Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.     

Changes in intracellular UDP-sugar levels during the cell cycle in a synchronous culture of Catharanthus roseus

UDP-sugar contents were measured using high performance liquid chromatography and gas chromatography during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-xylose and UDP-arabinose could be determined, and 75–90% of the UDP-sugars were UDP-glucose. The contents of UDP-glucose and UDP-galactose increased in the late G2-M and the late S-M phases, respectively, whereas UDP-glucoronic acid and UDP-arabinose increased in amount in the G1 phase. These changes in the levels of UDP-sugars during the cell cycle generally correlated well with the changes in cell wall constituents and in the activities of the enzyme involved in synthesis and interconversion of UDP-sugars reported by S. Amino et al. (Physiol. Plant. 1985. 64: 111–117).     

Synthesis of Flavonoid O-Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.     

Uridine diphosphate-sugar metabolism by sycamore (Acer pseudoplatanus) cambial tissue

Summary Extracts of sycamore cambial tissue convert UDP-glucose into UDP-glucuronic acid, and the latter into UDP-xylose and UDP-rhamnose. None of the corresponding galactose series of monosaccharides was formed indicating the absence of epimerases, postulated as an important feature of differentiation. UDP-glucose is formed in greater quantities than any of its nucleoside analogues and it is suggested that UDP-glucose plays a more important role in carbohydrate metabolism in this tissue.     

Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae

UDP-D-glucuronic acid and UDP-D-xylose are required for the biosynthesis of glycosaminoglycan in mammals and of cell wall polysaccharides in plants. Given the importance of these glycans to some organisms, the development of a system for production of UDP-D-glucuronic acid and UDP-D-xylose from a common precursor could prove useful for a number of applications. The budding yeast Saccharomyces cerevisiae lacks an endogenous ability to synthesize or consume UDP-D-glucuronic acid and UDP-D-xylose. However, yeast have a large cytoplasmic pool of UDP-D-glucose that could be used to synthesize cell wall beta-glucan, as a precursor of UDP-D-glucuronic acid and UDP-D-xylose. Thus, if a mechanism for converting the precursors into the end-products can be identified, yeast may be harnessed as a system for production of glycans. Here we report a novel S. cerevisiae strain that coexpresses the Arabidopsis thaliana genes UGD1 and UXS3, which encode a UDP-glucose dehydrogenase (AtUGD1) and a UDP-glucuronic acid decarboxylase (AtUXS3), respectively, which are required for the conversion of UDP-D-glucose to UDP-D-xylose in plants. The recombinant yeast strain was capable of converting UDP-D-glucose to UDP-D-glucuronic acid, and UDP-D-glucuronic acid to UDP-D-xylose, in the cytoplasm, demonstrating the usefulness of this yeast system for the synthesis of glycans. Furthermore, we observed that overexpression of AtUGD1 caused a reduction in the UDP-D-glucose pool, whereas coexpression of AtUXS3 and AtUGD1 did not result in reduction of the UDP-D-glucose pool. Enzymatic analysis of the purified hexamer His-AtUGD1 revealed that AtUGD1 activity is strongly inhibited by UDP-D-xylose, suggesting that AtUGD1 maintains intracellular levels of UDP-D-glucose in cooperation with AtUXS3 via the inhibition of AtUGD1 by UDP-D-xylose.     

UDP-glucuronate decarboxylase, a key enzyme in proteoglycan synthesis: cloning, characterization, and localization

UDP-glucuronate decarboxylase (UGD) catalyzes the formation of UDP-xylose from UDP-glucuronate. UDP-xylose is then used to initiate glycosaminoglycan biosynthesis on the core protein of proteoglycans. In a yeast two-hybrid screen with the protein kinase Akt (protein kinase B), we detected interactions with a novel sequence, which we cloned and expressed. The expressed protein displayed UGD activity but did not display the activities of homologous nucleotide sugar epimerases or dehydratases. We did not detect phosphorylation of UGD by Akt nor did we detect any influence of Akt on UGD activity. Effects of UGD on Akt kinase activity were also absent. Northern blot and Western blot analyses revealed the presence of UGD in multiple tissues and brain regions. Subcellular studies and histochemistry localized UGD protein to the perinuclear Golgi where xylosylation of proteoglycan core proteins is known to occur.     

Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase.

Hemicellulose is a major component of primary plant cell walls. Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose. The enzyme controlling the biosynthesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22), was cloned from soybean (Glycine max [L.] Merr.) by an antibody screening procedure. This enzyme is surprisingly homologous to the bovine sequence, which is the only other eukaryotic UDP-glucose dehydrogenase sequence known. The characteristic motifs of the enzyme, the catalytic center, a NAD-binding site, and two proline residues for main chain bends, are conserved within the prokaryotic and eukaryotic sequences. The soybean full-length cDNA clone encodes a protein of 480 amino acids with a predicted size of 52.9 kD. The enzyme is highly expressed in young roots, but lower expression levels were observed in expanding tissues of the epicotyl and in young leaves. The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver

1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6–7.2. Michaelis–Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg²⁺; Ca²⁺ was slightly less, and Mn²⁺ slightly more, stimulatory than Mg²⁺. Of the activities found in standard assay systems containing Mg²⁺, 58–78% (substrate UDP-glucose) and 0–38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg²⁺-dependent activities against the concentration of added Mg²⁺ were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.