Identification, properties and genetic control of UDP-glucose: isovitexin 7-O-glucosyltransferase isolated from petals of Melandrium album

Summary In extracts of petals of M. album, an enzyme has been demonstrated which catalyzes the transfer of the glucosyl moiety of UDP-glucose to the 7-hydroxylgroup of isovitexin.This enzyme is controlled by a dominant gene G; in plants with the recessive genotype no glucosyltransferase activity could be detected.The enzyme was purified 16-fold by (NH₄)₂SO₄ fractionation and Sephadex-chromatography.The glucosyltransferase had a pH optimum of pH 7.5, was not stimulated by divalent metal ions, and had a true Km value of 1.2x10^-4 M for UDP-glucose and a true Km value of 4.6x10^-4 M for isovitexin.Several flavones with an apigenin hydroxylation pattern could serve as glucosyl acceptors. The highest activity was found, however, with isovitexin.The enzyme was unable to catalyze the transfer of xylose from UDP-xylose to the 7-hydroxylgroup of isovitexin, although isovitexin 7-O-xyloside has been found in petals of M. album plants.ADP-glucose could not serve as a glucosyl donor.The transferase activity was also present in leaves and calyces of GG plants. In these organs the transferase uses another flavone as a substrate. Neither isovitexin 7-O-glucoside nor isovitexin could be detected in these organs.     

Biosynthesis and genetic control of isovitexin 7-O-xyloside in the petals of melandrium album

An enzyme was detected in petal extracts of Melandrium album which catalysed the transfer of the xylose moiety of UDP-xylose to the 7-hydroxyl group of isovitexin. Genetical analysis revealed that the presence of the dominant allele g^x was necessary for enzymic activity. This activity was independent of the residual genetic background. Xylosyltransferase activity is also present in extracts of g^Gg^x plants, in which the product of the enzyme is not detectable. Maximal activity was found between pH 7·0 and 7·5; MnCl₂ inhibited this transfer. The enzyme had an ‘apparent K_m' value of 1·0 mM for UDP-xylose and of O·04 mM for isovitexin.     

The 2″-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes

Two separate genes, Fg and Vg, which govern the presence of isovitexin 2″-O-glucoside and vitexin 2″-O-glucoside respectively in the petals of Silene alba control different glucosyltransferases. In Vg/Vg,fg/fg plants no isovitexin 2″-O-glucosyltransferase was present and in vg/vg,Fg/Fg plants no vitexin 2″-O-glucosyltransferase activity could be detected. The Fg-controlled UDP-glucose: isovitexin 2″-O-glucosyltransferase has a pH optimum of8.5, while the Vg-controlled vitexin 2″- O-glucosyltransferase has a pH optimum of7.5. Both glucosyltransferases are stimulated by the divalent cations Ca²⁺, Co²⁺, Mn²⁺ and Mg²⁺. For isovitexin 2″-O-glucosylation, however, much higher concentrations are needed than for vitexin 2″-O-glucosylation.For UDP-glucose a ‘true K_m’ value of0.3 mM with the Fg-controlled and of 0.2 mM with the Vg-controlled enzyme was found. For isovitexin and vitexin these values are respectively 0.09 and 0.01 mM.     

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.     

Identification,properties, and genetic control of UDP-glucose: Cyanidin-3-rhamnosyl-(1→6)-glucoside-5-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 5-hydroxyl group of cyanidin-3-rhamnosyl-(1→6)-glucoside has been demonstrated in petal extracts of Silene dioica plants. This glucosyltransferase activity was not detectable in green parts of these plants. The enzyme activity is controlled by a single dominant gene M; no glucosyltransferase activity could be demonstrated in petals of m/m plants. The enzyme was purified eightyfold by PVP and Sephadex G50 chromatography. The glucosyltransferase had a pH optimum of 7.4, had a molecular weight of about 55,000, was stimulated by divalent metal ions, and had a “true K_m” value of 0.5×10^?3 m for UDP-glucose and 3.6×10^?3 m for cyanidin-3-rhamnosylglucoside. Pelargonidin-3-rhamnosylglucoside also could serve as acceptor. The enzyme did not catalyze the glucosylation of the 5-hydroxyl group of cyanidin-3-glucoside, although in petals of M/- n/n mutants cyanidin-3,5-diglucoside is present. ADP-glucose could not serve as a glucosyl donor.     

Variation in the substrate specificity of allozymes catalyzing flavone-O-glucoside biosynthesis in Silene plants

The 7-O- and 2-O-glycosylation of the flavone isovitexin (6-C-glucosylapigenin) in the petals of Silene plants is accomplished by allozymes which differ in their specificity toward the sugar to be transferred. The g locus controls the 7-O-glycosylation; allele gG controls the binding of glucose, and allele gX that of xylose. In the present paper it is shown that at least two different forms of gG exist. The enzyme activities encoded by these two different alleles differ with respect to the flavone acceptor to which glucose is transferred. Allele gGm encodes a 7-O-glucosyltransferase that transfers glucose to isovitexin but that is not able to glycosylate isovitexin 2-O-rhamnoside. The 7-O-glucosyltransferase encoded by allele gGd preferentially transfers glucose to isovitexin 2-O-rhamnoside and not to isovitexin. The allozymes encoded by gGm and gGd were partly purified. Linearity of incorporation, pH optimum, effect of divalent cations and EDTA, apparent molecular weight, substrate specificity, and Michaelis enzyme kinetic parameters were determined for both enzyme activities. The simultaneous presence within a plant of gene glR, which controls the biosynthesis of isovitexin 2-O-rhamnoside, with either gGm or gGd leads to different glycosylation types. In gGm/glR plants two monoglycosides accumulate in the petals, isovitexin 7-O-glucoside and isovitexin 2-O-rhamnoside, respectively, whereas in gGd/glR plants the corresponding diglycoside, isovitexin 7-O-glucose 2-O-rhamnoside, is synthesized. The distribution of the two alleles over chemical races of Silene pratensis in Europe is described; possible evolutionary relations between the various glycosyltransferases in Silene are discussed.These investigations were supported by the Foundation for Fundamental Biological Research (BION; Grant 14-15-01), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

The genetic control of isovitexin-glycosylation in the petals of Melandrium album

Summary The glycosylation of flavones in the petals of Melandrium album is shown to be controlled by the genes G, X and A. In the presence of the recessive alleles of these genes, only the aglycone isovitexin (6-C-glucosylapigenin) is found in the petals. The gene G controls the transfer of glucose, the gene X the transfer of xylose to the 7-hydroxyl group of isovitexin. The gene G is epistatic over X. In the presence of the gene A arabinose is coupled to the carbon-carbon bound glucose of isovitexin. In the presence of both G and A, or both X and A the corresponding di-glycosides are formed.The petals of the plants in which all genes for the flavone glycosylation are present in the homozygous recessive form are of a particular phenotype.     

Genetic control and biosynthesis of two new flavone-glycosides in the petals of Melandrium album

In a chemicogenetic analysis of the geographical distribution of flavone-glycosides in the petals of Melandrium album, we found two unknown flavone-glycosides in ten Hungarian and four German populations. By means of classical techniques for the identification and structure determination of flavonoids, the structure of these flavones turned out to be 6-C-glucosylglucosylapigenin and 7-O-glucosyl-6-C-glucosylglucosylapigenin, respectively. Genetic analysis showed that the coupling of glucose to the carbon-carbon bound glucose of isovitexin (6-C-glucosylapigenin) was controlled by a single dominant gene, Fg. Fg controls a UDP-glucose: isovitexin 6-C-glucosylglucosyltransferase. By means of ammonium sulfate fractionation and Sephadex chromatography, the enzyme was purified sixfold. The partly purified enzyme had a pH optimum between 8.0 and 8.5. The apparent K _m value for UDP-glucose in the presence of 1.0 mm isovitexin was 2.2 mm. The apparent K _m value for isovitexin in the presence of 1.8 mm UDP-glucose was 0.08 mm. The glucosyltransferase activity was stimulated by the divalent cations Mn, Mg, Co, and Ca. Neither 7-O-glucosyl nor 7-O-xylosyl isovitexin could serve as an enzyme substrate. Therefore, the biosynthesis of 7-O-glucosyl-6-C-glucosylglucosylapigenin found in the petals of M. album proceeds in a sequential manner: first the formation of 6-C-glucosylglucosylapigenin, followed by 7-O-glucosylation. Isovitexin 6-C-glucosylglucosyltransferase activity controlled by gene Fg could also be demonstrated in leaves of Fg plants. The enzyme probably uses another substrate in these green parts of the plant, because both isovitexin and isovitexin-glycosides are absent.     

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).     

A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ;mapping' in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5'-dithiobis-(2-nitrobenzoate). 5. NAD(+), UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4-8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.     

Identification and properties of UDP-glucose: Cyanidin-3-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been demonstrated in petal extracts of Silene dioica mutants with cyanidin-3-O-glucoside in the petals. This transferase activity was also present in young rosette leaves and calyces of these plants. The highest glucosyltransferase activity was found in petals of opening flowers of young plants. The enzyme was purified ninetyfold by PVP and Sephadex chromatography. The glucosyltransferase had a pH optimum of 7.5, had a “true K_m value” of 4.1×10^?4 m for UDP-glucose and 0.4×10^?4 m for cyanidin chloride, and was not stimulated by divalent metal ions. Both p-chloromercuribenzoate and HgCl₂ inhibited the enzyme activity. Pelargonidin chloride and delphinidin chloride at reduced rates also served as substrates. The enzyme did not catalyze the glucosylation of the 3-hydroxyl group of flavonols or the 5-hydroxyl group of anthocyanins. ADP-glucose could not serve as a glucosyl donor. The results of Sephadex G150 chromatography suggest that the glucosyltransferase can exist as dimer of about 125,000 daltons and as active monomers of 60,000 daltons. The genetic control of the glucosyltransferase activity is discussed.     

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.     

The pH dependent substrate specificity of udp-glucose: isovitexin 2″-O-glucosyltransferase in Silene alba

In Silene alba plants the dominant allele of gene Fg controls an enzyme which catalyses the formation of isovitexin 2″-O-glucoside both in petals and green parts. Both isovitexin and isoorientin can act as substrate. K_mvalues for the isovitexin glucosylation are 0.09 mM for isovitexin and 0.3 mM for UDP-glucose, V_max 0.17 nmol min^?1 mg protein^?1. For the isoorientin glucosylation K_m values of 0.45 mM for isoorientin, of 0.75 mM for UDP-glucose and V_max of 0.27 nmol min^?1 mg protein^?1 are found. The pH optima for both substrates differ markedly. For the substrate with one hydroxyl in the B-ring, isovitexin, the pH optimum is pH 8.5. For isoorientin, which has two hydroxyls in the B-ring, a pH optimum of 7.5 is found. These results suggest that the B-ring hydroxylation pattern influences the pH at which the substrate has optimal affinity for the enzyme. The location of the carbon-carbon bound glucose on a the flavonoid skeleton is of importance for enzyme activity as well. Vitexin, which has glucose at the 8-position, was not a substrate. The glucosylation of vitexin could, however, be demonstrated in enzyme extracts of petals of plants, grown from seed collected in Armenia; in these petals apart from isovitexin glycosides, vitexin glycosides are found as well.     

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.     

厚叶算盘子的化学成分研究(Ⅱ)

从厚叶算盘子(Glochidion hirsutum)的乙醇提取物中,用硅胶和凝胶柱层析分离得到8个化合物,通过波谱学方法鉴定为bergenin(1),isovitexin(2),isovitexin 7-O-xyloside(3),decoumaroylibotanolide(4),n-butyl-α-D-fructofuranoside(5),n-butyl-β-D-fructofuranoside(6),4-O-ethylgallic acid(7),3-O-methylgallic acid(8)。以上化合物均为首次从该植物中分离得到。     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

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.     

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.     

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).     

Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.