Myosin-linked calcium regulation in vascular smooth muscle.

Rat liver microsomal membranes have been shown to contain a biosynthetic pathway of UDP-glucose. In addition, they are able to bind UDP-glucose in a reversible manner, As UDP-glucose is also metabolized in these membranes, the study of the binding has been performed with a microsomal Triton X 100 extract. This reversible binding depends on pH (maximum at pH 8.1) and manganous ions, and disappears at pH 6.5. It exhibits a high affinity (K-diss equals 3 mu M), and a narrow specifity for UDP-glucose. Proteolytic digestion inhibits the binding up to 90%, showing that the UDP-glucose receptor has a proteic nature. These binding characteristics have been also found in the membranes themselves, indicating that the detergent solubilization does not destroy the protein binding capacity.     

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.     

Characterization of the <Emphasis Type="Italic"> ugpG </Emphasis>gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer <Emphasis Type="Italic"> Sphingomonas paucimobilis </Emphasis>ATCC 31461

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.     

Présence des enzymes catalysant la biosynthèse d'UDP glucose dans une fraction légère de l'appareil de Golgi des hépatocytes de chat

A Golgi-rich fraction is prepared from cat hepatocytes by the means of a four-step sucrose density gradient. The material applied to this gradient is composed either of smooth microsomes prepared from healthy animals, or of total microsomes prepared from cat treated by 50 per cent ethanol (0.6 g/100 g body weight, administered by stomach tube).A light fraction (d : 1.10) is obtained by the two procedures. It does not show any glucose-6-phosphatase activity, but is enriched in sialyltransferase, known as a marker enzyme for Golgi apparatus. It also contains the three enzymes implicated in the biosynthetic pathway for UDP-glucose (glucokinase, phosphoglucomutase and UTP : glucose-1-phosphate uridylyltransferase).UDP-glucose being the ultimate substrate in membranous glucosylation reactions, these results could support the hypothesis that sugar-nucleotides necessary for the glycoprotein biosynthesis are produced in the Golgi vesicles directly.     

Presence of enzymes catalyzing UDP-glucose biosynthesis in a low density Golgi fractions of cat hepatocytes]

A Golgi-rich fraction is prepared from cat hepatocytes by the means of a four-step sucrose density gradient. The material applied to this gradient is composed either of smooth microsomes prepared from healthy animals, or of total microsomes prepared from cat treated by 50 per cent ethanol (0.6 g/100 g body weight, administered by stomach tube). A light fraction (d : 1.10) is obtained by the two procedures. It does not show any glucose-6-phosphatase activity, but is enriched in sialyltransferase, known as a marker enzyme for Golgi apparatus. It also contains the three enzymes implicated in the biosynthetic pathway for UDP-glucose (glucokinase, phosphoglucomutase and UTP : glucose-1-phosphate uridylyltransferase). UDP-glucose being the ultimate substrate in membranous glucosylation reactions, these results could support the hypothesis that sugar-nucleotides necessary for the glycoprotein biosynthesis are produced in the Golgi vesicles directly.     

The molecular architecture of galactose mutarotase/UDP-galactose 4-epimerase from Saccharomyces cerevisiae

The metabolic pathway by which beta-D-galactose is converted to glucose 1-phosphate is known as the Leloir pathway and consists of four enzymes. In most organisms, these enzymes appear to exist as soluble entities in the cytoplasm. In yeast such as Saccharomyces cerevisiae, however, the first and last enzymes of the pathway, galactose mutarotase and UDP-galactose 4-epimerase, are contained within a single polypeptide chain referred to as Gal10p. Here we report the three-dimensional structure of Gal10p in complex with NAD(+), UDP-glucose, and beta-D-galactose determined to 1.85-A resolution. The enzyme is dimeric with dimensions of approximately 91 A x 135 A x 108 A and assumes an almost V-shaped appearance. The overall architecture of the individual subunits can be described in terms of two separate N- and C-terminal domains connected by a Type II turn formed by Leu-357 to Val-360. The first 356 residues of Gal10p fold into the classical bilobal topology observed for all other UDP-galactose 4-epimerases studied thus far. This N-terminal domain contains the binding sites for NAD(+) and UDP-glucose. The polypeptide chain extending from Glu-361 to Ser-699 adopts a beta-sandwich motif and harbors the binding site for beta-D-galactose. The two active sites of Gal10p are separated by over 50 A. This investigation represents the first structural analysis of a dual function enzyme in the Leloir pathway.     

Glucose starvation results in UDP-glucose deficiency and inactivation of glycogen synthase

The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to approximately 10% of those in cells cultured with glucose. GS activity, protein and mRNA amounts in pyruvate-cultured cells were decreased to approximately 35, 60, and 60%, respectively, of values in glucose-cultured cells. Incubation of extracts from glucose-cultured cells with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS. However, binding in immunoprecipitates from pyruvate-cultured cells was decreased to approximately 25% of values in glucose-cultured cells. These data indicate that glucose starvation and the subsequent depletion of UDP-glucose result in: (1) inactivation of GS, owing to a decrease in its ability to bind UDP-glucose, and (2) decreased amount of GS protein, owing to a decrease in the levels of GS mRNA.     

The primary in vivo steroidal alkaloid glucosyltransferase from potato

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of α-solanine was increased and α-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.     

A novel UDP-glucose transferase is part of the callose synthase complex and interacts with phragmoplastin at the forming cell plate

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein--protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-beta-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein--UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.     

UDP-glucose pyrophosphorylase from potato tuber: purification and characterization

UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.     

Selective induction of P2Y14 receptor by RANKL promotes osteoclast formation

The purinergic receptor P2Y, G protein coupled, 14 (P2Y14) receptor for UDP-glucose and other UDP-sugars has been implicated in the regulation of the stem cell compartment as well as neuroimmune function. However, the role of P2Y14 in osteoclast formation is completely unknown. We found that RANKL selectively induced P2Y14 among seven mammalian P2Y receptors when analysed at both the mRNA and protein level, but inhibitors of the mitogenactivated protein (MAP) kinase pathway suppressed induction of P2Y14 proteins. Extracellular addition of UDP-sugars such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetyl glucosamine promoted RANKL-induced osteoclastogenesis, while P2Y14 downregulation by RNA interference inhibited osteoclast formation. Taken together, these results suggest that P2Y14 may act as the receptor for UDP-sugars in osteoclast precusors and may regulate RANKL-induced osteoclastogenesis.     

Galactosyl-sucrose metabolism and UDP-galactose pyrophosphorylase from Cucumis melo L. fruit

In muskmelon ( Cucumis melo L.), sink tissues receive stachyose, raffinose and sucrose through phloem translocation of carbohydrates that are formed as products of leaf photosynthesis. Melon fruits accumulate sucrose massively during the final stages of maturation. This sucrose is derived partially from the catabolism of raffinose saccharides. Rapid galactose metabolism is required, because liberation of free galactose is the first step in the metabolic utilization of the raffinose sugars. The current study demonstrates that the enzyme UDP-glucose-hexose-1-P uridylyltransferase (EC 2.7.7.12), the central enzyme in the classical Lelior pathway, is not the central enzyme in galactose metabolism in muskmelon fruit. Rather, a broad substrate specificity UDP-galactose pyrophosphorylase (PPase) serves the same functional role. This enzyme accepts either UDP-galactose or UDP-glucose as a substrate and is different from a UDP-glucose PPase with more strict substrate specificity for UDP-glucose that is also present in melon tissue. UDP-galactose PPase was purified 113-fold from melon tissue and was shown to be a 54 kDa (size exclusion chromatography) to 68 kDa (SDS-PAGE) protein that is enzymatically active as a monomer. We also present evidence that the enzyme likely accepts UDP-galactose and UDP-glucose at the same catalytic site. Polyclonal antibodies prepared against this protein reacted with numerous other antigens in melon extracts, apparently as a result of the presence of common antigenic epitopes.     

Structure and function of a chlorella virus-encoded glycosyltransferase

Paramecium bursaria chlorella virus-1 encodes at least five putative glycosyltransferases that are probably involved in the synthesis of the glycan components of the viral major capsid protein. The 1.6 A crystal structure of one of these glycosyltransferases (A64R) has a mixed alpha/beta fold containing a central, six-stranded beta sheet flanked by alpha helices. Crystal structures of A64R, complexed with UDP, CMP, or GDP, established that only UDP bound to A64R in the presence of Mn(2+), consistent with its high structural similarity to glycosyltransferases which utilize UDP as the sugar carrier. The structure of the complex of A64R, UDP-glucose, and Mn(2+) showed that the largest conformational change occurred when hydrogen bonds were formed with the ligands. Unlike UDP-glucose, UDP-galactose and UDP-GlcNAc did not bind to A64R, suggesting a selective binding of UDP-glucose. Thus, UDP-glucose is most likely the sugar donor for A64R, consistent with glucose occurring in the virus major capsid protein glycans.     

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase,a key gene of hemicellulose/pectin formation

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.     

Glycogen biosynthesis via UDP-glucose in the ruminal bacterium Prevotella bryantii B1(4).

Prevotella bryantii is an important amylolytic bacterium in the rumen that produces considerable amounts of glycogen when it is grown on maltose. Radiolabel studies indicated that glucose-1-phosphate was converted to UDP-glucose, and this latter intermediate served as the immediate precursor for glycogen synthesis. High levels of UDP-glucose pyrophosphorylase activities (> 1,492 nmol/min/mg of protein) were detected in cells grown on maltose, cellobiose, glucose, or sucrose, and activity was greatly stimulated (by approximately 60-fold) by the addition of fructose-1,6-bis phosphate (half-maximal activation concentration was 240 microM). However, ADP-glucose pyrophosphorylase activity was not detected in any of the cultures. Glycogen synthase activity in maltose-grown cultures (48 nmol/min/mg of protein) was higher than that in cellobiose-, sucrose-, and glucose-grown cultures (< 26 nmol/min/mg of protein). This is the first report of a bacterium that exclusively uses UDP-glucose to synthesize glycogen. The elucidation of this unique glycogen biosynthesis pathway provides information necessary to further investigate the role of bacterial glycogen accumulation in rumen metabolism.     

Sterol biosynthetic capability of purified membrane fractions from maize coleoptiles

Membrane fractions were isolated from etiolated maize coleoptiles by differential and sucrose density gradient centrifugation. Specific membrane components were identified by using marker enzyme activities. Fractions were also tested for α-naphthylacetic acid (NAA).and N-naphthylphthalamic acid (NPA) binding activities. Evidence is presented for the isolation of a plasma-membrane fraction containing specific binding sites for NPA, a high concentration of sterols, and most of the total UDP-glucose sterol glucosyltransferase activity. A fraction rich in endoplasmic reticulum is shown to contain most of the binding sites for NAA and all of the activity of both S-adeno-Syl-L-methionine-Δ24 cycloartenol methyltransferase and cycloeucalenol-obtusifoliol isomerase.     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

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.     

Nature of the periplastidial pathway of starch synthesis in the cryptophyte Guillardia theta

The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch synthase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V(max) measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose-synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch.