Properties of uridine diphosphate glucose pyrophosphorylase from Golgi apparatus of liver

Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.     

A source of apparent pyrophosphate:fructose 6-phosphate phosphotransferase activity in rabbit muscle phosphofructokinase

In the presence of UDPglucose, rabbit muscle phosphofructokinase appeared to use PPi as a phosphoryl donor, as reported previously (Biochem. Biophys. Res. Commun. 121, 842-847). This apparent activity was due to conversion of UDPglucose and PPi to glucose 1-phosphate and UTP, the latter being metabolized by phosphofructokinase. Auxiliary enzymes used in the assays were contaminated by UDPglucose pyrophosphorylase. This contamination was sufficient to account for, and had similar properties to, the apparent PPi-dependent activity. Without auxiliary enzymes phosphofructokinase could not use PPi. These findings indicate that the apparent interconversion of phosphofructokinase and PPi:fructose 6-phosphate phosphotransferase must be re-assessed.     

Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.     

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.     

Evidence for coupling between transport of UDP-glucose and its synthesis by membrane-bound pyrophosphorylase in Golgi apparatus of cat liver

Incubation of sealed vesicles of cat-liver Golgi apparatus with UDP[14C]glucose showed that the vesicles accumulated radioactivity. After Triton X-100 treatment or sonication of washed vesicles, soluble radiolabeled species were released and identified by paper chromatography as UDP[14C]glucose, [14C]glucose 1-phosphate and free glucose. In the incubation medium, UDPglucose was effectively protected by addition of dimercaptopropanol and UTP. Presence of glucose 1-phosphate and glucose within the vesicles most probably arose from luminal pyrophosphatase and phosphatase. A portion of the [14C]glucose moiety became covalently linked to endogenous acceptors. Uptake of UDPglucose was saturable and dependent on time and on the concentration of sugar nucleotide. Together, these results were consistent with a transport system for UDPglucose in Golgi vesicles. Furthermore, penetration rate was considerably higher with UDPglucose synthetized in situ from glucose 1-phosphate by membrane-bound pyrophosphorylase than from added UDPglucose: Vmax values were respectively 10 and 2 pmol/15 min per mg protein. This result allows the conclusion that a coupling between translocase and synthetase is involved in UDPglucose transport through Golgi apparatus membranes. The mechanism of this 'kinetic advantage' is discussed.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

Regulation of ADPglucose pyrophosphorylase in cucumber plants infected with the cucumber mosaic virus

ADPglucose pyrophosphorylase level and mechanisms regulating its activity were studied in cucumber plants infected with the cucumber mosaic virus at the stage of chronic infection. Studies carried out with partially purified preparations of the enzyme have shown that there was no substantial difference in the regulatory influence of the ratio 3-PGA/P₁, or in the number of binding sites of the effectors on the enzyme, but that the virus infection reduced the level of the enzyme in the tissues to 74% of the control and the 3-PGA/P₁ ratio to one half which resulted in a further decrease in ADPglucose pyrophosphorylase activity. In crude homogenate prepared from diseased plants, activity of the enzyme was reduced to 42% of the healthy control. The level of UDPglucose pyrophosphorylase was three times higher in cucumber leaf tissues than the level of ADPglucose pyrophosphorylase which was inhibited by both 3-PGA and P₁. Inhibitory effects of both these effectors were cumulated. The enzyme isolated from healthy plants was inhibited by inorganic phosphate more strongly than the enzyme isolated from diseased plants. UDPglucose pyrophosphorylase activity was increased in crude homogenate of diseased plants to 127% of the healthy control when the level of the enzyme was the same in the tissues of both healthy and diseased plants which was presumably connected with the enhanced rate of sucrose catabolism.     

Exploring the catalytic mechanism of skeletal muscle UDP-glucose pyrophosphorylase: identification of a hyperreactive cysteine at the enzyme active site

1. The involvement of cysteine residues in the catalytic mechanism of UDP-glucose pyrophosphorylase was suggested by the rapid inactivation of the enzyme by N-ethylmaleimide, even at 1:1 reagent/enzyme stoichiometric ratios. 2. The inactivation is largely prevented by uridine substrates (UDP-glucose and UTP) in agreement with the assumption that the reactive cysteine is located at the active site.     

Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase.

A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.     

Subcellular distributions of UDP-galactose:polysaccharide transferase and UDP-glucose pyrophosphorylase involved in biosynthesis of prespore-specific acid mucopolysaccharide in Dictyostelium discoideum

During the development of a cell aggregate of Dictyostelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UDPgalactose:polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 X g supernatant gave two main peaks. When the profiles wee compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in each developmental stage.     

Developmental regulation of multiple forms of UDPglucose pyrophosphorylase of Dictyostelium

Uridine diphosphoglucose pyrophosphorylase (UTP:α-d-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) is a developmentally regulated enzyme in Dictyostelium discoideum essential for the completion of its life cycle. During vegetative growth and the early stages of differentiation the specific activity of the enzyme remains constant. However, it increases threefold by the time fruiting bodies are formed. We have identified a developmentally specific form of uridine diphosphoglucose pyrophosphorylase, altered in both isoelectric point and apparent molecular weight, by resolving crude extracts of cells on two-dimensional denaturing polyacrylamide gels, renaturing the protein in situ, and localizing active enzyme with a histochemical stain. Quantitation of the amount of enzyme stain deposited in the gels shows that the activity in the new form can account for the increase observed in development. The appearance of the developmental form of the enzyme requires de novo protein synthesis since it is inhibited by cycloheximide. Immunoprecipitation of uridine diphosphoglucose pyrophosphorylase from in vivo and in vitro synthesized proteins has revealed heterogeneity not previously detected in the enzyme from both vegetative and developed cells. Two different proteins are synthesized in vitro by mRNA from either vegetative or developed cells. These two proteins are also found in vivo in developed cells. Only one of the two proteins is found in vegetative cells. Enzyme protein synthesized in vivo appears to be modified after translation. Therefore, the observed heterogeneity in uridine diphosphoglucose pyrophosphorylase found in vivo appears due both to post-translational modification and to synthesis of two polypeptides from one or more species of mRNA.     

Subcellular distributions of UDP-galactose: Polysaccharide transferase and UDP-glucose pyrophosphorylase involved in biosynthesis of prespore-specific acid mucopolsaccharide in Dictyostelium discoideum

During the development of a cell aggregate of Dictystelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UCPgalactose: polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 × g supernatant gave two main peaks. When the profiles were compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in eac developmental stage.     

Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate. The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study.     

Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.     

UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties.

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K_m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 mm, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.     

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 ( Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PP_i. We show that, although the GalF protein is expressed in vivo , GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF  ⁺/ galU ⁺ and galF/galU  ⁺ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo . We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.     

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.     

UDPGLUCOSE PYROPHOSPHORYLASE ACTIVITY IN THE DIATOM CYCLOTELLA CRYPTICA. PATHWAY OF CHRYSOLAMINARIN BIOSYNTHESIS 1

UDPglucose pyrophosphorylase activity was detected in cell-free extracts of the diatom Cyclotella cryptica TI3L Reimann, Lewin and Guillard. When assayed in the direction of UDPglucose formation, the enzyme had maximal activity at pH 7.8 and was stimulated by Mg²⁺and Mn²⁺ions. 3-Phosphoglycerate and inorganic phosphate had little effect on enzymatic activity, and the enzyme was relatively insensitive to feedback inhibition from UDPglucose (K, > I millimolar). A glucan was formed from UDP-[¹⁴C]glucose in cell-free extracts of C. cryptica. This glucan had a median molecular weight of 4600 (as determined by gel filtration chromatograbhy) and could be hydrolyzed by laminarinase. Partial acid hydrolysis of the glucan resulted in the formation of glucose and laminaribiose. but not cellobiose. These results suggest that the synthesis of chrysolaminarin (the major storage carbohydrate of diatoms) occurs via the activity of UDPglucose pyrophosphorylase. followed by glucosyl transfer from UDPglucose to the growing β-(1–3)-linked glucan.     

Investigations on the structural perturbations induced by the binding of mercuric chloride to L-glutamate dehydrogenase.

Three mercuric chloride binding sites are identified on l-glutamate dehydrogenase. In the presence of EDTA, the binding of two mercuric chloride molecules per subunit induces the dissociation of the polyhexamers into hexamers. The physical and catalytic properties of this modified hexamer are similar to those of the native enzyme. This induced dissociation of the enzyme is probably the result of an exclusive binding of the mercurial to the free hexamer, and the dissociation velocity does not appear to be rate limited by the binding reaction of the mercurials. The third mercuric chloride binding site is protected by both EDTA and l-glutamate. The binding of HgCl₂ to this site leads to the complete inactivation of the protein. There is no overlap between these modifications of l-glutamate dehydrogenase and the two previously described modifications of the enzyme by mercurial.