Nucleoside Diphosphate Glucose Research Articles

Genome-wide analysis of the invertase genes in strawberry (Fragaria×ananassa)

Sugar is an important material basis in fruit development, and strawberry fruit flavour and sweetness largely depend on the sugar content and variety. Invertases (INVs) play an important role in the regulation of sugar accumulation because they irreversibly catalyse the hydrolysis of sucrose into the corresponding nucleoside diphosphate-glucose, glucose or fructose in fruit. In this work, we provided a comprehensive analysis of the INV gene family in octoploid strawberry (Fragaria×ananassa), including the gene structure, chromosomal locations, conserved domains, and gene evolution and expression profiles during strawberry fruit development. Our study revealed that polyploid events resulted in the abundant amplification (almost three- or four-fold) of the INV gene in the F.×ananassa genome, and these amplified INV genes showed dominant expression in strawberry fruit. More than half of the FaINVs transcripts with low expression had incomplete coding sequences by alternative splicing. Previous studies have shown that cell wall invertases (CWINV) are involved in the regulation of phloem unloading and sink strength establishment. The expression of FaCWINV1 was markedly upregulated during fruit development and strongly expressed in ripe fruit. Moreover, a significant correlation was observed between the total sugar content and the FaCWINV1 expression level. These findings suggest that FaCWINV1 may be involved in sugar accumulation in strawberry fruit. Taken together, the results of our study will be beneficial for further research into the functions of INVs in the regulation of fruit ripening.

On the stability of nucleoside diphosphate glucose metabolites: implications for studies of plant carbohydrate metabolism.

Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo- and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP- and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specific enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are significantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADP-glucose produced by sucrose synthase in the cytosol.

Enhancing Sucrose Synthase Activity Results in Increased Levels of Starch and ADP-Glucose in Maize (Zea mays L.) Seed Endosperms

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. In cereal endosperms, it is widely assumed that the stepwise reactions of SuSy, UDPglucose pyrophosphorylase and ADPglucose (ADPG) pyrophosphorylase (AGP) take place in the cytosol to convert sucrose into ADPG necessary for starch biosynthesis, although it has also been suggested that SuSy may participate in the direct conversion of sucrose into ADPG. In this study, the levels of the major primary carbon metabolites, and the activities of starch metabolism-related enzymes were assessed in endosperms of transgenic maize plants ectopically expressing StSUS4, which encodes a potato SuSy isoform. A total of 29 fertile lines transformed with StSUS4 were obtained, five of them containing a single copy of the transgene that was still functional after five generations. The number of seeds per ear of the five transgenic lines containing a single StSUS4 copy was comparable with that of wild-type (WT) control seeds. However, transgenic seeds accumulated 10-15% more starch at the mature stage, and contained a higher amylose/amylopectin balance than WT seeds. Endosperms of developing StSUS4-expressing seeds exhibited a significant increase in SuSy activity, and in starch and ADPG contents when compared with WT endosperms. No significant changes could be detected in the transgenic seeds in the content of soluble sugars, and in activities of starch metabolism-related enzymes when compared with WT seeds. A suggested metabolic model is presented wherein both AGP and SuSy are involved in the production of ADPG linked to starch biosynthesis in maize endosperm cells.

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124-13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1-4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

Structural Insights on the New Mechanism of Trehalose Synthesis by Trehalose Synthase TreT from Pyrococcus horikoshii

Many microorganisms produce trehalose for stability and survival against various environmental stresses. Unlike the widely distributed trehalose–biosynthetic pathway, which utilizes uridine diphosphate glucose and glucose-6-phosphate, the newly identified enzyme trehalose glycosyltransferring synthase (TreT) from hyperthermophilic bacteria and archaea synthesizes an α,α-trehalose from nucleoside diphosphate glucose and glucose. In the present study, we determined the crystal structure of TreT from Pyrococcus horikoshii at 2.3 Å resolution to understand the detailed mechanism of this novel trehalose synthase. The conservation of essential residues in TreT and the high overall structural similarity of the N-terminal domain to that of trehalose phosphate synthase (TPS) imply that the catalytic reaction of TreT for trehalose synthesis would follow a similar mechanism to that of TPS. The acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in TPS. The observation of a wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis. The structural analysis and biochemical data in this study provide a molecular basis for understanding the synthetic mechanism of trehalose, or the nucleotide sugar in reverse reaction of the TreT, in extremophiles that may have important industrial implications.

Catalytic reversibility of Pyrococcus horikoshii trehalose synthase: Efficient synthesis of several nucleoside diphosphate glucoses with enzyme recycling

The trehalose synthase (TreT) from Pyrococcus horikoshii represented reversible catalysis in alternative synthesis of trehalose and nucleoside 5′-diphosphate-glucose (NDP-Glc), depending on the substrates involved. TreT from P. horikoshii had differential preferences on NDP-Glc as a donor for trehalose synthesis, in which guanosine 5′-diphosphate (GDP)-Glc was the most favored in terms of reaction specificity, k cat/ K m. Uridine 5′-diphosphate (UDP)- and adenosine 5′-diphosphate (ADP)-Glcs were employed with less preferences. This enzyme reversely cleaved trehalose to transfer the glucosyl moiety to various NDPs, efficiently producing NDP-Glcs. Although ADP-Glc was the least favorable donor, the counterpart, ADP, was the most favorable acceptor for the reverse synthesis of NDP-Glc in k cat/ K m. GDP and UDP were less preferred, compared to ADP. In a batch reaction of 12 h, the molar yield of NDP-Glc per NDP used was decreased approximately in the order of ADP-Glc > GDP-Glc > cytidine 5′-diphosphate (CDP)-Glc or UDP-Glc. The overall productivity of the enzyme was largely improved in a gram scale for NDP-Glcs using repetitive batch reactions with enzyme recycling. Thus, it is suggested that TreT from P. horikoshii may be useful for the regeneration of NDP-Glc from NDP, especially for ADP-Glc from ADP, with trehalose as a glucose resource.

Enhancing Sucrose Synthase Activity in Transgenic Potato (Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: Molecular cloning and characterization

A gene (ORF PH1035), annotated to encode an uncharacterized hypothetical protein in Pyrococcus horikoshii, was first cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by Ni–NTA affinity chromatography and its molecular mass was determined to be 49,871 Da by MALDI-TOF mass spectrometry. When the purified enzyme was reacted with nucleoside diphosphate–glucoses including UDP–glucose as a donor and glucose, rather than glucose-6-phosphate, as an acceptor, it specifically created a free trehalose. The enzyme was also able to partly hydrolyze the trehalose to glucose. The optimum pH was 5.5 and the enzyme was highly stable from pH 6 to 8. The deduced amino acid sequence showed a high homology with that of the glycosyl transferase group 1 (Pfam00534) in the BLAST search. The results suggest that the enzyme is a novel glycosyltransferase catalyzing the synthesis of the trehalose in the archaeon.

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.

Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions.

The trehalose-P synthase was purified to near homogeneity from the cytoplasmic fraction of Mycobacterium smegmatis. At the final stage of purification, the enzyme preparation showed one major band of 59 kDa on SDS gels. The 59 kDa band became labeled with N3-UDP[32P]-glucose, and this labeling was inhibited in a concentration-dependent manner by either unlabeled UDP-glucose or GDP-glucose. The native enzyme also had a molecular weight of about 60 kDa by gel filtration, indicating that the active enzyme is a monomer. The 59 kDa protein was subjected to endoproteinase Lys-C digestion, and three peptides isolated by HPLC were sequenced. The sequences of 56 amino acids in these three peptides showed 60% identity to the trehalose-P synthases of Saccharomyces cerevesiae and Schizosaccharomyces pombe. The purified mycobacterial enzyme catalyzed the synthesis of trehalose-P from glucose-6-P and a variety of nucleoside diphosphate glucose derivatives, depending on whether a polyanion was absent or present. Thus, UDP-glucose and GDP-glucose were the best glucosyl donors, but maximum activity with UDP-glucose required the presence of a polyanion such as heparin, whereas activity with GDP-glucose was relatively independent of polyanion. The presence of heparin in the incubation mixture increased the affinity of the enzyme for UDP-glucose by a factor of 100, or more. However, the affinity for GDP-glucose was only twofold better in the presence of heparin. The purified synthase also utilized ADP-glucose and CDP-glucose, but the K(m) for these glucosyl donors was quite high even in the presence of polyanion. The effect of heparin on UDP-glucose activity was dose-dependent and maximum at about 1-2 micrograms of heparin/incubation. However, the size of the heparin molecule (i.e., the number of monosaccharide residues) was critical for activation, and only those heparins with 18 or more monosaccharide units were effective in stimulating activity.

New and specific nucleoside diphosphate glucose substrates for glycogenin

Glycogenin, the autocatalytic, self-glucosylating primer for glycogen synthesis by glycogen synthase, is presumed, in vivo, to use UDP-glucose as the source of the glucose residues it adds to itself. When we tested its ability to utilize other nucleoside diphosphate glucoses, it emerged that purine nucleotides are not utilized but two pyrimidine nucleotides are used, in addition to UDP-glucose. These are CDP-glucose and TDP-glucose. CDP-glucose is utilized at 70% of the rate of UDP-glucose. While there is no evidence that CDP-glucose is a natural substrate for glycogenin, it has the advantage over UDP-glucose in that it can be used specifically to detect and assay glycogenin in the presence of glycogen synthase because CDP-glucose, unlike UDP-glucose, is not a substrate for the synthase.

Utilization of Nucleoside Diphosphate Glucoses in Developing Cotton Fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-(14)C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-(14)C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.

Nucleoside Diphosphate Glucose Pyrophosphorylase Isoenzymes of Developing Normal, Brittle‐2, and Shrunken‐2 Endosperms of Zea mays L.1

Maize (Zea mays L.) endosperms from normal, brittle‐2, and shrunken‐2 near‐isogenic sublines of the Oh43 inbred were assayed for nucleoside diphosphate glucose pyrophosphorylase activity at 10, 20, and 30 days post‐pollination. Polyethaleneamine‐cellulose thin layer chromatography and polyacrylamide gel electrophoresis were used for product assay and identification of isoenzyme patterns from crude enzyme preparations, respectively.Evidence is presented for the existence in corn endosperm of pyrophosphorylose activity with guanosine triphosphate and thymidine triphosphate in addition to adenosine triphosphate, cytidine triphosphate, and uridine triphosphate. Two adenosine diphosphate glucosepyrophosphorylase isoenzyme bands from normal and shrunken‐2 endosperms were detected with the same pattern, suggesting that the shrunken‐2 gene may be regulatory in nature. Brittle‐2 showed an altered mobility of the adenosine diphosphate glucose pyrophosphorylase banding pattern, which indicates that brittle‐2 may be a structural gene. Three isoenzymes for uridine diphosphate glucose pyrophosphorylase were found in normal and mutant endosperms. Evidence indicated that guanosine triphosphate may be utilized by adenosine diphosphate glucose pyrophosphorylase. Thymidine triphosphate and possibly cytidine triphosphate may be utilized by uridine diphosphate glucose pyrophosphorylase.There were no differential appearances of the pyrophosphorylase isoenzymes during corn endosperm development. However, quantitative differences in the isoenzyme bands were observed.

The Purification and Properties of Sucrose Synthetase from Etiolated Phaseolus aureus Seedlings

Abstract UDP-glucose:d-fructose 2-glucosyltransferase (EC 2.4.1.14), commonly called sucrose synthetase, has been purified from etiolated Phaseolus aureus seedlings to homogeneity by electrophoretic, immunological, and ultracentrifugal criteria. Molecular weight determinations by low speed equilibrium centrifugation yields a value of 375,000 ± 12,000. A value of 405,000 ± 20,000 was obtained by gel filtration. Other physical determinations for the enzyme yielded the following data: s20,w = 12.4 x 10-13 sec-1; diffusion constant (D20,w) = 3.03 x 10-7 cm-2 sec-1; and f/f0 = 1.49. Subunit analyses indicate that the enzyme is a tetramer composed of identical protein subunits of approximate molecular weight 94,000. An equilibrium constant of 0.15 for the sucrose synthetase reaction at pH 7.5 and 25° in the direction of uridine diphosphate glucose synthesis was experimentally determined. The kinetic parameters of the enzyme in the proposed physiological direction, that of nucleoside diphosphate glucose synthesis, have been measured. When UDP is present at saturating levels, the Km for sucrose was determined to be 17 mm. A similar value (29 mm) was obtained when ADP was the second substrate. Upon complete purification the enzyme retains activity for UDP, ADP, TDP, CDP, or GDP as substrates. Km values for these substrates, determined in the presence of saturating sucrose, are 0.19, 0.19, 0.30, 0.44, and 0.17 mm, respectively. The Vmax in the presence of these substrates are, in relative numbers, 100, 28.6, 5.8, 3.4, and 2.8, respectively. Kinetic data indicate a random mechanism for the enzyme.

The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.The data suggest that sucrose-phosphate synthetase and sucrose phosphatase are the enzymes responsible for the biosynthesis of sucrose from photosynthetically fixed CO(2), and that the major function of sucrose synthetase is to catalyze the synthesis of UDP-, ADP-, dTDP-, CDP-, and GDP-glucose from translocated sucrose in nonphotosynthetic tissues; in photosynthetic tissues the pyrophosphorylases may replace sucrose synthetase in catalyzing the synthesis of these nucleoside diphosphate glucoses. We offer the suggestion that sucrose synthetase and UDP-glucose pyrophosphorylase play a major role in the uptake and metabolism of sucrose in nonphotosynthetic tissues. Results are presented from preliminary studies on the conversion in vitro of sucrose to glucose 1-phosphate by the coupled reactions of sucrose synthetase and UDP-glucose pyrophosphorylase with highly purified preparations of these enzymes.

Sucrose Synthetase from Phaseolus aureus Seedlings

Abstract Sucrose synthetase from mung bean (Phaseolus aureus) has been purified by a combination of ammonium sulfate precipitation, gel filtration, and sucrose density gradient centrifugation. The molecular weight of sucrose synthetase was determined to be about 106 by gel filtration chromatography and sucrose density gradient centrifugation. The density of the sucrose synthetase particle was determined by equilibrium sedimentation to be 1.20 g per cc. The ability to catalyze the synthesis of sucrose from each of the nucleoside diphosphate glucoses containing uridine, adenosine, thymidine, cytidine, and guanosine remains associated with the purified particle. Attempts to subdivide the particle into catalytically active units have been unsuccessful. The Michaelis constants of the substrates for the sucrose synthetase particles and the maximum velocities of sucrose synthesis in the presence of each of the nucleoside diphosphate glucoses have been determined. In competition experiments between pairs of nucleoside diphosphate glucoses, uridine diphosphate glucose inhibits the synthesis of sucrose from the other nucleoside diphosphate glucoses far more effectively than would be expected on the basis of competition between the substrates for the same catalytic sites. On the other hand, the adenosine-, thymidine-, cytidine-, and guanosine-containing nucleoside diphosphate glucoses do not inhibit sucrose synthesis from uridine diphosphate glucose as effectively as would be anticipated on the basis of a simple competition mechanism. It is concluded that uridine diphosphate glucose is able to control the synthesis of the other nucleoside diphosphate glucoses from sucrose by causing a modification of sucrose synthetase.

The Metabolism of Glyceride Glycolipids: I. BIOSYNTHESIS OF MONOGLUCOSYL DIGLYCERIDE AND DIGLUCOSYL DIGLYCERIDE BY GLUCOSYLTRANSFERASE PATHWAYS IN STREPTOCOCCUS FAECALIS

Abstract Particulate enzyme preparations of Streptococcus faecalis ATCC 9790 catalyze the synthesis of 1,2-di-O-acyl-3-O-(α-d-glucopyranosyl)-sn-glycerol (monoglucosyl diglyceride), 1,2-di-O-acyl-3-O-(2-O-α-d-glucopyranosyl-α-d-glucopyranosyl)-sn-glycerol (diglucosyl diglyceride), and a third glucolipid of unknown structure from two common precursors, UDP-glucose and 1,2-diglyceride. The three glucolipids appear to be formed sequentially. Isolated, radiochemically pure monoglucosyl diglyceride is converted to diglucosyl diglyceride in a reaction dependent upon UDP-glucose and enzyme. The biosynthetic enzyme or enzymes displayed a relatively high degree of specificity for UDP-glucose (the best of five nucleoside diphosphate glucose substrates tried) and for the 1,2 stereoconfiguration of the diglyceride substrate. 14C-Galactose of UDP-galactose-14C is converted to glucose prior to incorporation into glycolipids of S. faecalis. Of seven 14C-diglycerides, 14C-dilinolein served best as a substrate for the synthesis of the three glucolipids. Magnesium ion stimulates the reaction. None of 18 surface active agents tried appeared to enhance the enzymatic reaction. Consistent and relatively uniform stimulation of the biosynthesis by exogenous diglyceride was best achieved by adsorbing the diglyceride substrate directly to the lyophilized enzyme preparation.

Nucleoside diphosphate glucose pyrophosphorylases in mast cell tumors

1. I. Mast cell tumors were shown to contain uridine diphosphate glucose pyrophosphorylase (UTP:α- d-glucose- i-phosphate uridylyltransferase, EC 2.7.7.9) and guanosine diphosphate glucose pyrophosphorylase (GTP:α- d-glucose- i-phosphate guanylyltransferase). The two activities were separated from each other by ammonium sulfate fractionation and are thus distinct enzymes. 2. 2. The GDP-glucose pyrophosphorylase fraction was purified further on DEAE-cellulose and some of its properties were studied. The enzyme was shown to catalyze the synthesis of GDP-glucose from d-glucose- i-phosphate and GTP. Other nucleoside triphosphates did not yield nucleoside diphosphate glucose when incubated with this enzyme preparation. 3. 3. The enzyme also catalyzed, to a lesser degree, the formation of guanosine diphosphate mannose from GTP and d-mannose- i-phosphate. Mannose i-phosphate also had an inhibitory effect on the synthesis of GDP-glucose. 4. 4. Fibrosarcomas and umbilical cord do not contain any detectable amount of GDP-glucose pyrophosphorylase although they do have UDP-glucose pyrophosphorylase activity.

Glycogen accumulation by wild-type and uridine diphosphate glucose pyrophosphorylase-negative strains of Escherichia coli

Several uridine diphosphate glucose-pyrophosphorylase (UDPG-PP)-negative mutants of Escherichia coli were found to accumulate intracellularly a polyglucose compound during the stationary phase caused by limiting nitrogen. The mutants accumulated as much or more of the polyglucose compound than their wild-type parents. The polyglucose compound was isolated and shown to be a true glycogen. No significant difference in structure was found between the glycogen synthesized by the mutants and that synthesized by their wild-type parents. These observations strongly suggest that the glycogen synthetase of E. coli utilizes as a substrate some nucleoside diphosphate glucose other than UDPG. Similarly, the results show that the inhibition of phosphorylase by UDPG is not a prerequisite for glycogen accumulation, although inhibition by other NDPG compounds such as TDPG and ADPG may play a role in vivo. One of the strains used in this study was a “leaky” threonine-requiring mutant. In the presence of exogenous threonine this mutant grows with a generation time of 70 minutes and a growth yield (based on glucose utilized) of about 48%. When threonine becomes limiting the growth rate and growth yield decrease markedly, but the specific rate of glucose utilization continues at a rate close to the original rate. During the first generation of this uncoupled growth phase, glycogen rapidly accumulates. This result is consistent with the idea that glycogen accumulation is a result of excess ATP production, over and above that necessary to maintain a given exponential growth rate (or macromolecule turnover rate for nitrogen-limited stationary cultures). The addition of chloramphenicol or p-fluorophenylalanine to nitrogen-limited stationary cultures strongly inhibits glycogen accumulation. The mechanism of this inhibition is unknown. A possible explanation is that some enzyme(s) involved in glycogen synthesis has an extremely high turnover rate, and that the steady-state level of the enzyme(s) decreases rapidly when protein synthesis is inhibited.