The effect of the seed coat, embryonic axis and aeration conditions on starch degradation hi cotyledons of Lens culinaris

The effect of exogenously applied galactose on the cell wall polysaccharide synthesis and UDP-sugar levels in oat ( Avena sativa L. cv. Victory I) coleoptile segments was studied to clarify the mechanism of inhibition of IAA-induced cell elongation by galactose, and the following results were obtained: (1) The inhibition of IAA-induced cell elongation by galactose became apparent after a 2 h-lag, while the lag was shortened to 1 h when galactose was added to the segments after more than 1 h of IAA application. (2) Galactose inhibited the [¹⁴C]-glucose incorporation into cellulosic and non-cellulosic fractions of the cell wall and the increase in net polysaccharide content in the fractions during long-term incubation. (3) The dominant sugar nucleotide in oat coleoptiles was UDP-glucose (2.1 nmol segment⁻¹). Galactose application caused a remarkable decrease in the UDP-glucose level, accompanying a strong accumulation of galactose-1-phosphate and UDP-galactose. (4) Galactose-1-phosphate competitively inhibited the UTP: a- d -glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) activity of the crude enzyme preparation from oat coleoptiles. From these results we conclude that galactose inhibits the IAA-induced cell elongation by inhibiting the formation of UDP-glucose, which is a key intermediate of cell wall polysaccharide synthesis.     

Studies on the metabolic determinants of D-galactose-induced neurotoxicity in the chick

The contents of galactose, galactitol, galactose 1-phosphate, UDP-galactose and UDP-glucose in the brains of chicks fed a diet containing 40 % (w/w) D-galactose were determined at regular intervals during a 48 h period which terminated in convulsive activity and death of the animals. Although levels of galactose and galactitol were markedly elevated, UDP-galactose and UDP-glucose levels were not significantly increased. The level of galactose 1-phosphate rose to 1-3 μg/g of fresh tissue by 14 h but gradually diminished until, at 48 h, the content was 0-25 μg/g. The metabolic turnover of these compounds, as shown by labelling experiments with inorganic [³²P]phosphate and [U-¹⁴C]galactose, indicated that galactose 1-phosphate and UDP-galactose were rapidly metabolized, yet relatively little galactose was utilized by the brain as a source of energy. These observations have prompted us to propose a mechanism for the turnover of galactose 1-phosphate that involves cyclical phosphorylation and dephosphorylation reactions in the brains of galactose-fed chicks. In support of this hypothesis, we have identified phosphatase activity which has a relatively low K_m value for galactose 1-phosphate (0-06-0-07 mM) in virtually all subcellular fractions of homogenates of chick brain. Maximum activity of the phosphatase is several-fold greater than that recorded for galactokinase (EC 2.7.1.6) and galactose 1-phosphate uridyltransferase (EC 2.7.710) from chicken brain.     

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.     

GALT deficiency causes UDP-hexose deficit in human galactosemic cells

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.     

Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose

Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

Cell wall modifications during auxin-induced cell extension in monocotyledonous and dicotyledonous plants

There are several differences between monocotyledonous and dicotyledonous plants. The sensitivity towards added galactose which inhibits auxin-induced coleoptile elongation but not stem elongation is one of the conspicuous differences between the two types of plants. InAvena coleoptile segments, galactose, probably as galactose-1-phosphate, inhibits the formation of UDP-glucose from glucose-l-phosphate. The inhibition of UDP-glucose formation due to galactose is not found inPisum epicotyl segments. InAvena UTP: α-D-glucose-1-phosphate uridyltransferase (EC 2.7.7.9) which catalyzes the reaction from glucose-1-phosphate to UDP-glucose seems to be inhibited by galactose-1-phosphate.     

D-glucosamine-induced changes in nucleotide metabolism and growth of colon-carcinoma cells in culture.

Human colon-carcinoma cells were exposed to D-glucosamine at 2.5, 5 and 10 mM, concentrations that were growth-inhibitory but not cytocidal in the presence of a physiological glucose concentration. Labelling of these HT-29 cells with D-[14C]-glucosamine, followed by nucleotide analyses, demonstrated that UDP-N-acetyl-hexosamines represented the major intracellular nucleotide pool and the predominant metabolite of the amino sugar. D-[14C]Glucosamine was not a precursor of UDP-glucosamine. After 4h exposure to D-glucosamine (2.5 mM), the pool of UDP-N-acetylhexosamines was increased more than 6-fold, whereas UTP and CTP were markedly decreased. UDP-glucuronate content increased by more than 2-fold, whereas purine nucleotide content was little altered. Uridine (0.1 mM) largely reversed the decrease in UTP, CTP, UDP-glucose and UDP-galactose, while intensifying the expansion of the UDP-N-acetylhexosamine pool. Uridine did not reverse the D-glucosamine-induced retardation of growth in culture. A 50% decrease in growth also persisted when uridine and cytidine, cytidine alone, or UDP, were added together with D-glucosamine. The growth-inhibitory effect of the amino sugar could therefore be best correlated with the quantitative change in the pattern of sugar nucleotides, and, in particular, with the many-fold increase in UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.     

Molecular cloning of the Leishmania major UDP-glucose pyrophosphorylase, functional characterization, and ligand binding analyses using NMR spectroscopy

The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the gamma-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

Nachweis von Enzymen des Galactosestoffwechsels in der Grünalge Acetabularia mediterranea

Summary The reason for interest in the galactose metabolism of the unicellular green alga Acetabularia mediterranea lies in the question of how the different galactose content in the cell wall of the stalk and the cap is formed. The galactose content is low in the cell wall of the stalk and high in the membrane of the cap (Werz, 1963). Progress in this question presupposes information about the enzymes of galactose metabolism in this organism. The occurrence of the whole series of enzymes which are concerned with the synthesis of UDP-galactose from fructose-6-phosphate could be demonstrated. These enzymes are as follows: Phosphoglucose-isomerase [E.C.5.3.1.9], phosphoglucomutase [E.C.2.7.5.1], UDP-glucose pyrophosphorylase [E.C.2.7.7.9], UDP-glucose-4-epimerase [E.C.5.1.3.2], nucleosidediphosphate kinase [E.C.2.7.4.6] and inorganic pyrophosphatase [E.C.3.6.1.1].

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Nachweis von Enzymen des Galactosestoffwechsels in der Grünalge Acetabularia mediterranea

Zusammenfassung In der einzelligen Grünalge Acetabularia mediterranea konnten die für die Synthese von UDP-galactose aus Fructose-6-phosphat notwendigen Enzyme Phosphoglucose-Isomerase, Phosphoglucomutase, UDP-glucose-Pyrophosphorylase und UDP-glucose-4-Epimerase nachgewiesen werden, außerdem Nucleosiddiphosphat-Kinase und anorganische Pyrophosphatase. Der Galactosestoffwechsel von A. mediterranea ist deshalb von Interesse, weil Stiel- und Hutmembran einen unterschiedlichen Galactosegehalt aufweisen.

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Herrn Professor Dr. J. Hämmerling zum 65. Geburtstag gewidmet.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.     

Labeling of precursor pools for glycosphingolipid biosynthesis. Incorporation of [3H]galactose by rat hepatocytes in primary culture

UDP-galactose and UDP-glucose are the immediate sources of monosaccharide residues in glycosphingolipid biosynthesis. The incorporation of [6-3H]D-galactose into these compounds was measured in primary cultures of rat hepatocytes, which take up and metabolize galactose rapidly. The UDP-glucose and UDP-galactose content of hepatocytes, determined enzymatically and by the HPLC-analysis of UDP-sugars, was 1.87 +/- 0.22 and 0.51 +/- 0.06 nmol/mg protein, respectively. Galactose concentrations in the medium of up to 7.5 microM did not influence the intracellular levels of UDP-glucose and UDP-galactose. Although the specific radioactivity of these precursor pools did not reach a constant plateau, conditions were defined that allow the calculation of rates of glycolipid synthesis from added labeled galactose. They include the replacement of glucose in the culture medium by sodium pyruvate and D-galactose.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Peculiarities of nucleotide and nucleic acid metabolism in hydroxythiamine-induced vitamin B1 deficiency

24 hours after administration of hydroxythiamine, the vitamin B1 antimetabolite, the rat liver pools of orotic acid, UDP-glucose and ATP show a decrease. The cellular energy charge calculated from the adenylic nucleotide concentrations also appears to be significantly diminished. The de novo pyrimidine synthesis lowers under these conditions, while the rates of formation and destruction of essential UDP-sugars remain unaffected. The nucleic acid content is at the control level. A comparison of specific activities of UTP and RNA allows one to conclude that the previously observed decrease in [14C]orotate incorporation into RNA under the action of hydroxythiamine reflects the inhibition of RNA synthesis.