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1.
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.  相似文献   

2.
Hydrolysis of sugar phosphates by crude and purified preparations of periplasmic hexose phosphatase from Salmonella typhimurium followed Michaelis-Menten kinetics. The enzyme bound glucose 1-phosphate with high affinity (Km = 10 microM) but bound glucose 6-phosphate with low affinity (Km = 2,000 microM). The order of substrate affinities was glucose 1-phosphate greater than mannose 1-phosphate = galactose 1-phosphate greater than fructose 1-phosphate greater than glucose 6-phosphate. These results and others suggest that the physiological function of the enzyme is the periplasmic hydrolysis of hexose 1-phosphates.  相似文献   

3.
Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis   总被引:2,自引:1,他引:1       下载免费PDF全文
A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [(14)C]galactose from uridine 5'-diphosphate (UDP)-[(14)C]galactose and of [(14)C]glucose from UDP-[(14)C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [(14)C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described.  相似文献   

4.
The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.  相似文献   

5.
When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.  相似文献   

6.
In tumoral islet cells (RINm5F line) the phosphorylation of D-fructose is catalyzed by hexokinase rather than fructokinase. Fructose 6-phosphate appears to be preferentially channelled into the pentose cycle, as suggested by a ratio of D-[1-14C]fructose/D-[U-14C]fructose oxidation close to 2.7, the failure to generate 14C-labelled lactate from D-[1-14C]fructose and a poor metabolic response to menadione. When the islet cells are exposed to both D-fructose and D-glucose, however, the metabolism of the former hexose is dramatically modified, fructose 6-phosphate being now formed at a lower rate and preferentially channelled into the glycolytic pathway. These findings illustrate the existence of regulatory steps in fructose catabolism located distally to its site of phosphorylation.  相似文献   

7.
Fructose, galactose, L-arabinose, gluconate, and several organic acids support rapid growth and N2 fixation of Azospirillum brasiliense ATCC 29145 (strain Sp7) as a sole source of carbon and energy. Growth of Azospirillum lipoferum ATCC 29707 (strain Sp59b) is also supported by glucose, mannose, mannitol, and alpha-ketoglutarate. Oxidation of fructose and gluconate by A. brasiliense Sp7 and of glucose, gluconate, and fructose by A. lipoferum Sp59b was achieved through inducible enzymatic mechanisms. Both strains exhibited all of the enzymes of the Embden-Meyerhof-Parnas pathway, and strain Sp59b also possesses all the enzymes of the Entner-Doudoroff pathway. Fluoride inhibited growth on fructose (strains Sp7 and Sp59b) or on glucose (strain Sp59b) but not on malate. There was no activity via the oxidative hexose monophosphate pathway in either strain. There was greater activity with 1-phosphofructokinase than with 6-phosphofructokinase in both strains. Strain Sp59b formed fructose-6-phosphate via hexokinase, an enzyme that is lacking in strain Sp7. A. brasiliense and A. lipoferum exhibited the enzymes both of the tricarboxylic acid cycle and of the glyoxylate shunt; iodoacetate, fluoropyruvate, and malonate were inhibitory. A. brasiliense Sp7 could not transport [14C]glucose and alpha-[14C]ketoglutarate into its cells.  相似文献   

8.
The fate of unlabelled D-glucose and D-[2-3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of D-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from D-[2-3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from D-[2-3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of D-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.  相似文献   

9.
Abstract: Oligodendroglial and neuronal perikarya have been isolated from fresh and frozen bovine brain and used to investigate the synthesis of cerebrosides from UDP-hexoses and ceramides. The radioactive cerebrosides produced have been identified by TLC of the intact lipids on borate-silica gel and by a combination of acid hydrolysis and ion-exchange chromatography of the liberated hexoses. Incubation of either neuronal or oligodendroglial homogenates with UDP-galactose (UDP-gal) and mixed ceramides (hydroxy plus nonhydroxy fatty acids) leads to the synthesis of hydroxy fatty acid galactocere-broside. This lipid is also formed by both neuronal and oligodendroglial homogenates if the UDP-gal is replaced by UDP-glucose (UDP-glu). Formation of glucocerebroside has been observed only in the presence of neuronal homogenates and UDP-glu. Electron microscopic examination of the isolated cell preparations has shown that, although the oligodendroglia are relatively pure, the neurones are contaminated with oligodendroglia, which may account for some of the hydroxy fatty acid galactocere-broside synthesis. When unlabelled UDP-gal is included in the incubations containing cell homogenates, mixed ceramides, and labelled UDP-glu, galactocerebroside formation is greatly reduced, but there is comparatively little effect on glucocerebroside synthesis. In addition, experiments using D-[14C]galactose 1-phosphate and UDP d-[6-3H]glucose simultaneously have shown that the rate of formation of tritiated cerebroside is much greater than that of the 14C-labelled compound. For these reasons it is suggested that galactocerebroside is formed from UDP-glu and ceramide by epimerisation of the UDP hexose to UDP-gal rather than by synthesis of UDP-gal from galactose 1-phosphate and UDP-glu.  相似文献   

10.
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 Km 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.  相似文献   

11.
Intact chicken embryo neural retina cells have been shown to catalyze the transfer of galactose-14C from uridine diphosphate galactose (UDP-galactose) to endogenous acceptors of high molecular weight as well as to exogenous acceptors. Four lines of evidence indicate that the galactosyltransferases catalyzing these reactions are at least partly located on the outside surface of the plasma membrane: (a) there is no evidence for appreciable uptake of sugar-nucleotides by vertebrate cells nor did unlabeled galactose, galactose 1-phosphate, or UDP-glucose interfere with the radioactivity incorporated during the reaction; (b) the cells remained essentially intact during the course of the reaction; (c) there was insufficient galactosyltransferase activity in the cell supernatants to account for the incorporation of galactose-14C into cell pellets; and (d) the intact cells could transfer galactose to acceptors of 106 daltons, and the product of this reaction was in the extracellular fluid. Appropriate galactosyl acceptors interfered with the adhesive specificity of neural retina cells; other compounds, which were not acceptors, had no effect. These results suggested that the transferase-acceptor complex may play a role in cellular recognition.  相似文献   

12.
Glycogen synthase activation by sugars in isolated hepatocytes   总被引:2,自引:0,他引:2  
We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.  相似文献   

13.
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.  相似文献   

14.
Compartmentation between glycolysis and gluconeogenesis in rat liver   总被引:8,自引:6,他引:2  
1. The specific radioactivity-time relationships of glucose, glucose 6-phosphate, glycerol 1-phosphate and UDP-glucose were determined in rat liver after the intravenous injection of [U-(14)C]fructose, and a kinetic analysis was carried out. The glucose 6-phosphate pool was found to be compartmented into gluconeogenic and glycolytic components, and evidence was obtained that the triose phosphates were similarly compartmented. The glycolytic pathway was fed by glycogenolysis and glucose phosphorylation. There was no direct evidence that glycogenolysis fed only the glycolytic pathway, but this interpretation would make the liver resemble other organs in this respect. 2. UDP-glucose was not formed solely from gluconeogenic glucose 6-phosphate, as there was some dilution of label in the intervening glucose 1-phosphate pool, probably from glycogenolysis, though other pathways cannot be excluded. 3. The data cannot be explained by isotopic exchange.  相似文献   

15.
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.  相似文献   

16.
In human erythrocytes, in which the fractional turnover rate of glucose 6-phosphate is rather low, menadione increases to almost the same relative extent the oxidation of D-[U-14C]glucose and D-[U-14C]galactose. However, in pancreatic tumoral islet cells (RINm5F line), in which the fractional turnover rate of glucose 6-phosphate is considerably higher, menadione increases the oxidation of D-[1-14C]glucose but not that of D-[1-14C]galactose. These results suggest that alpha-D-glucose 6-phosphate generated from exogenous D-galactose is channeled preferentially into the glycolytic rather than pentose phosphate pathway. Such was no more the case, however, when the RINm5F cells were exposed simultaneously to both D-glucose and D-galactose.  相似文献   

17.
A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.  相似文献   

18.
1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose].  相似文献   

19.
Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.  相似文献   

20.
We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.  相似文献   

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