Identification of D-sorbitol 3-phosphate in the normal and diabetic mammalian lens

The 31P NMR spectra of mammalian lenses show, in addition to previously identified compounds such as ATP, Pi, glycerophosphorylcholine, and alpha-glycerophosphate, an unusual phosphorylated metabolite resonating at 6.5 ppm. The concentration of this compound increases manyfold in the lenses of diabetic rats concomitant with activation of the aldose reductase pathway. We have purified this material and have identified it as D-sorbitol 3-phosphate. It appears likely that this unusual metabolite of sorbitol may reflect or be part of the metabolic aberration of diabetes in the lens.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Conversion of glucose to sorbitol and fructose by liver-derived cells in culture.

Conversion of glucose to fructose and sorbitol is documented in rat hepatoma-derived cultured cells (HTC cells). After addition of 5.5 mM [U-14C]glucose to incubation medium, labeled sorbitol and fructose accumulated intracellularly at a linear rate over a period of 60 min. The sugars were isolated, identified, and quantitated by paper chromatography, gas-liquid chromatography, and enzymatic phosphorylation of fructose. Primary culture of adult rat hepatocytes was analyzed similarly and demonstrated no significant accumulation of labeled fructose or sorbitol. The basis for this difference between HTC cells and primary hepatocyte culture was examined both in terms of enzyme activities that mediate the formation of sorbitol and fructose and in terms of the catabolism of these sugars. Both types of culture (as well as extracts of intact rat liver) exhibited enzymatic activities catalyzing the conversion of glucose to sorbitol (aldose reductase) and sorbitol to fructose (sorbitol dehydrogenase). However, the cultures differed strikingly with regard to the catabolism of sorbitol and fructose. The conversion of labeled sorbitol to metabolites in HTC cells was negligible; by contrast, hepatocytes in primary culture utilized the sugars at rates comparable to that of glucose, which may account for the lack of their accumulation in primary culture. The findings suggest that the conversion of glucose to sorbitol and fructose by HTC cells may represent a retained normal liver function, one which is amplified by the inability of HTC cells to dispose of these sugars.     

Thermic and pH modulation of phosphofructokinase during trout (Salmo gairdneri R.) haemopoiesis: influence of fructose 6-phosphate

Thermic and pH modulation of phosphofructokinase (EC 2.7.1.11) activity with respect to fructose 6-phosphate has been studied comparatively in trout (Salmo gairdneri R.) haemopoietic cells and erythrocytes. Phosphofructokinase of both cellular populations displays a biphasic kinetic behaviour with respect to fructose 6-phosphate at two values of pH and temperature. In haemopoietic cells, when pH decreases the enzyme-substrate affinity increase while an opposite effect is found in erythrocytes. Decreases in temperature act as a positive modulator in haemopoietic cells while in erythrocytes this effect is observed only at low fructose 6-phosphate concentrations. Therefore a different pH and temperature modulation of phosphofructokinase during trout haemopoiesis has been established.     

Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes

Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D-Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D-glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon. Both fructose and D-glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2-3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D-glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1-phosphate mediates the effects of fructose, D-glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein.     

Formation of sorbitol 6-phosphate by bovine and human lens aldose reductase, sorbitol dehydrogenase and sorbitol kinase

Formation of sorbitol 6-phosphate by bovine and human lens aldose reductase and sorbitol dehydrogenase by the reduction of glucose 6-phosphate and fructose 6-phosphate, respectively, has been demonstrated. The reaction product has been identified by Dowex-formate column chromatography, gas chromatography and mass spectrometry. Sorbitol 6-phosphate can also be formed by the phosphorylation of sorbitol by lens sorbitol kinase in the presence of ATP.     

Enzymes related to fructose utilization in Pseudomonas cepacia

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.     

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Essential fructosuria: increased levels of fructose 3-phosphate in erythrocytes.

Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.     

The role of calcium in the stimulation of gluconeogenesis by catecholamines

In hepatocytes isolated from fasted normal rats and incubated without albumin or gelatin, norepinephrine stimulated gluconeogenesis from fructose or dihydroxyacetone only in the absence of added calcium and from sorbitol or glycerol only in the presence of added calcium. The effects of calcium, norepinephrine, or calcium in combination with norepinephrine on the concentration of intermediary metabolites were therefore studied in hepatocytes metabolizing fructose or sorbitol as the representative oxidized or reduced substrate, respectively. With fructose as the substrate, addition of calcium increased the concentrations of lactate, pyruvate, glyceraldehyde 3-phosphate, and β-hydroxybutyrate, but decreased the concentrations of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, glucose 6-phosphate, malate, citrate, and α-oxoglutarate. With sorbitol as the substrate, calcium increased the concentrations of pyruvate, malate, β-hydroxybutyrate, and glucose. With either substrate, calcium caused a decrease in the lactate/ pyruvate ratio and an increase in the β-hydroxybutyrate/acetoacetate ratio, indicating the stimulation of transfer of reducing equivalents from cytosol to mitochondria. With sorbitol as the substrate, and with calcium present, norepinephrine promoted further electron transfer from cytosolic to mitochondrial NAD. Enhanced cytosolic calcium concentrations, when cells are exposed to catecholamines in the presence of medium calcium, stimulate the mitochondrial α-glycerophosphate dehydrogenase and thus the transfer of electrons between cell compartments.     

Existence of two alternative pathways for fructose and sorbitol metabolism in Bacillus subtilis Marburg.

Strains of Bacillus subtilis mutated for fructose phosphotransferase system (fruA), fructose-1-phosphate kinase (fruB), fructokinase (frucC) have been tested for their catabolism of sorbitol and fructose. It is shown that the previously known pathways of sorbitol and fructose degradation in B. subtilis, e.g.: (see article) may metabolize intracellular fructose produced either by sorbitol oxidation or by fructose-1-phosphate dephosphorylation. The intracellular fructore degradation via fructose-1-phosphate kinase has been found to require the fructose phosphotransferase system which ensures a vectorial transport of fructose.     

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.     

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.     

Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.     

Hepatic fructose-metabolizing enzymes and related metabolites: role of dietary copper and gender

The purpose of this study was to further examine the hypothesis that variations in hepatic fructose-metabolizing enzymes between males and females might account for the differences in the severity of copper (Cu) deficiency observed in fructose-fed male rats. Weanling rats of both sexes were fed high-fructose diets either adequate or deficient in copper for 45 days. Cu deficiency decreased sorbitol dehydrogenase activity and dihydroxyacetone phosphate levels and increased glyceraldehyde levels in both sexes. Gender effects were expressed by higher activities of glycerol 3-phosphate dehydrogenase and aldehyde dehydrogenase in male than in female rats and higher levels of dihydroxyacetone phosphate and fructose 1,6-diphosphate (F1,6DP) in female than in male rats. The interactions between dietary Cu and gender were as follows: alcohol dehydrogenase activities were higher in female rats and were further increased by Cu deficiency in both sexes; aldehyde dehydrogenase activities were decreased by Cu deficiency only in male rats; sorbitol levels were higher in male rats and were further increased by Cu deficiency in male rats; fructose 1-phosphate (F1P) levels were increased by Cu deficiency in both sexes, but to a greater extent in male rats; glyceraldehyde 3-phosphate levels were higher in female rats, but were decreased by Cu deficiency in female and increased in male rats. Though most of the examined hepatic fructose-metabolizing enzymes and metabolites showed great differences between rats fed diets either adequate or deficient in Cu, it is the activity of fructokinase and aldolase-B, and the concentrations of their common metabolites, F1P and notably F1,6DP, that could be in part responsible for differences in the severity of pathologies associated with Cu deficiency observed between female and male rats.     

Multiple enzyme forms of glyceraldehyde-3-phosphate dehydrogenase in Pseudomonas aeruginosa PAO

Both NAD- and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (EC 1.2.1.12) activities were detected in glucose-grown cells of Pseudomonas aeruginosa strain PAO. After growth on gluconeogenic substrates such as citrate, the activity of the NAD-G3PDH was reduced severalfold in contrast to little change for the NADP-G3PDH. The two G3PDH activities could be separated by ammonium sulphate fractionation. PAGE revealed the presence of two G3PDH isoenzymes of 140 (NADP-specific) and 315 (NAD-specific) kDa. Slight differences were observed in the thermostabilities and pH optima of the two enzymes whereas the regulation of their activities by various compounds varied strongly. The NADP-G3PDH enzyme was activated by ATP, reduced NAD, and fructose 6-phosphate. It was inhibited by fructose 1,6-diphosphate and 6-phosphogluconate. The NAD-G3PDH enzyme was inhibited by ATP, reduced NAD, and 6-phosphogluconate; it was slightly activated by reduced NADP. The possible roles of these isoenzymes in the control of hexose catabolism and gluconeogenesis in P. aeruginosa are discussed.     

Fructose and related phosphate derivatives impose DNA damage and apoptosis in L5178Y mouse lymphoma cells

Glycation between reducing sugars and amino groups of long-lived macromolecules results in an array of chemical modifications that may account for several physiological complications. The consequences of the reaction are directly related to the reactivity of the sugars involved, whether aldoses or ketoses, phosphorylated or non-phosphorylated. So far, most studies have been focused on glucose, while fructose, a faster glycating agent, attracted minor attention. We have recently demonstrated that under in vitro conditions fructose and its phosphate derivatives can modify plasmid DNA faster than glucose and its phosphate metabolites. In the present study we provide further evidences suggesting that fructose and its phosphate metabolites, at the tested conditions, are cytotoxic and inflict deleterious DNA modifications to L5178Y cells in culture. Damage was verified by viable cell counts, MTT assay, colony forming ability, induction of mutation in the thymidine kinase gene, internucleosomal DNA cleavage, and single strand breaks. The intensity of the tested sugars to impose damage increased significantly in the following order: sucrose = glucose 1-phosphate < glucose < glucose 6-phosphate < fructose 1-phosphate = fructose < fructose 6-phosphate. Aminoguanidine, an inhibitor of the glycation reaction, inhibited internucleosomal DNA cleavage. Taken together, these results suggest that fructose triggers deleterious modification in cultured cells through the glycation process, and thus should deserve more attention as an agent that may induce physiological complications.     

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.     

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.