The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5''[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The stereochemical course of phosphoryl transfer catalysed by glucokinase.

Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an 'in-line' mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.     

Interaction of D-glucose with alkaline-earth metal ions. Synthesis, spectroscopic, and structural characterization of Mg(II)- and Ca(II)-D-glucose adducts and the effect of metal-ion binding on anomeric configuration of the sugar

The interaction of D-glucose with the hydrated alkaline-earth metal halides has been studied in solution, and adducts of the type Mg(D-glucose)X2.4 H2O, Ca(D-glucose)X2.4 H2O, and Ca(D-glucose)2X2.4 H2O, where X = Cl- and Br-, have been isolated, and characterized by means of F.t.-i.r. and 1H-n.m.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. Spectroscopic and other evidence suggested that the Mg(II) ion in the Mg(D-glucose)X2.4 H2O adducts six-coordinate, binding to a D-glucose molecule (possibly via O-1 and O-2 atoms) and to four H2O molecules, whereas, in the corresponding 1:1 Ca-D-glucose adduct, the Ca(II) ion is possibly seven-coordinate, binding to a sugar moiety (through the O-1, O-2, and other sugar donor atoms) and to four H2O molecules. In 1:2 Ca(D-glucose)2X2.4 H2O, the calcium ion may be eight-coordinate, binding to two D-glucose molecules (possibly via the O-1 and O-2 atoms of each sugar moiety) and to four H2O molecules. The strong, sugar H-bonding network is rearranged upon D-glucose adduct-formation, and the alpha-anomeric configuration is favored by these metal cation coordinations.     

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.     

Structure of the O-specific polysaccharide from the lipopolysaccharide of Serratia marcescens O8

Structural studies have been carried out on the O-specific polysaccharide from the lipopolysaccharide of the reference strain (CDC 1604-55) for serogroup O8 of Serratia marcescens. The polymer has a branched, tetrasaccharide repeating unit of D-galactose(Gal),D-glucose(Glc), and 2-acetamido-2-deoxy-D-glucose(GlcNAc) with the following structure: (Formula: see text). The anomeric configuration assigned to the glucose residue differs from that (beta) previously proposed [Tarcsay, L., Wang, C. S., Li, S.-C. and Alaupovic, P. (1973) Biochemistry 12, 1948-1955]. The structure of the O8 polymer is identical with that of one of two polymers present in the cell envelope of a strain (CDC 1783-57) of S. marcescens O14.     

Glucose transfer across the intact guinea-pig placenta

Experiments were carried out in anaesthetized pregnant guinea-pigs. Following the maternal injection of a bolus containing 14C-hexose and 3H2O, blood was sampled from the fetal umbilical vein during a single circulatory transit. A placental transfer index was calculated from the ratio of the tracers in the fetal whole blood divided by that in maternal plasma. The transfer index for D-glucose, 0.66 +/- 0.03 (SEM), greatly exceeded that for L-glucose, 0.013 +/- 0.004. Elevation of the maternal plasma D-glucose concentration, with unlabelled D-glucose, resulted in saturation of D-glucose transfer with an apparent Km of 1.2 x 10(-2) mol/l mean maternal plasma D-glucose. Phlorizin at maternal plasma concentrations of approximately 10(-3) mol/l inhibited D-glucose transfer by 40%. Phloretin did not affect D-glucose transfer at levels estimated to be 10(-4) mol/l. Specificity studies with substituted D-glucose analogues showed that alpha-methyl-D-glucoside is not transported by a facilitated pathway; 2-deoxy-D-glucose and 3-O-methyl-D-glucose share the D-glucose carrier and D-galactose has a partial affinity for the D-glucose carrier.     

Crabtree effect in tumoral pancreatic islet cells

The relationship between glycolysis and respiration was examined in a model of pancreatic B-cell dysfunction, namely in tumoral insulin-producing cells of the RINm5F line. A rise in D-glucose concentration from 2.8 to 16.7 mM increased the utilization of D-[5-3H]glucose and production of [14C]lactate from D-[U-14C]glucose, whereas decreasing the oxidation of either D-[U-14C]glucose or D-[6-14C]glucose. Whereas 2.8 mM D-glucose augmented O2 uptake above basal value, a further rise in D-glucose concentration to 16.7 mM decreased respiration, which remained higher, however, than basal value. Whether at low or high concentration, D-glucose exerted a pronounced sparing action upon the oxidation of endogenous nutrients in cells prelabeled with either L-[U-14C]glutamine or [14C]palmitate and, nevertheless, augmented above basal value the rate of lipogenesis, ATP/ADP content, adenylate charge, and cytosolic NADH/NAD+ and NADPH/NADP+ ratios. The generation of ATP resulting from the catabolism of either exogenous D-glucose or endogenous nutrients was not affected by the rise in hexose concentration from 2.8 to 16.7 mM. Thus, in sharp contrast with the situation found in normal islet cells, a rise in D-glucose concentration, instead of stimulating mitochondrial oxidative events, caused, through a Crabtree effect, inhibition of hexose oxidation and O2 consumption in tumoral islet cells.     

Effects of titanium compounds on a D-glucose-D-glucose oxidase assay system.

Titanium compounds affect the measurement of D-glucose oxidase (and therefore D-glucose) by the D-glucose-D-glucose oxidase-peroxidase-2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) assay system. The validity of measurement of the activity of D-glucose oxidase immobilised on supports based on titanium oxides is affected by complexation of the intermediate hydrogen peroxide with the support, and such supports may prove to be unsuitable for the immobilisation of D-glucose oxidase. The formation of titanic peroxides is among the reasons discussed for the various interactions encountered. The use of the assay system for the determination of D-glucose oxidase contaminated with catalase and for the determination of hydrogen peroxide is also described.     

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3

Lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3 have been isolated and characterized. 6-Deoxy-L-altrose residues were shown to be the main constituents of lipopolysaccharides isolated in addition to residues of L-rhamnose, D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid being minor components of sugar chains. Mild hydrolysis of lipopolysaccharides with acetic acid furnished O-specific polysaccharides, which are composed of 6-deoxy-L-altrose. Using 13C-NMR spectroscopy and methylation data, the structural features of backbones have been elucidated as follows: ----2)-6d-L-Altp(beta 1----2)-6d-L-Altp(beta 1----3)-6d-L-Altp)(beta 1----for serovars O:1,2a,3 and O:2a,2b,3;----2)-6d-L-Altp(beta 1----for serovar O:3. In addition, O-polysaccharide of serovar O:2a,2b,3 was found to contain an O-acetyl group at the C-3 position of some 1,2-linked sugar residues.     

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.     

Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase.

Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.     

Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.     

Hexose metabolism in pancreatic islets stimulation by D-glucose of [2-3H]glycerol detritiation

1. In pancreatic islets, a rise in glucose concentration is known to increase the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization. The opposite situation was found to prevail in parotid cells. 2. In rat pancreatic islets, D-glucose caused a concentration-related stimulation of 3H2O production from [2-3H]glycerol, but failed to affect 3H2O production from [1(3)-3H]glycerol or 14CO2 production from [U-14C]glycerol. At the low concentration used in most of these experiments (i.e. 1.0 mM), glycerol failed to affect D-[U-14C]glucose oxidation. 3. These findings suggest that the preferential stimulation by D-glucose of mitochondrial oxidative events in pancreatic islets represents an unusual situation in secretory cells and involves an accelerated circulation in the glycerol phosphate shuttle.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: distinction of the 1-monodeutero-isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy.

The 1H NMR spectrum obtained with the alpha- and beta-anomers of D-[1-2H]fructose 6-phosphate generated from D-glucose 6-phosphate sequentially exposed in D2O to phosphoglucoisomerase, phosphofructokinase and fructose-1,6-diphosphatase differed from that recorded when the deuterated ketohexose phosphate was produced from D-mannose 6-phosphate sequentially exposed in D2O to phosphomannoisomerase, phosphofructokinase and fructose-1,6-diphosphatase. The identification of the 2 isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy provides a new tool to assess the relative extent of interconversion of hexose phosphates in the reactions catalyzed by phosphoglucoisomerase and phosphomannoisomerase, respectively.     

Synthesis of L-2'-deoxypentofuranonucleoside derivatives of thymine from D-glucose

Convergent synthesis of L-2'-deoxypentofuranonucleoside derivatives of thymine was carried out from D-glucose via 6-O-toluoyl-3-deoxy-1,2-O-isopropylidene-beta-L-lyxo-hexofuranose as a key intermediate.     

Protective effect of D-glucose,L-leucine and fetal calf serum against oxidative stress in neonatal pancreatic islets

B-cell destruction during the onset of diabetes mellitus is associated with oxidative stress. In this work, we investigated the mechanisms of defense against oxidative stress present in neonatal islets and their modulation by D-glucose, L-leucine and fetal calf serum (FCS). Culturing neonatal rat islets in the presence of low D-glucose concentrations (2.8-5.6 mmol/l) and 1 mmol/l H(2)O(2) increased the D-glucose uptake by islets sixfold compared to control levels. This effect was dose-dependently inhibited by D-glucose or FCS and by high concentrations of L-leucine. These supplements allowed islets to increase cytoplasmic catalase (CAT) activity only in response to H(2)O(2), with no decrease in NO formation. Although L-leucine increased CAT activity and restored D-glucose uptake, it did not prevent damage to the islets. These data indicate that the most important H(2)O(2) scavenger system in the islets is CAT and that this system can be modulated by metabolic substrates.     

Structure of the glycerol phosphate-containing O-specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14)

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.