Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.     

Hydrolysis of beta-D-glucopyranosyl fluoride to alpha-D-glucose catalyzed by Aspergillus niger alpha-D-glucosidase

Aspergillus niger alpha-D-glucosidase, crystallized and free of detectable activity for beta-D-glucosides, catalyzes the slow hydrolysis of beta-D-glucopyranosyl fluoride to form alpha-D-glucose. Maximal initial rates, V, for the hydrolysis of beta-D-glucosyl fluoride, p-nitrophenyl alpha-D-glucopyranoside, and alpha-D-glucopyranosyl fluoride are 0.27, 0.75, and 78.5 mumol.min-1.mg-1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from beta-D-glucosyl fluoride and not from a contaminating trace of alpha-D-glucosyl fluoride, and is catalyzed by the alpha-D-glucosidase and not by an accompanying trace of beta-D-glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase. Present findings provide new evidence that alpha-glucosidases are not restricted to alpha-D-glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.     

Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride

Rhizopus niveus glucoamylase and Arthrobacter globiformis glucodextranase, which catalyze the hydrolysis of starch and dextrans, respectively, to form D-glucose of inverted (beta) configuration, were found to convert both alpha- and beta-D-glucosyl fluoride to beta-D-glucose and hydrogen fluoride. Each enzyme directly hydrolyzes alpha-D-glucosyl fluoride but utilizes th beta-anomer in reactions that require 2 molecules of substrate and yield glucosyl transfer products which are then rapidly hydrolyzed to form beta-D-glucose. Various D-glucopyranosyl compounds serve as acceptors for such reactions. Mixtures of beta-D-glucosyl fluoride and methyl-alpha-D-glucopyranoside[14C], incubated with either enzyme, yielded both methyl-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-[14C]glucopyranoside and methyl-alpha-D-glucopyranosyl-(1 leads to 6)-alpha-D-[14C]glucopyranoside. Glucoamylase produced more of the alpha-maltoside; glucodextranase produced more of the alpha-isomaltoside. Thus, both "exo-alpha-glucan hydrolases" emerge as glucosylases that catalyze stereospecifically complementary hydrolytic and transglucosylative reactions with glucosyl donors of opposite configuration. These reactions not only provide a new view of the catalytic capabilities of these supposedly strict hydrolases; they also furnish a basis for defining a detailed mechanism for catalysis. Present results, together with those of several recent studies from this laboratory (especially similar findings obtained with beta-amylase acting on alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950), provide strong new evidence for the functional flexibility of the catalytic groups of carbohydrases.     

Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride

Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic versatility of trehalase: Synthesis of α-d-glucopyranosyl α-d-xylopyranoside from β-d-glucosyl fluoride and α-d-xylose

Trehalase was previously shown (see ref. 5) to hydrolyze α-d-glucosyl fluoride, forming β-d-glucose, and to synthesize α,α-trehalose from β-d-glucosyl fluoride plus α-d-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. α-d-Glucopyranose and α-d-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with β-d-glucosyl fluoride. As little as 0.2mm added α-d-glucose (0.4mm α-d-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mm β-d-glucosyl fluoride at 0°. Digest of β-d-glucosyl fluoride plus α-d-xylose yielded the α,α-trehalose analog, α-d-glucopyranosyl α-d-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when β-d-glucosyl fluoride is the donor, and for water when α-d-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma

Phosphorylation of alpha- and beta-D-glucose by glucokinase from rat liver or a radiation-induced, transplantable insulinoma was investigated. Glucokinase partially purified by ion exchange chromatography on DEAE-Cibacron blue F3GA agarose was incubated for brief periods (1 or 3 min) with glucose anomers. Glucokinase from both liver and insulinoma tissue had a higher affinity for alpha-D-glucose (S0.5 = 6-7 mM) than beta-D-glucose (S0.5 = 12-14 mM). The maximum velocity was 15-20% lower for alpha-D-glucose than beta-D-glucose. Cooperative rate dependence with respect to glucose concentration was observed with both anomers (nH = 1.4). These kinetic data imply that both anomers of glucose are phosphorylated by glucokinase, however, at the physiological range of glucose concentrations below 15 mM, the higher affinity of alpha-D-glucose results in higher rates than with beta-D-glucose. At clearly pathological glucose concentrations exceeding 20 mM, the observed velocities are slightly higher with beta- than alpha-D-glucose. Glucokinase is thought to be the glucose sensor of pancreatic beta cells. The present data indicating a preferential phosphorylation of alpha-D-glucose compared to beta-D-glucose by glucokinase, supports the glucokinase-glucose sensor hypothesis, because it parallels the well established greater potency of alpha-D-glucose as a stimulant of insulin release.     

Conformations in solution of alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-mannopyranoside, and their 1-thioglycosyl analogs, and a tentative correlation of their behaviour with respect to the enzyme trehalase

The conformation in solution of alpha-D-glucopyranosyl alpha-D-glucopyranoside (alpha,alpha-trehalose, 1), alpha-D-glucopyranosyl alpha-D-mannopyranoside (3) and their corresponding 1-thioglycosyl analogs, alpha-D-glucopyranosyl 1-thio-alpha-D-glucopyranoside (1-thio-alpha,alpha-trehalose, 2) and alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (4) were established from high-resolution 1H-NMR and 13C-NMR measurements. These experimental results are in good agreement with the conformations as inferred from hard-sphere calculations. The dihedral angles phi H and psi H are not significantly different for the O-glycosyl disaccharides 1 and 3 compared with their 1-thioglycosyl analogs 2 and 4; however, the internuclear H-1--H-1' and H-1--H-5' distances appear to be longer for 1-thiodisaccharides. This may account for the differences in affinities of cockchafer trehalase which have been observed. This enzyme exhibits less affinity for the competitive inhibitor alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (4) than for its O-glycosyl analog 3 (Ki 0.055 mM versus 0.0057 mM). From the similarity in Ki between 1-thio-alpha, alpha-trehalose and alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (0.050 mM versus 0.055 mM), it is possible to assume a similar decrease in the enzymic affinity between the natural substrate (1) and the corresponding 1-thioglycosyl inhibitor (2), which can together be ascribed to the aforementioned difference in the conformation of the molecules.     

Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions.

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.     

Partial purification and characterization of trehalase from axenically grown myxamoebae of Dictyostelium discoideum

Lysosomal trehalase from the myxamoebae of Dictyostelium discoideum has been partially purified. The behavior of the enzyme under different chromatographic and electrophoretic conditions reveals its close similarities to other lysosomal enzymes that have been studied earlier. The cellular trehalase, which is electrophoretically homogeneous, appears as two peaks of activity when subjected to hydroxyapatite and gel filtration chromatography. The enzyme has isoelectric points of 4.0 and less than 2.5. Among natural disaccharides tested, the purified trehalase showed absolute specificity for trehalose with an apparent Km of 1.15 mM. However, the enzyme efficiently utilized the synthetic sugar alpha-D-glucosyl fluoride as a substrate. Various methods were employed to estimate the apparent molecular weight, which was found to lie in the range of 30-162 kDa.     

The physical properties of glycosyl diacylglycerols. Calorimetric, X-ray diffraction and Fourier transform spectroscopic studies of a homologous series of 1,2-di-O-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols.

We have synthesized a homologous series of saturated 1,2-di-O-n-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols with odd- and even-numbered hydrocarbon chains ranging in length from 10 to 20 carbon atoms, and have investigated their physical properties using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy. The DSC results show a complex pattern of phase behaviour, which in a typical preheated sample consists of a lower temperature, moderately energetic lamellar gel/lamellar liquid-crystalline (L(beta)/L(alpha)) phase transition and a higher temperature, weakly energetic lamellar/nonlamellar phase transition. On annealing at a suitable temperature below the L(beta)/L(alpha) phase transition, the L(beta) phase converts to a lamellar crystalline (L(c1)) phase which may undergo a highly energetic L(c1)/L(alpha) or L(c1)/inverted hexagonal (H(II)) phase transition at very high temperatures on subsequent heating or convert to a second L(c2) phase in certain long chain compounds on storage at or below 4 degrees C. The transition temperatures and phase assignments for these galactolipids are supported by our XRD and FTIR spectroscopic measurements. The phase transition temperatures of all of these events are higher than those of the comparable phase transitions exhibited by the corresponding diacyl alpha- and beta-D-glucosyl glycerols. In contrast, the L(beta)/L(alpha) and lamellar/nonlamellar phase transition temperatures of the beta-D-galactosyl glycerols are lower than those of the corresponding diacyl phosphatidylethanolamines (PEs) and these glycolipids form inverted cubic phases at temperatures between the lamellar and H(II) phase regions. Our FTIR measurements indicate that in the L(beta) phase, the hydrocarbon chains form a hexagonally packed structure in which the headgroup and interfacial region are undergoing rapid motion, whereas the L(c) phase consists of a more highly ordered, hydrogen-bonded phase, in which the chains are packed in an orthorhombic subcell similar to that reported for the diacyl-beta-D-glucosyl-sn-glycerols. A comparison of the DSC data presented here with our earlier studies of other diacyl glycolipids shows that the rate of conversion from the L(beta) to the L(c) phase in the beta-D-galactosyl glycerols is slightly faster than that seen in the alpha-D-glucosyl glycerols and much faster than that seen in the corresponding beta-D-glucosyl glycerols. The similarities between the FTIR spectra and the first-order spacings for the lamellar phases in both the beta-D-glucosyl and galactosyl glycerols suggest that the headgroup orientations may be similar in both beta-anomers in all of their lamellar phases. Thus, the differences in their L(beta)/L(c) conversion kinetics and the lamellar/nonlamellar phase properties of these lipids probably arise from subtly different hydration and H-bonding interactions in the headgroup and interfacial regions of these phases. In the latter case, such differences would be expected to alter the ability of the polar headgroup to counterbalance the volume of the hydrocarbon chains. This perspective is discussed in the context of the mechanism for the L(alpha)/H(II) phase transition which we recently proposed, based on our X-ray diffraction measurements of a series of PEs.     

Reassessment of the catalytic mechanism of glycogen debranching enzyme

The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Environmental modulation of the anomeric specificity of glucose metabolism in normal and tumoral cells

In rat pancreatic islets and erythrocytes, alpha-D-glucose (2.8-5.6 mM) is better metabolized than beta-D-glucose, as judged from the conversion of D-[5-3H]glucose to 3H2O, augmentation in lactic acid production (or output) or oxidation of D-[U-14C]glucose. In tumoral cells, however, whether of the insulin-producing or lymphocytic leukemia type, the anomeric preference for alpha-D-glucose utilization is no longer present when the cells are incubated at comparable glucose concentrations (2.8-4.0 mM). Nevertheless, the tumoral insulin-producing cells are able to display preference for either alpha-D-glucose (at very low glucose concentrations in the 0.14-0.82 mM range) or beta-D-glucose (in the presence of 16.7 mM glucose). These findings indicate that the anomeric specificity of glucose metabolism may differ in distinct cell types, and can be modulated by the ambient glucose concentration. ambient glucose concentration.     

Enzymatic synthesis of a library of beta-(1-->4) hetero- D-glucose and D-xylose-based oligosaccharides employing cellodextrin phosphorylase

Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1-->4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.49) as the enzyme catalyst, with alpha-D-glucose 1-phosphate (1) or alpha-D-xylose 1-phosphate (2) as the donor substrates, and cellobiose (3), xylobiose (4), betaGlc-(1-->4)-Xyl (5), or betaXyl-(1-->4)-Glc (6) as the acceptor substrates. All enzymatic reactions were performed at pH 7.0 and the products purified by gel-filtration chromatography. We successfully synthesized all six hetero-trisaccharides and 10 of the 14 possible hetero-tetrasaccharides. It was not found possible to synthesize the four tetrasaccharides with a Xyl-->Glc sequence at their non-reducing ends employing this method. The stereochemistries of the isolated products were assessed by analysis of their 2D NMR spectra (DQF-COSY, TOCSY, HSQC, HMBC), confirming that all of the glycosidic bonds in the products were beta-(1-->4) linkages.     

Anomeric preference of glucose phosphorylation and glycolysis in human erythrocytes

Lactate output from the alpha and beta anomers of glucose was measured in intact human erythrocytes at 37 degrees C; and glucose anomer phosphorylation, in human erythrocyte homogenates. The rates of both glucose metabolism (lactate output) and phosphorylation were higher in the presence of beta-D-glucose as distinct from alpha-D-glucose at three glucose concentrations used (2, 5, and 10 mM). Thus, the v beta/v alpha ratios of metabolism and phosphorylation of glucose at 2 mM were 1.24 and 1.22, respectively. The results indicate that the beta preference of hexokinase, a rate-limiting enzyme in glycolysis, is reflected in beta-preferential glycolysis.     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Metabolism of tritiated D-glucose anomers in rat erythrocytes

It was recently proposed that alpha-D-glucose 6-phosphate may undergo enzyme-to-enzyme channelling between glucokinase and phosphoglucoisomerase in rat pancreatic islets. The present study aims at exploring whether a different situation prevails in cells deprived of glucokinase, namely in erythrocytes. At anomeric equilibrium, the ratio between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH was lower in rat erythrocytes incubated for 60 min at 4 degrees C in the presence of 2.8 mM, rather than 8.3 mM, D-glucose. This coincided with both a greater relative increase in beta-D-[5-3H]glucose, as compared to alpha-D-[5-3H]glucose, conversion to 3HOH and an increase in the beta/alpha ratio for 3HOH generation from D-[5-3H]glucose in response to an increase in the anomeric concentration from 2.8 to 8.3 mM, the suppression of the difference between the beta/alpha ratios for 3HOH generation from D-[2-3H]glucose and D-[5-3H]glucose in the erythrocytes incubated at 8.3 mM, as distinct from 2.8 mM, alpha- and beta-D-glucose, and a [2-3H]/[5-3H] ratio for 3HOH generation lower than unity in erythrocytes exposed to alpha-D-glucose but not significantly different from unity in the presence of beta-D-glucose. These findings emphasize the relevance of alpha-D-glucose 6-phosphate channelling between hexokinase and phosphoglucoisomerase as a determinant of the difference between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH, and reveal that the regulation of such a tunnelling process by the concentration of the D-glucose represents, in rat erythrocytes, a mirror image of that observed in rat pancreatic islets. The regulation of this process thus tightly depends on the identity of the hexokinase enzyme mainly responsible for the phosphorylation of D-glucose in distinct cell types.     

Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.     

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.