Isomerization of d-glucose with sodium aluminate: Mechanism of the reaction

A mechanism for the isomerization of d-glucose to d-fructose by sodium aluminate is proposed, involving transformation of a β-d-glucopyranose-1,3-aluminate complex into an α-d-fructofuranose-1,3,6-aluminate complex through an enolaluminate complex that inhibits the formation of a d-mannose-aluminate complex. The α-d-fructofuranose-1,3,6-aluminate further reacts to form a d-psicose-aluminate complex in substantial yield. Constant degradation of the 6-carbon sugars occurred during the reaction because of the high pH of the solution. The C₆ sugars were analyzed chromatographically but the degradation products were not identified.     

Effects of Mannose on the Growth of N2-Fixing Azotobacter vinelandii

Mannose is not a suitable substrate for N₂-fixing Azotobacter vinelandii. However, when H₂ gas is provided, A. vinelandii can grow mixotrophically with H₂ as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N₂-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N₂-fixing processes.     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

Detection of nanosecond time scale side-chain jumps in a protein dissolved in water/glycerol solvent

In solution, the correlation time of the overall protein tumbling, τ _R , plays a role of a natural dynamics cutoff—internal motions with correlation times on the order of τ _R or longer cannot be reliably identified on the basis of spin relaxation data. It has been proposed some time ago that the ‘observation window’ of solution experiments can be expanded by changing the viscosity of solvent to raise the value of τ _R . To further explore this concept, we prepared a series of samples of α-spectrin SH3 domain in solvent with increasing concentration of glycerol. In addition to the conventional ¹⁵N labeling, the protein was labeled in the Val, Leu methyl positions (¹³CHD₂ on a deuterated background). The collected relaxation data were used in asymmetric fashion: backbone ¹⁵N relaxation rates were used to determine τ _R across the series of samples, while methyl ¹³C data were used to probe local dynamics (side-chain motions). In interpreting the results, it has been initially suggested that addition of glycerol leads only to increases in τ _R , whereas local motional parameters remain unchanged. Thus the data from multiple samples can be analyzed jointly, with τ _R playing the role of experimentally controlled variable. Based on this concept, the extended model-free model was constructed with the intent to capture the effect of ns time-scale rotameric jumps in valine and leucine side chains. Using this model, we made a positive identification of nanosecond dynamics in Val-23 where ns motions were already observed earlier. In several other cases, however, only tentative identification was possible. The lack of definitive results was due to the approximate character of the model—contrary to what has been assumed, addition of glycerol led to a gradual ‘stiffening’ of the protein. This and other observations also shed light on the interaction of the protein with glycerol, which is one of the naturally occurring osmoprotectants. In particular, it has been found that the overall protein tumbling is controlled by the bulk solvent, and not by a thin solvation layer which contains a higher proportion of water.     

Solution thermodynamic approach to analyze protein stability in aqueous solutions

Protein thermal stability was analyzed by a solution thermodynamic approach. The small energetic differences in hydrogen-bonds (HB) among amino acid resdues and water molecules were proved to be amplified by the large number of HB involved to bring about the equilibrium shift from folding to unfolding of proteins. In aqueous solutions, water activity (A_w) plays a key role in protein stability. Therefore, A_w was precisely determined for various solutions and its relationship with solution structure was discussed. Wyman-Tanford analysis based on A_w showed linear regressions, without exception, between protein unfolding-ratio and A_w for lysozyme, ribonuclease A, and α-chymotrypsinogen A in various solutions with sugars, osmolytes, alcohols, and protein denaturant. From this linear regression, the free energy difference, ΔΔG, for a protein in a solution and in pure water, was easily obtained. Protein stability in a solution was proved to be determined by a balance between hydration and solute-binding effects to the protein and also by solution structure, which indirectly affects the hydrophobic interaction in a protein molecule. Temperature dependence of HB on protein stability suggested its interrelationship with hydrophobic interaction.     

Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO₃⁻)-α(1-3)-l-Fucp2,4(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H₂O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space ¹H–¹H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.     

Strong coupling effects during <Emphasis Type="Italic">X</Emphasis>-pulse CPMG experiments recorded on heteronuclear <Emphasis Type="Italic">ABX</Emphasis> spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as ¹³C _i –¹³C _j –¹H, where ¹³C _i and ¹³C _j are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of ¹H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2′ position, deuterated at all other sugar positions and ¹³C labeled at all sugar carbons is presented to illustrate the problems that strong ¹³C–¹³C coupling introduces and a simple solution is proposed.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

Correlation between the Carbon Isotope Discrimination in Leaf Starch and Sugars of C(3) Plants and the Ratio of Intercellular and Atmospheric Partial Pressures of Carbon Dioxide

Carbon isotope discrimination (Δ) was analyzed in leaf starch and soluble sugars, which represent most of the recently fixed carbon. Plants of three C₃ species (Populus nigra L. × P. deltoides Marsh., Gossypium hirsutum L. and Phaseolus vulgaris L.) were kept in the dark for 24 hours to decrease contents of starch and sugar in leaves. Then gas exchange measurements were made with constant conditions for 8 hours, and subsequently starch and soluble sugars were extracted for analysis of carbon isotope composition. The ratio of intercellular, p_i, and atmospheric, p_a, partial pressures of CO₂, was calculated from gas exchange measurements, integrated over time and weighted by assimilation rate, for comparison with the carbon isotope ratios in soluble sugars and starch. Carbon isotope discrimination in soluble sugars correlated strongly (r = 0.93) with p_i/p_a in all species, as did Δ in leaf starch (r = 0.84). Starch was found to contain significantly more ¹³C than soluble sugar, and possible explanations are discussed. The strong correlation found between Δ and p_i/p_a suggests that carbon isotope analysis in leaf starch and soluble sugars may be used for monitoring, indirectly, the average of p_i/p_a weighted by CO₂ assimilation rate, over a day. Because p_i/p_a has a negative correlation with transpiration efficiency (mol CO₂/mol H₂O) of isolated plants, Δ in starch and sugars may be used to predict differences in this efficiency. This new method may be useful in ecophysiological studies and in selection for improved transpiration efficiency in breeding programs for C₃ species.     

An R1ρ expression for a spin in chemical exchange between two sites with unequal transverse relaxation rates

An analytical expression is derived for the rotating frame relaxation rate, R _1ρ , of a spin exchanging between two sites with different transverse relaxation times. A number of limiting cases are examined, with the equation reducing to formulae derived previously under the assumption of equivalent relaxation rates at each site. The measurement of a pair off-resonance R _1ρ values, with the carrier displaced equally on either side of the observed correlation, forms the basis of one of the approaches for obtaining signs of chemical shift differences, Δω, of exchanging nuclei. The results presented here establish that this method is relatively insensitive to differential transverse relaxation rates between the exchaning states, greatly simplifying the calculation of optimal parameters in R _1ρ based experiments that are used for measurement of signs of Δω.     

Cloning of Glucuronokinase from Arabidopsis thaliana, the Last Missing Enzyme of the myo-Inositol Oxygenase Pathway to Nucleotide Sugars

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K_m of 0.7 mm. It requires ATP as phosphate donor (K_m 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.     

Effects of temperature and field strength on the NMR relaxation times of 23Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.     

Monitoring conformational dynamics with solid-state R 1ρ experiments

A new application of solid-state rotating frame (R _1ρ) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R _1ρ relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d ₆ -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins could represent a new site-specific probe of conformational dynamics.     

Synthesis and characterization of a macrocyclic Schiff base Gd complex as a relaxation agent for a faster acquisition of H NMR spectra of ethanol

Quantitative ²H NMR analysis at the natural abundance represents a well-recognized and efficient method for the identification of the origin of ethanol from different sources. An intrinsic limitation of the protocol used is the long time required, about 8 h, because of the long T₁ values of the ²H resonances. In this work we propose the use a paramagnetic relaxation agent that significantly catalyzes the relaxation times and reduces the total time of the analysis. This agent is the macrocyclic Schiff base complex [Gd(H₂L)(H₂O)₃(EtOH)](Cl)₃ · 2 EtOH (H₂L is the [1 + 1] macrocycle derived from the condensation of 3,3^′-(3-oxapentane-1,5-diyldioxy)bis(2-hydroxybenzaldehyde) with 1,5-diamino-3-azamethylpentane), which is highly stable and soluble in the alcoholic solution used. Elemental analysis, IR and mass spectrometry have characterized this complex. The homogeneity of the complex and the correct Gd:Cl=1:3 ratio was established by SEM-EDS measurements. Further characterization of the paramagnetic complex has been achieved by measuring the magnetic field dependence of the ¹H longitudinal nuclear magnetic relaxation time of a 1 mM solution in CH₃OD with a field-cycling relaxometer. The Gd^III ion accommodates up to four methanol molecules in its inner coordination sphere, whose rapid exchange with the bulk provides an efficient relaxation mechanism. The addition of about 37 mg of the complex to a solution of ethanol (3.0 g) and tetramethylurea (TMU) (1.5 g) results in the reduction of the experimental time of more than 50% with a S/N ratio compatible with that required for this application.     

A new method for the determination of hydraulic conductivity and cell volume of plant cells by pressure clamp

Internodes of Chara corallina were used for experiments in which cell turgor pressure was clamped by means of the pressure probe technique. Essentially, the procedure consisted of a combination of volume and turgor pressure relaxations. This technique permits the determination of the cell volume by nonoptical means. The values obtained are in agreement with the ones determined by optical means. Furthermore, the hydraulic conductivity (L_p) was determined from the initial slope of the volume relaxation; the values thus obtained are in agreement with those calculated from the half-times of pressure relaxations. The determination of L_p from volume relaxation measurements has the advantage that the cell volume, the volumetric elastic modulus of the cell wall, and the internal osmotic pressure do not have to be known. Furthermore, the half-time of volume relaxation is longer than that of pressure relaxation, as shown by theory and experiment. This may be used to enhance the resolution of the relaxation measurement and, thus, to improve the accuracy of L_p determinations for higher plant cells which exhibit a very fast pressure relaxation.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Studies on the electrical potential profile across rabbit ileum. Effects of sugars and amino acids on transmural and transmucosal electrical potential differences

When isolated strips of mucosal rabbit ileum are bathed by physiological electrolyte solution the electrical potential difference (PD) across the brush border (ψ_mc) averages 36 mv, cell interior negative. Rapid replacement of Na in the mucosal solution with less permeant cations, Tris or choline, results in an immediate hyperpolarization of ψ_mc. Conversely, replacement of choline in the mucosal solution with Na results in an abrupt depolarization of ψ_mc. These findings indicate that Na contributes to the conductance across the brush border. The presence of actively transported sugars or amino acids in the mucosal solution brings about a marked depolarization of ψ_mc and a smaller increase in the transmural PD (Δψ_ms). It appears that the Na influx that is coupled to the influxes of amino acids and sugars is electrogenic and responsible for the depolarization of ψ_mc. Under control conditions Δψ_ms can be attributed to the depolarization of ψ_mc together with the presence of a low resistance transepithelial shunt, possibly the lateral intercellular spaces. However, quantitatively similar effects of amino acids on ψ_mc are also seen in tissues poisoned with metabolic inhibitors or ouabain. Under these conditions Δψ_mc is much smaller than under control conditions. Thus, the depolarization of ψ_mc might not account for the entire Δψ_ms, observed in nonpoisoned tissue. An additional electromotive force which is directly coupled to metabolic processes might contribute to the normal Δψ_ms.     

Long-lived spin state of a tripeptide in stretched hydrogel

The longitudinal (T ₁), transverse (T ₂), and singlet state (T _s) relaxation times of the geminal backbone protons (CH₂) of l-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D₂O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T ₂ and T _s of the CH₂ protons correspondingly decreased (T _s/T ₂ ~ constant), while the change in T ₁ was relatively small. The largest observed T _s/T ₁ value was 3.3 at 46 % w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D₂O in ²H NMR spectra, and residual dipolar splitting of the CH₂ resonance in ¹H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T ₁, T ₂, and T _s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T ₂ is shorter than T ₁ in the presence of non-zero residual dipolar couplings.