Solvent and solute effects on “inside-outside” conformations and rotation about the peptide bond in a miniature protein model

The physicochemical parameters affecting protein unfolding in relation to peptide bond rotations are briefly reviewed. As a suitable model for the study of solvent and solute effects on amide rotation and inside-outside conformations, the 2,2′-biphenyl analog of N-benzoyl-l-phenylalanine methyl ester (I) was synthesized and resolved enzymatically with α-chymotrypsin. The optically pure substance exists as conformer I_a (R,S configuration) with an axial methoxycarbonyl in the crystalline state. Rotation about the biphenyl axis leads to the equatorial conformer I_b (S,S configuration) in various solvents. In polar solvents, rotation about the amide is rate limiting. Accurate measurements of this rotation were accomplished by following the rate of change in the maximum amplitude of the biphenyl Cotton band at 256 nm. The high sensitivity of the method allowed rate and equilibrium measurements at 10^?3M in the absence of intermolecular association. Small differences of the order of 100 cal/mole in ΔG^≠ or ΔG_eq could thus be detected accurately. It was found that k_obs or k₁ (forward step) for equilibration was linearly related (correlation coefficient of 0.96 for k_obs) with E_T, the solvent polarity index on Reichardt and Dimroth's scale. Rotation was slowest in water and fastest in carbon tetrachloride, δΔG^≠, being 2.4 kcal/mole. Chaotropic anions, cations, and guanidinium chloride accelerated the rate in water. However, the inside-outside (axial-equatorial; I_aI_b) ratio at equilibrium did not correlate in any simple manner with the solvent E_T values. Rather, correlation within groups of solvents appeared to exist. It was suggested that solvent association with the amide differs quantitatively in the inside and outside conformations. The position of the equilibrium in water was affected by chaotropic ions but not by urea or quanidinium chloride. Some possible mechanisms are briefly outlined.     

Solvent Effect and Anchimeric Assistance on α-Glycosylation

The condensation reaction of 3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-d-glucopyranosyl chloride, 6-acetamido-2,3,4-tri-O-benzyl-6-deoxy-d-glucopyranosyl chloride and 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl chloride were performed by a modified Königs-Knorr method. The rapid conversion of the benzyl halogeno derivative of 3-acetamido-3-deoxy-d-glucose to a stable intermediate caused a poor yield in the glucoside formation with complex aglycons at the presence of dioxane. For the benzyl halogeno derivative of 6-acetamido-6-deoxy-d-glucose, the C-6 acetamido group was favorable to the α-glucoside formation by its anchimeric assistance. A favorable effect of dioxane was observed for the α-glucoside formation of benzyl halogeno derivative of d-glucose.     

Atmospheric deposition and solute export in giant sequoia — mixed conifer watersheds in the Sierra Nevada,California

Atmospheric depostion and stream discharge and solutes were measured for three years (September 1984 — August 1987) in two mixed conifer watersheds in Sequoia National Park, in the southern Sierra Nevada of California. The Log Creek watershed (50 ha, 2067–2397 m elev.) is drained by a perennial stream, while Tharp's Creek watershed (13 ha, 2067–2255 m elev.) contains an intermittent stream. Dominant trees in the area include Abies concolor (white fir), Sequoiadendron giganteum (giant sequoia), A. magnifica (red fir), and Pinus lambertiana (sugar pine). Bedrock is predominantly granite and granodiorite, and the soils are mostly Pachic Xerumbrepts. Over the three year period, sulfate (SO₄ ^2–), nitrate (NO₃ ^–), and chloride (Cl^–1) were the major anions in bulk precipitation with volume-weighted average concentrations of 12.6, 12.3 and 10.0 eq/1, respectively. Annual inputs of NO₃-N, NH₄-N and SO₄-S from wet deposition were about 60 to 75% of those reported from bulk deposition collectors. Discharge from the two watersheds occurs primarily during spring snowmelt. Solute exports from Log and Tharp's Creeks were dominated by HCO₃ ^–, Ca²⁺ and Na⁺, while H⁺, NO₃ ^–, NH₄ ⁺ and PO₄ ^3– outputs were relatively small. Solute concentrations were weakly correlated with instantaneous stream flow for all solutes (r² <0.2) except HCO₃ ^– (Log Cr. r² = 0.72; Tharp's Cr. r² = 0.38), Na⁺ (Log Cr. r² = 0.56; Tharp's Cr. r² = 0.47), and silicate (Log Cr. r² = 0.71; Tharp's Cr. r² = 0.49). Mean annual atmospheric contributions of NO₃-N (1.6 kg ha^–1), NH₄-N (1.7 kg ha^–1), and SO₄-S (1.8 kg ha^–1), which are associated with acidic deposition, greatly exceed hydrologic losses. Annual watershed yields (expressed as eq ha^–1) of HCO₃ ^– exceeded by factors of 2.5 to 37 the annual atmospheric deposition of H⁺.     

Intracellular salt and solute concentrations in Ectothiorhodospira marismortui: glycine betaine and Nα-carbamoyl glutamineamide as osmotic solutes

Ectothiorhodospira marismortui, a moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring, contains glycine betaine and N-carbamoyl glutamineamide (CGA) as the main intracellular osmotic solutes, with sucrose as a minor component. The concentration of glycine betaine was found to increase with increasing salt concentration of the medium, from 0.47 M to 1.29 M in cells grown from 0.85 to 2.56 M NaCl, while the estimated CGA concentration rose from about 0.2 M to 0.5 M. The concentration of sucrose remained constant at a value of around 0.05 M. Intracellular sodium and potassium concentrations were relatively low (around 0.5 and 0.3 M, respectively, at an external NaCl concentration of 1.8 M). The concentration of the novel compound N-carbamoyl glutamineamide was enhanced when l-glutamine was added to the growth medium, suggesting that glutamine served as a precursor for the synthesis of the compound.Abbreviations CGA N-carbamoyl glutamineamide     

Solvent perturbation spectra of yellow mealworm α-amylase and of wheat protein inhibitors

• 1.1. The number of tyrosine and tryptophan residue-equivalents on the surfaces of the α-amylase and of two of its protein inhibitors has been determined.
• 2.2. The solvent-exposed tyrosine and tryptophan residue-equivalents in the dimeric inhibitor are respectively four and two times as much as those ones of the monomeric inhibitor.
• 3.3. On the basis of the homology among their polypeptide chains, it is suggested that the outer tryptophan residues in either inhibitors are located at the positions 4 and 51 of the primary structure.
• 4.4. On the interaction of the monomeric inhibitor with the amylase, two tryptophan residue-equivalents became no more accessible to the solvent.

     

How do transporters couple solute movements?

Abstract

The availability of high-resolution atomic structures for transport proteins provides unprecedented opportunities for understanding their mechanism of action. The details of conformational change can be deduced from these structures, especially when multiple conformations are available. However, the singular ability of transporters to couple the movement of one solute to that of another requires even more information than what is supplied by a crystal structure. This short commentary discusses how recent biochemical and biophysical studies are beginning to reveal how solute coupling is achieved.     

Effect of Biomass Pre-Treatment and Solvent Extraction on β-Carotene and Lycopene Recovery from Blakeslea trispora Cells

Abstract

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being β-carotene, lycopene, and γ-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C_max values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44–56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in β-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.     

Approximate analytical solutions of root surface nutrient uptake flux and rhizosphere solute concentrations北大核心CSCD

Aims We use the Nye-Tinker-Barber model to obtain approximate analytical solutions of the root surface nutrient uptake flux and the rhizosphere solute concentrations. Methods The rhizosphere is divided into the distant field and the close-range field. The outer solution is obtained by the similarity variable method, and the inner solution is obtained by the rescaling method. Expanding the outer solution from root surface and matching it with the inner solution, we finally obtain the approximate analytical solutions of the root surface nutrient uptake flux and rhizosphere solute concentrations based on Nye-Tinker-Barber model. Numerical simulations are performed on the uptake fluxes of six elements (N, K, P, Mg, S and Ca) and the solute concentrations of two elements (N and K). Comparisons are made among the Nye-Tinker-Barber model approximate analytical solutions, Roose’s analytical solutions and the numerical solutions. Important findings The approximate analytical solutions of the root surface uptake fluxes based on the diffusion Nye-Tinker-Barber model are similar to those of the Roose's analytical solutions; both approaches produced higher values than the numerical solutions. The approximate analytical solutions of the rhizosphere N and K concentrations based on the diffusion Nye-Tinker-Barber model are similar to the Roose’s analytical solutions and coincide with the trend of changes in the numerical solutions. The approximate analytical solutions of all elements but N, of the root surface, uptake fluxes based on the convection-diffusion Nye-Tinker-Barber model are more similar to the numerical solutions than the Roose’s analytical solutions, and the approximate analytical solutions of the rhizosphere N and K concentrations are coincide with the trend of changes in the numerical solutions. © 2018 Editorial Office of Chinese Journal of Plant Ecology. All Rights Reserved.     

Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Solvent production from starch: effect of pH on α-amylase and glucoamylase localization and synthesis in synthetic medium

Summary The fermentation of starch by Clostridium acetobutylicum ATCC 824 has been reviewed in an optimised synthetic medium. A progressive increase of pH from 4.4 to 5.2 led to a higher production of extracellular -amylase whereas glucoamylase was poorly affected. A portion of these enzymes was cell-associated and on increasing the pH from 4.4 to 5.8 a decrease was noted in cell-bound enzymes. The association was higher for the glucoamylase than for the -amylase. The highest rate of starch consumption was at pH 5.2 whereas due to the earlier shift to solvent production at low pH, the highest solvent production was at pH 4.4. This study suggested that the level of -amylase and then the rate of starch hydrolysis was the limiting step of sugar catabolism in C. acetobutylicum ATCC 824. Correspondence to: P. Soucaille     

Water and solute regulation in Procephalothrix spiralis Coe and Clitellio arenarius (Müller) III. Long-term acclimation to diluted seawaters and effect of putative neuroendocrine structures

When acutely transferred to diluted seawater (SW), Procephalothrix spiralis and Clitellio arenarius regulate water content (g H₂O/g solute free dry wt = s.f.d.w.) via loss of Na and Cl (µmoles/g.s.f.d.w.). The present study extends these observations to a greater range of salinities and determines the effects of long-term, stepwise acclimation to diluted seawaters. Final exposure to a given experimental seawater (70, 50, 30, 15%) was 48 hours. Osmolality (mOsm/kg H₂O) and Na, K, and Cl ion concentrations (mEq/l) were determined in total tissue water and in the extracellular fluid of C. arenarius. Extracellular volume was determined as the ¹⁴C-polyethylene glycol space. Both species behaved as hyperosmotic conformers in diluted seawaters. However, reduction of the osmotic gradient between worm and medium occurred in P. spiralis, but not C. arenarius, in 30 and 15% SW. In both species, osmolality and Na, Cl, and K concentrations in total tissue water decreased with increased dilution of the SW. Water content increased with dilution of the medium but was lower than that which would be predicted based on approximation of the van't Hoff relation. This indicated the occurrence of regulatory volume decrease (RVD). In P. spiralis, in 70 or 50% SW, RVD was accompanied by loss of Na and Cl contents. However, in 30 or 15% SW, Na and Cl contents increased and in worms in 15% SW K content decreased. The latter movements of Na, Cl and K are indicative of cellular hysteresis and were associated with decreased viability, indicating the lower limits of regulatory ability in this species. In comparison, RVD in C. arenarius occurred in all diluted seawaters and was accompanied by loss of Na and Cl contents. In C. arenarius, evidence for reduced viability was absent. Removal of the supra- and subesophageal ganglia of C. arenarius resulted in retention of water, Na and Cl (g H₂O or µmoles/g s.f.d.w.) in worms acclimated to 70% SW. Removal of the cerebral ganglia and cephalic glands of P. spiralis did not significantly influence regulation of water content.     

Light and Life in Baltimore—and Beyond

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.     

Veneers and plywood—their manufacture and use

Two closely related industries that depend, to a major degree, upon domestic sources of red gum, Douglasfir, black walnut, red and white oaks, sugar maple, Sitka spruce, Ponderosa pine and Port Orford cedar; and upon foreign sources of mahogany, rosewood, zebrawood, avodiré, bossé, ebony and other exotic species.     

Vegetative growth,compatible solute accumulation,ion partitioning and chlorophyll fluorescence of ‘Malas-e-Saveh’ and ‘Shishe-Kab’ pomegranates in response to salinity stress

The present research was conducted to assess physiological responses of ‘Malas-e-Saveh’ (Malas) and ‘Shishe-Kab’ (Shishe) pomegranates to water of different salt content and electrical conductivity (1.05, 4.61, and 7.46 dS m^?1). Both cultivars showed a reduced trunk length due to salinity. Relative water content and stomatal conductivity of both cultivars were significantly reduced under salt stress, but ion leakage increased. In both cultivars, total chlorophyll (Chl) and carbohydrates decreased with rise in salinity, while proline accumulation increased. With salinity increment, the Chl fluorescence parameters (maximum photochemical efficiency of PSII and effective quantum yield of PSII) declined significantly in both cultivars, with higher reduction observed in Shishe. Generally, more Na⁺ accumulated in shoots and more Cl^? was observed in leaves. Cl^? accumulation increased by salinity in leaves of Malas, but it was reduced in Shishe. The K⁺/Na⁺ ratio in leaves decreased in both cultivars by salinity increment. Malas was less affected by osmotic effects of NaCl, but it accumulated more Cl^? in its leaves. Thus, Malas might be more affected by negative effects of salinity.