Solid phase (9-fluorenylmethyloxycarbonyl) synthesis and characterization of [[1-13C]Phe11]gramicidin B1

The solid phase synthesis of [[1-¹³C]Phe¹¹]Gramicidin B was successfully achieved using the 9-Fluorenylmethyloxycarbonyl protecting group. There was at least a 30% drop in yield during the adition of the three valines in positions 8, 7 and 6, which is likely due to steric hindrance caused by the steric constraints of the valine side chains. Still, the overall yield of the peptide was comparable with that obtained using thetert-butyloxycarbonyl group for protection. The synthetic Gramicidin B was completely characterized by high-pressure liquid chromatography, circular dichroism, and¹³C nuclear magnetic resonance spectra. Also reported are the single-channel conductance properties, which compare favorably with those reported earlier and demonstrate the interesting multiplicity of conductance states to be distinquishable from that of Gramicidin A. Thus, these several useful characterizations have been carried out on the same preparation.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Mn²⁺ and Cu²⁺ to the cyclitol, cis-inositol. Resonance linewidths and the electron nuclear relaxation rates [(T₁^e)^?1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.     

Analysis of tert.-butyldimethylsilyl [1-C]palmitic acid in stool samples by gas chromatography–mass spectrometry with electron impact ionisation: comparison with combustion isotope-ratio mass spectrometry

The use of ¹³C-labelled compounds to study lipid metabolism is increasing. Typically less than 40% of the orally administered label is recovered in breath CO₂. The remainder must be either absorbed and not oxidised or not absorbed and remain in the faeces. Two methods of determining how much tracer passes through the body, and is present in the stool, were compared. Compound specific analysis of tert.-butyldimethylsilyl [¹³C]hexadecanoic acid by gas chromatography–mass spectrometry (GC–MS) with electron impact ionisation was compared with bulk analysis of whole stool and lipid extract by continuous flow isotope ratio mass spectrometry (CF–IRMS) with a combustion interface. The mean difference between the IRMS and GC–MS methods was −0.02 mmol ¹³C d⁻¹ with a mean excretion of 14.2 mmol ¹³C d⁻¹. Combustion IRMS is both simpler and cheaper, when the objective is to determine how much administered dose appears in stool, and information about the form of the label is not required.     

Comparison of the effects of calcium and magnesium on the conformation of tetra cycline in Me2SO solution

The effects of anhydrous Ca(NO₃)₂ on the ¹³C nuclear magnetic resonance (nmr) and circular dichroism spectra of tetracycline in Me₂SO-d₆ solution have been investigated in order to make a comparison with the results of a previous study in which the effects of Mg(NO₃)₂ were determined under the same conditions. The results of experiments described in this article provide strong evidence that Ca²⁺ and Mg²⁺ bind tetracycline at the same sites in Me₂SO and that both ions induce the same change in molecular conformation of tetracycline upon binding. The Ca²⁺ complex, in contrast to the Mg²⁺ complex, has a lifetime that is short on the nmr time scale.     

A nuclear magnetic resonance study of the d-[C6]glucose metabolism of Mesocestoides corti tetrathyridia in the absence and presence of monensin

Novak M. and Blackburn B. J. 1988. A nuclear magnetic resonance study of the d-[¹³C₆]glucose metabolism of Mesocestoides corti tetrathyridia in the absence and presence of monensin. International Journal for Parasitology18: 1029–1033. The effect of monensin on the glucose metabolism of Mesocestoides corti tetrathyridia was studied using ¹H and ¹³C nuclear magnetic resonance (n.m.r.) spectroscopy. Signals due to lactate, succinate, acetate and alanine were identified in the spectra of the excretory products of tetrathyridia fed d-[¹³C₆]glucose in vitro for 120 min. Monensin, at a concentration of 10 μm, inhibited glucose uptake across the brush border of the tetrathyridia, as indicated by a higher level of labelled hexose and lower levels of metabolic end products in ionophore-containing culture medium. The possible action of monensin on the glucose transport mechanism is discussed.     

Purification of gramicidin C.

Gramicidin C can be purified from a mixture of gramicidins by preparative thin-layer chromatography. A maximum R_f difference, 0.24, was attained on aluminum oxide plates. The antibiotic was identified by amino acid analysis, nuclear magnetic resonance spectroscopy, and uv absorbance spectroscopy.     

Conformation and activity in angiotensin II peptides

Angiotensin II and its competitive inhibitor [Sar¹, Ile⁸]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d₆-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.     

Stereochemistry of Gd3+ and Mn2+ interactions with D-gluconamide derivatives by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Gd³⁺ and Mn²⁺ to the polyol portion of several synthetic D-gluconamides. The results indicate that Gd³⁺ forms a single, unique binding structure requiring three oxygen atoms. The binding of Mn²⁺ to the polyol portion of these compounds appears to be nonspecific. The carbohydrate containing model compounds studied may be used to design new metal-ion chelating agents.     

Discontinuities in the Temperature Function of Transmembrane Water Transport in Chara: Relation to Ion Transport

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P _d ) were observed delimiting a region of low activation energy (E _a around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high E _a (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15–30°C and lipid matrix-mediated transport beyond this region. The K⁺ channel inhibitor, tetraethylammonium as well as the Cl⁻ channel inhibitor, ethacrynic acid, diminished P _d in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K⁺ transport in Chara, only increased P _d below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K⁺ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K⁺ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K⁺ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29°C. Received: 12 August 1998/Revised: 13 November 1998     

Synthesis of chiral N-protected amino acid esters by the use of UNCAs

Summary An easy synthesis of N-protected amino acid esters, including tert-butyl esters, is described by the use of urethane N-protected carboxyanhydrides (UNCAs). Treating UNCAs with tert-butanol in the presence of potassium bicarbonate at 45°C yielded the corresponding N-protected amino acid tert-butyl esters in a very simple way. Benzyloxycarbonyl and tert-butyloxycarbonyl N-protected amino acid tert-butyl esters have been obtained by this procedure. Once more, this reaction showed the great reactivity of UNCAs.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

Secondary structure of peptidese: 13. 15N and 13C n.m.r. spectroscopic investigation of the helix stability of poly(l-alanine) in acidic solutions

¹⁵N-enriched poly(l-alanines) of various molecular weights were prepared from $\text{l}\text{-alanine}\text{-N-}\text{carboxyanhydride}$ (l-Ala-NCA) and their helix/coil equilibrium in trifluoroacetic acid (TFA) investigated by means of 40.5 MHz ¹⁵N nuclear magneic resonance (n.m.r.), 22.3 MHz ¹³C n.m.r. and circular dichroism (c.d.) spectra. The ¹⁵N n.m.r. spectra exhibit at least three peaks, and the dependence of their intensities on molecular weight, molecular weight distribution and temperature, as well as dynamic nuclear Overhauser effect (NOE) measurements, indicate that the high-field peak represents the helix fraction. All three spectroscopic methods agree that a helix→coil transition takes place with decreasing concentration. Furthermore, poly(l-alanines) containing d-alanine or glycine in various mole ratios were synthesizsed by copolymerizations of N-carboxyanhydrides (NCAs). The ¹⁵N n.m.r. spetra demonstrate that one d-Ala unit per 100 l-Ala units suffices to affect significantly the helix/coil equilibrium in TFA. In other words, the helix content under equilibrium conditions is highly sensitive to racemization. Furthermore, ¹³ C n.m.r. cross-polarization/magic angle spinning (CP/MAS) spectra demonstrate that the presence of d-Ala units also affects the α-helix content in the solid state.     

Deuterium and carbon-13 tracer studies of ethanol metabolism in the rat by 2H, 1H-decoupled 13C nuclear magnetic resonance

[1-¹³C, 1,1-²H₂] ethanol and [2,2,2-²H₃] ethanol were administered to bile fistula rats. A new technique, ²H, ¹H-decoupled ¹³C nuclear magnetic resonance, was used in attempting to account for the distribution of the isotopic species along the steroid skeleton of 3–45 mg of isolated bile acids. The technique revealed ²H incorporation at many carbon sites unambiguously, but has limitations as a quantitative ²H assay at these levels of sample availability.     

Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 m in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from -helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

Decomposition and superoxide dismutase activity of the copper complex of D-penicillamine (Cu(II)6Cu(I)8 (D-penicillamine)12 Cl)5−

The possible time- and/or light-dependent decomposition of the purple Cu(I), Cu(II)-complex of D-penicillamine (Cu(II)₆Cu(I)₈(D-penicillamine)₁₂Cl)^5? was examined. Superoxide dismutase activity of the freshly prepared complex was assayed using the nitroblue tetrazolium assay. The formazan colour formation was inhibited by 50% in the presence of approximately 500 μM copper. Ageing of the copper complex, especially in the light, resulted in a marked increase of EDTA-sensitive activity. Upon gel chromatography of the aged samples the original low inhibitory activity was restored. All EDTA-sensitive inhibitory activity was found in a clearly separated low M_r copper-containing fraction. Aerobic irradiation with a tungsten lamp at 30 °C accelerated the decomposition of (Cu(II)₆Cu(I)₈(D-penicillamine)₁₂Cl)^5?. ?_Cu518 = 1800 M^?1 cm^?1 dropped to ?_Cu640 = 60 M^?1 cm^?1. The photochemical conversion of (Cu(II)_6? Cu(I)₈(D-penicillamine)₁₂Cl)^5? was complete within 48 h. Due to the identical electronic absorption profile of both, the decomposition product and Cu(II) D-penicillamine disulphide the latter complex was assigned to be the unknown low M_r copper-compound. Circular dichroism and electron paramagnetic resonance measurements support this conclusion.     

Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens

The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-¹⁴C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of ¹⁸O₂, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD circular dichroism - DMF N,N-dimethyl-formamide - GC/MS gas chromatography/mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - ODS octadecylsilane - PAH polycyclic aromatic hydrocarbon - TLC thin-layer chromatography - TMS tetramethylsilane - UV ultraviolet     

Cr6+对人工湿地薏苡光合特性和微量元素吸收的影响

为深入了解Cr⁶⁺胁迫对人工湿地植物薏苡光合特性和微量元素吸收的影响,该文通过构筑微型垂直流薏苡(Coix lacryma-jobi)人工湿地,采用1/2 Hoagland’s营养液配制的含0、5、20、40 mg·L^-1 Cr⁶⁺废水为灌溉用水,研究铬胁迫对人工湿地植物生长、光合特性、抗氧化酶活性和微量元素吸收等的影响。结果表明：(1)低浓度(5 mg·L^-1)Cr⁶⁺对薏苡的株高、茎粗和分蘖影响不显著,高浓度(20、40 mg·L^-1)Cr⁶⁺则显著抑制薏苡的生长。(2)低浓度Cr⁶⁺处理下,薏苡叶片净光合速率(P_n)、气孔导度(G_s)和蒸腾速率(T_r)有不同程度的提高,提高幅度分别为6.8%～54.8%、13.0%～40.3%和9.1%～36.4%。高浓度Cr⁶⁺处理下,叶片P_n、G_s     

Use of synthetic gramicidins in the determination of channel structure and mechanism

The syntheses of (1-13C) Trp9 gramicidin A (GA), (1-13C) Trp11 GA, (1-13C) Trp13 GA, (1-13C) Trp15 GA, and D . Leu2 GA are verified by means of high performance liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and characterization of transport properties. The use of these and other synthetic gramicidins is discussed in terms of determining ion binding sites within the channel, helix sense of the channel, the basis of monovalent vs divalent cation selectivity, and means of modulating channel conductance.     

Kinetic Studies of a (Na++ K++ Mg2+) ATPase in Sugar Beet Roots

Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na⁺ and K⁺ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na⁺ and K⁺ are suggested. 2) In the absence of K⁺, Na⁺ acts as an uncompetitive modifier raising the apparent K_m and V_max for MgATP. 3) In the absence of Na⁺, K⁺ activates non-competitively with respect to MgATP. Thus K⁺ increases V_max but does not affect the apparent affinity constant. 4) K⁺ and Na⁺ double the rate constants. 5) In the presence of Na⁺ or K⁺, Mg²⁺ in excess acts as a weak inhibitor to Na⁺ and/or K⁺ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.