Recent progress in polar metabolite quantification in plants using liquid chromatography-mass spectrometry

Metabolite analysis or metabolomics is an impor-tant component of systems biology in the post-genomic era. Although separate liquid chromatography (LC) methods for quantification of the major classes o...     

米曲霉氨基酰化酶的双水相萃取研究

本文利用聚乙二醇和磷酸盐组成的水溶液双相系统,从米曲霉(Aspergillus oryzae)中提取氨基酰化酶。通过实验对影响氨基酰化酶分配的各参数进行了研究,确定了最适体系:PEC-1540 10％(W/V),K_2HPO_418％(W/V),pH8.5;PEG-154010％(W/V),K_2HPO_412％(W/V),NaCl 0.5mol/L,pH8.5。二步萃取收率90％,纯化倍数9。为实验室和工业上采用双水相系统萃取氨基酰化酶提供了一个新方法。     

Amphotericin B Kills Unicellular Leishmanias by Forming Aqueous Pores Permeable to Small Cations and Anions

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3′ dipropylthiadicarbocyanine (DiSC₃(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations (≤0.1 μm) induced a polarization potential, indicating K⁺ leakage, but no movement of cations and anions was allowed until AmB concentrations greater than 0.1 μm were added. In agreement with these data, it was found that AmB altered the negative membrane potential held across LPs in a manner consistent with the differential cation/anion selectivity exhibited by the channels formed in liposomes. Thus, LPs suspended in an iso-osmotic sucrose solution did not exhibit any AmB-induced membrane depolarization effect brought about by efflux of anions until 0.1 μm or higher AmB concentrations were added. By contrast, LPs suspended in an iso-osmotic NaCl solution and exposed to 0.05 μm AmB exhibited a nearly total collapse of the negative membrane potential, indicating Na⁺ entry into the cells. The concentration dependence of the AmB-induced permeability to different salts was also measured across vesicles derived from the plasma membrane of leishmanias (LMVs), by using a rapid mixing technique. At concentrations above 0.1 μm, AmB induced the formation of aqueous pores across LMVs with a positive cooperativity, yielding Hill coefficients between 2 to 3. Measured anion selectivity across such aqueous pores followed the sequence: SCN > NO3 > Cl > I > Br > acetate (SO²⁻ ₄ being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (≤0.1 μm), AmB was found to be nonlethal against LPs but, above this concentration, leishmanias were rapidly killed. The rate and extent of such an effect were found to be dependent on the type of cation and anion present in the external aqueous solution. For both NH⁺ ₄ and Na⁺ salts, the measured rank order of AmB cell killing followed the same sequence that was determined for AmB-induced salt permeation across LMVs. Further, replacement of either extracellular Na⁺ by choline or Cl⁻ by SO²⁻ ₄, or its partial substitution by sucrose, in iso-osmotic conditions, led to a complete inhibition of the killing effect exerted by otherwise lethal AmB concentrations. Finally, it was shown that tetraethylammonium (TEA⁺), an organic cation that is known to block AmB-induced salt permeation across LMVs was able to retard the time lag observed for EB incorporation across LPs, indicating that this parameter can be taken to represent the time taken for salt accumulation inside the parasites. The present results thus indicate clearly that low AmB concentrations (≤0.1 μm) were able to form across LPs, cation channels that collapsed the parasite membrane potential but are not lytic. At high concentrations (<≥0.1 μm), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB. Received: 25 September 1995/Revised: 11 March 1996     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Fractionation of wheat proteins by counter-current distribution using aqueous two-phase systems containing propionic acid

Wheat proteins, soluble in diluted acid (glutenins), have been fractionated by counter-current distribution (CCD) using an aqueous two-phase system. The phase system is based on poly(ethylene glycol) and dextran but contains also 1% propionic acid and 6 mM magnesium sulfate. Approximately half of the bulk proteins partitioned to the upper phase while starch and other particles were recovered only into the lower phase. Whole wheat flour could be applied as sample for the CCD and 57 transfers were carried out. Starch and insoluble proteins remained stationary, while proteins followed the mobile phase to various degrees giving rise to a distribution pattern. The CCD pattern of the proteins showed distinct differences when various kinds of wheat flour were analysed. The patterns indicate that at least six subpopulations of proteins can be obtained by using two-phase extraction.     

Radiolysis of aqueous solutions of hydrogen cyanide (pH∼6): Compounds of interest in chemical evolution studies

Summary Oxygen-free aqueous solutions of hydrogen cyanide, 0.1 M and pH∼6, were exposed to gamma rays from a⁶⁰Co source, the mixture of nonvolatile products was fractionated and the fractions were analysed. It has been found that the complex mixture contains oligomers and polymers with molecular weights up to 20,000 daltons, mainly polyamides with urea and peptidic fragments. Among the constituents are carbamyl glycinonitrile and carbamyl glycinamide that represent 6.4% and 3.1% of the total of unfractionated material respectively. Urea content is 2.6%, but the derivatives of urea are more abundant. Acid hydrolysis releases several amino acids. Glycine is the most abundant (75% or more of total amino acid content), and its concentration considerably increases in some fractions when the hydrolysis is carried out at 130°C. The role of free radicals in reactions leading to the formations of radiolytic products is considered. Some comparisons are made between findings in the present work, at initial pH∼6, and an earlier study of ammonium cyanide at pH 9.     

Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation

The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH₄CN (pH 9) and HCN (pH 6), after their exposure to rays from a⁶⁰Co source (15–20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.     

The radiolysis of aqueous propionitrile: Compounds of interest to chemical evolution studies

Summary Oxygen-free aqueous solution of CH₃CH₂CN (0.1 M, pH 6) were exposed to gamma rays from a⁶⁰Co source and the mixture of radiolytic products fractionated. The separated fractions were analysed by magnetic resoncance methods (EPR, NMR), spectrophotometry (UV-VIS-IR), gas chromatography and amino acids analysis. About 70% of radiolytic products consist of non volatile material. A large variety of compounds was detected: propionaldehyde, acetaldeyde, sixteen carboxylic acids and, in the hydrolysate, eleven protein and nonprotein amino acids. About 20% of the nonvolatile radiolytic products have a hydrophobic character and can be extracted with chloroform. Among them is a long-living nitroxide free radical which is stable for months at room temperature. It has been suggested that the nitroso compounds are formed as radiolytic products, and that they act as spin-traps by converting some of the short living radicals to the observed nitroxide radical. This and other experimental findings are discussed in the light of free radical reactions induced by ionizing radiation.     

Molecular mobilities and the lowered osmolality of the chromaffin granule aqueous phase

Carbon-13 spin-lattice relaxation times, T₁, have been measured in whole adrenal medullary tissue slices, in suspensions of isolated chromaffin granules, in the reconcentrated chromaffin granule lysate, and in various model solutions containing catecholamines, ATP, chromogranins and Ca²⁺. Reorientational correlation times have been calculated at 10°C using T₁ data and nuclear Overhauser enhancemments for protonated carbons on both catecholamines and nucleotides. Correlation times in all media are relatively short and characteristic of highly fluid aqueous phases. Adrenalin and ATP exhibit substantial differences in correlation times in all media, however, the ratio γ_R(ATP):γ_R(catecholamine) ranging from 2.4 in simple 3:1 adrenalin-ATP solutions to 4 in intact chromaffin granules. This difference, as well as the relatively high absolute reorientational mobilities of both components, confirms the importance of labile ionic interactions between ATP and catecholamines, but rules out the presence of high concentrations of base-stacked structures. Participation of the chromogranins in ternary complexes with catecholamines and ATP appears to be of minor importance. Ionic interactions to the protein are not reflected in either ¹³C T₁ values or chemical shifts of arginine or glutamate sidechain resonances, or in the ¹³C chemical shifts of ATP or catecholamines. Very labile protein-ATP binding appears to be reflected in the correlation time measurements, however, which show selective immobilization of ATP relative to catecholamine in the presence of soluble protein. Osmotic measurements indicate that solutions containing adrenaline, ATP and Ca²⁺ are highly nonideal, but probably not sufficiently so to account fully for the osmotic stabilization of the chromaffin granule aqueous phase. Even in the absence of specific intermolecular complexation, the chromogranins, through their polyelectrolyte properties, exert a significant influence on the intragranular osmolality. The osmotic lowering due to polyion-counterion interactions has been estimated semiquantitatively using a theory developed by Oosawa.     

Vibrational circular dichroism of carbohydrate films formed from aqueous solutions

Vibrational circular dichroism (VCD) spectra in the entire 2000-900 cm(-1) region have been recorded, for the first time, for films of carbohydrates prepared from aqueous solutions. Eight different carbohydrates, alpha-D-glucopyranosyl-(1-->4)-D-glucose, cyclomaltohexaose, alpha-D-glucopyranosyl alpha-D-glucopyranoside, beta-D-glucopyranosyl-(1-->6)-D-glucose, beta-D-glucopyranosyl-(1-->4)-D-glucose, D-glucose, and both enantiomers of 6-deoxygalactose and of allose, were investigated. The VCD spectra obtained for films are found to be identical to the corresponding spectra obtained for aqueous solutions of carbohydrates. These measurements demonstrate several advantages of significant importance. The strong infrared absorption of water has prevented, in the past, the pursuit for routine applications of VCD in determining the structures of carbohydrates in aqueous solutions. This limitation is not present for film studies because water solvent is removed in the process of preparing the films. Also, strong infrared absorption of water at 1650 cm(-1) requires the use of very short-pathlength (6 microm) cells for measurements on aqueous solutions. This requirement and concomitant inconveniences (such as laborious assembling of a demountable liquid cell or purchasing an expensive variable pathlength liquid cell) have been eliminated for film measurements. The removal of interfering water absorption in film studies resulted in higher light throughput and better signal-to-noise ratios for VCD measurements. Another point of significance is that the amount of carbohydrate sample required for VCD measurements on films is approximately one to two orders of magnitude smaller than that required for corresponding VCD measurements on aqueous solutions. Since carbohydrate samples can now be studied as films, VCD spectroscopy becomes much more broadly applicable for carbohydrates than previously believed. The present work, in combination with other film measurements in our laboratory, indicate that VCD studies on films can be used more generally, providing a convenient and powerful approach for probing structural information for biologically important compounds.