Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.     

An efficient and highly selective deprotection of N-Fmoc-alpha-amino acid and lipophilic N-Fmoc-dipeptide methyl esters with aluminium trichloride and N,N-dimethylaniline.

A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N-dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N-Fmoc-protected amino acid methyl esters and N-Fmoc-protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.     

Kinetics of receptor modification. The multiply methylated aspartate receptors involved in bacterial chemotaxis

A method for determining the extent of methyl esterification of each of the four potential sites on the aspartate receptors involved in chemotaxis in Escherichia coli and Salmonella typhimurium is presented. In this procedure, radioactive methyl esters are incorporated into the receptors, the receptors are cleaved by trypsin and the V8 protease from Staphylococcus aureus, and the four fragments containing sites of methylation are separated by high performance liquid chromatography. Using this technique, we find that the rate of methyl esterification increases at all four sites after stimulation with the "attractant" aspartate, suggesting that all four sites of modification are involved in adaptation to aspartate. We also find that the rate of methyl esterification at each site is correlated with the homology between the protein sequence at that site and the "consensus" sequence, Glu-Glu-X-X-Ala-Thr/Ser.     

Quantitative release of fatty acids from lipids by a simple hydrolysis procedure

Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCl in acetonitrile-water 9:1 (by vol) for 45 min at 100 degrees C or 4 hr at 70 degrees C. After hydrolysis, free fatty acids (FFA) are recovered in chloroform and separated by thin-layer chromatography. More than 95% of the radioactivity from labeled phospholipids is recovered as FFA, and more than 97% of the lipid phosphorus is recovered as water-soluble phosphate. The yields of FFA, including polyunsaturated acids, after hydrolysis are as good as or better than those obtained for methyl esters using methanolysis catalyzed by acid, alkali, or BF3. High recoveries of FFA from glycerophospholipids, sphingomyelin, and neutral glycerides are attained. The procedure is quantitative, simple, inexpensive, and produces no methyl esters as secondary products.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

A column procedure for the esterification of organic acids with diazomethane at the microgram level

A simple procedure is described in which micro- and submicrogram amounts of organic acids can be converted to methyl esters with diazomethane. The gas, generated in only micromole amounts, contacts the acids which have been applied to a column of Celite contained in a capillary. The resultant esters are then eluted for chromatographic analysis. The entire procedure can be done in less than 5 min.     

Cell cycle-dependent methyl esterification of lamin B

Previous work from this laboratory has shown that approximately 24 proteins are reversibly modified by methyl esterification in a mouse lymphoma cell line. Here, we analyze several mouse tissues as well as other mouse, hamster, and human cell lines and find that many protein-methyl esters are ubiquitous while others show apparent tissue specificity. One of the modified proteins is identified by cellular localization and immunological detection as lamin B, a nuclear envelope structural protein which undergoes depolymerization during mitosis. The average stoichiometry of methylation is at least 0.5 methyl groups per lamin B molecule as determined by radioactive incorporation. By immunoblotting, however, demethylation appears to result in a gain of two negative charges suggesting the loss of two neutral methyl esters producing two carboxylic acid groups per molecule. By comparing mitotic and interphase cells, lamin B is found to be demethylated in mitosis while most other methyl esterified proteins show no appreciable cell cycle dependence. In addition to the correlation with cell cycle, it is shown that lamin B does not incorporate radioactive methyl esters in intact mouse brain tissue yet can do so if the cells are lysed. Analysis of lamin B charge by immunoblotting after isoelectric focusing indicates that this protein is fully methylated in brain suggesting that turnover of methyl groups in intact brain tissue is inhibited. We propose that methylation of lamin B may be involved in the control of disassembly and reassembly of the nuclear envelope during mitosis. If this were the case, the apparent lack of methyl group turnover in brain would be consistent with the inability of those cells to divide.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry

Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.     

In Vacuo Esterification of Carboxyl Groups in Lyophilized Proteins

A new method is described for the esterification of carboxyl groups in proteins by reaction of the lyophilized protein in vacuo with gaseous alcohol and HCI catalyst. Carboxyl groups are rapidly esterified with no protein degradation. 13C-Methyl or 13C-ethyl esters of the alpha-, gamma- and delta-carboxyl groups could be distinguished by the distinct chemical shifts of their resonances. Within the class of gamma- or delta-esters, the chemical shifts have little variation; however, the chemical shift of a C-terminal esterified alpha-carboxyl group shows a strong dependence on the nature of the C-terminal amino acid and sequence. Iodomethane reacts with deprotonated carboxyl groups in lyophilized proteins to form methyl esters, but unlike the reaction with gaseous methanol/HCI, it does not selectively methylate carboxyl groups. The procedure permits the cost-effective incorporation of isotopic labels and provides a new approach using 13C-NMR spectroscopy for determining the number of different C-termini present in a protein preparation.     

Gas--liquid chromatography of silylated mixtures from lipolysis of triglycerides containing unusual fatty acyl groups

A convenient and rapid procedure involving methylation, silylation, and temperature-programmed gas-liquid chromatography (GLC) is described for analyzing unfractionated products from the hydrolysis of triglycerides with pancreatic lipase. The conditions employed for GLC were selected to provide maximum and rapid separation of silylated monoglycerides in which the acyl moieties differ in chain length or degree of substitution with oxygen-containing functional groups. Derivatives differing only in the number of double bonds present were not separated. In the GLC curves obtained, peaks representing methyl esters are generally readily distinguishable from those of other components. Therefore, the extent of lipolysis and the composition (with respect to chain length and substitution of acyl groups) of monoglycerides formed are determined simultaneously. The accuracy of the new method was demonstrated with standard mixtures and by comparison of results for several lipolysis products with data obtained by conventional procedures.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

Synthesis and characterization of long-chain 1,2-dioxo compounds

A series of long-chain methyl esters with vicinal oxo groups (1,2-diones; 1,2-diketones) were synthesized by potassium permanganate-based oxidation of methyl esters of mono-unsaturated fatty acids. The presence of two additional carbonyl groups may facilitate the synthesis of other derivatives. The starting materials were selected in such a fashion to give the 1,2-dioxo moiety in consecutive positions from the methyl ester group. The compounds were characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. In mass spectrometry, both electron and chemical ionization (methane as reagent gas) were investigated. The position of the dioxo moiety can be determined in both ionization modes, however, in electron ionization mode the corresponding fragment ions are considerably stronger. In electron ionization mode, a fragmentation mechanism depending on the position of the 1,2-dioxo moiety occurs while the spectra derived from chemical ionization mode are mainly characterized by peaks around the molecular ion with both ionization modes appearing suitable.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Demethylation of protein carboxyl methyl esters: a nonenzymatic process in human erythrocytes?

We have compared the demethylation rate of protein carboxyl methyl esters from isolated human erythrocyte membranes with the corresponding rate of metabolic turnover of these same methyl groups in the intact erythrocyte. Surprisingly, the apparent spontaneous demethylation of these membrane protein methyl esters was significantly faster at physiological pH than the corresponding rate determined by pulse-chase analysis of intact cells incubated with L-[methyl-3H]methionine. Readdition of erythrocyte lysate to purified membranes did not increase the rate of demethylation, as might be expected if there were cytosolic or membrane-bound protein methylesterase activity, but resulted instead in an apparent stabilization of these methyl esters. Thus, the metabolic lability of these protein methyl esters in intact cells may be quantitatively explained by spontaneous, rather than enzymatic, demethylation reactions. A model is presented in which a rapid but nonenzymatic intramolecular demethylation reaction results in the formation of a polypeptide imide or anhydride intermediate. The metabolic fate of these hypothetical intermediates is unknown but may lead to the repair or degradation of protein D-aspartyl and L-isoaspartyl residues, which appear to be the substrates for the initial transmethylation reaction.     

Design and development of chiral reagents for the chromatographic e.e. determination of chiral alcohols

(S)-Trolox methyl ether is known as a powerful chiral reagent for the e.e. determination of chiral alcohols by separation of the corresponding diastereoisomeric esters on achiral GC and SFC columns. In order to further improve his methodology, five possible candidates resultings from variation of structural elements of parent reagent have been tested for derivatization with selected alcohols and subsequent analysis of the diastereoisomeric pairs of esters. The results of this optimization procedure showing the ways to new potent reagents are discussed. © 1996 Wiley-Liss, Inc.     

Nucleophilic addition of glycine esters to Gd@C82

The first investigation of nucleophilic addition of glycine esters to Gd@C₈₂ is reported and hydroxyl was found to compete with glycine esters in the reaction. The multiple adducts containing either mere glycine esters or both glycine esters and hydroxyl group were identified by MALDI TOF mass spectrometry and characterized with UV-Vis-NIR spectrometry. Because glycine ethyl ester is more soluble than glycine methyl ester in alcohol and toluene, as many as eight glycine ethyl ester groups can be added to the metallofullerene cage while the maximal number for glycine methyl ester groups is only four.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique

By condensing 3 alpha,21-dihydroxy-5 beta-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate in the Koenigs-Knorr reaction beta-D-glucosiduronates 10, 4, and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12, 6 and 8. Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3 alpha,21-dihydroxy-5 beta-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones (14, 20 and 26). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11 beta-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18, 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.