Dissociation and reconstitution of membranes synthesizing the peptidoglycan of Escherichia coli. Lipid dependence of the synthetic enzymes

The peptidoglycan synthetic enzymes can be dissociated with cholate and LiCl into components with mobilities on a gel filtration column in the same ranges as bovine serum albumin. The active enzymes can be separated further from the lipids necessary for synthesis by precipitation with ammonium sulfate. The needed lipids stable to hydrolysis with base. A protein needed for peptidoglycan polymerization can be separated from the other synthetic enzymes by hydroxylapatite chromatography.     

Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin-layer chromatography on small plates followed by microchemical assays

Methods are described for the rapid separation of the major individual phospholipids and neutral lipids of tissues by thin-layer chromatography on small glass plates (75 × 75 mm), and for the specific microchemical estimation of separated lipids and for determination of fatty acid composition and radioactivity. The overall method, involving tissues extraction, thin-layer chromatographic separation and assay has been evaluated using pure standards and biological samples and gives good reproducibility and almost complete recovery of lipids.     

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well. An improved method of lipid extraction from chlorophyll-containing membranes is presented. This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction. The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography. The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography. The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis. None of the separated lipids alone was able to produce a well-coupled system. The best results were obtained with the native lipid mixture. The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol. Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included. Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C. The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.     

Characterization of Alkylgalactolipids from Calf Brain by High Performance Liquid Chromatography

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11 :9. After perbenzoylation. they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor components were oleyl. myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.     

Biological activity of lipids of pine pollen on platelet aggregation in correlation with the platelet activating factor

Pollen lipids of a pine species were separated by thin layer chromatography systems. The purified neutral and polar lipid classes were examined for their possible platelet aggregation activity and for their effect on Platelet Activating Factor activity. The lipid fraction comigrating on thin layer chromatography with glycerylether standards was shown to have a remarkable inhibition of Platelet Activating Factor activity on washed rabbit platelets in a concentration of 4.5.10(-6) M. At a ten fold higher concentration these lipids also induced platelet aggregation.     

Preparative scale separation of neutral lipids and phospholipids by centrifugally accelerated thin-layer chromatography

Mixtures of lipids and phospholipids were separated by centrifugally accelerated thin-layer chromatography on a preparative scale (300-500 mg lipid mixture per run). The isolated lipids and phospholipids were identified by 1H and 13C NMR spectroscopy and their fatty acid composition was determined by GLC and GLC-MS of their methyl esters.     

Turnover of brain mitochondrial membrane lipids

1. The turnover of lipids, of myelin and other brain subcellular particles has been studied in double-labelling experiments on intact rats. 2. Overall metabolism of brain mitochondrial lipids was three times slower than that of the liver. 3. Individual lipids of brain mitochondria and myelin were also separated and their metabolism was studied. 4. All myelin lipids examined undergo very slow turnover. Two pools of brain mitochondrial lipid were identified. The slowly metabolized lipids were cholesterol, cardiolipin plus phosphatidic acid and possibly sphingomyelin; the remaining phosphatides underwent more rapid turnover. 5. The possible significance of these results is discussed.     

Chromatographic separation of plasmalogenic, alkyl-acyl, and diacyl forms of ethanolamine glycerophosphatides

The plasmalogenic, alkyl-acyl, and diacyl forms of ethanolamine glycerophosphatides were completely separated from each other as methylated dinitrophenyl derivatives by thin-layer chromatography on Silica Gel G. The relatively high resolving power needed was obtained by multiple unidimensional development with solvents that give very low mobility to the lipids. Under these conditions the plasmalogens moved fastest, the alkyl-acyl lipids were intermediate, and the diacyl lipids were the slowest. The presence of all these forms of lipids in the ethanolamine phosphatides of hen's eggs, ox brain, and human blood plasma could be directly demonstrated with the new method.     

CBF 3 基因过量表达的拟南芥细胞质膜组分的变化

通过提取过量表达CBF3基因和对照的拟南芥[Arabidopsis thaliana L．Heyn．(Columbia)]茎叶的质膜．分离并分析其脂类成分和蛋白质含量,从中探讨CBF3对膜脂成分的影响及与抗冷适应的关系。研究结果表明,过量表达CBF3植株的质膜膜脂总量和膜蛋白总量分别是对照的227％和190％,磷脂为105％,与冷适应诱导的效果相似。因此CBF3表达的变化可能对冷适应过程中质膜组成的改变起重要作用。     

Study of Lipid Components of Lupinus mutabilis Meal and Isolates

Two types of protein isolates have been obtained from defatted Lupinus mutabilis meal. Both, isolate-Α and -B, were obtained by alkaline extraction with 0.2% NaOH, and 0.25% bisulfite, followed by precipitation at the isoelectric point (pH = 4.8). Total lipids were extracted with 86% ethanol, and neutral lipids were separated using a Florisil column. The lipids found in the isolates were similar to those found in the original meal, and the following types of compounds were separated, identified, and measured: hydrocarbons, waxes, methylesters, triglycerides, free fatty acids, diglycerides, and free sterols.     

Liquid chromatographic analysis of sebum lipids and other lipids of medical interest

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid classes present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography—mass spectrometry, is very easy.Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.     

The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant.

By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph chol-1 of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes. Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 (2-propyl-2,5,5-trimethyloxazolidine-N-oxyl), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium.     

Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography

Two one-dimensional systems for separation of glycolipids from total lipid extracts of tissues by thin-layer chromatography are described. System I used, as adsorbent, an alkaline mixture of silica gel without CaSO(4) binder (75%) and magnesium silicate (25%), and the lipids were "developed" with three successive solvent mixtures. The separated compounds (from the fastest to the slowest moving) were: ceramide, ceramide monohexosides, sulfatides, ceramide dihexosides, psychosine, ceramide trihexosides, and ceramide N-acetylhexosamine trihexosides. In system II a two-step development was used on an adsorbent consisting of silica gel without CaSO(4) binder (80%) and magnesium silicate (20%). The separated compounds were: ceramides, ceramide monohexosides, and ceramide dihexosides. Psychosine and sulfatides as well as ceramide trihexosides and ceramide N-acetylhexosamine trihexosides were not separated. In both systems all neutral lipids moved to the very top of the chromatogram and phospholipids stayed at the origin. Application of systems I and II for separation of glycolipids was demonstrated on total lipid extracts from animal tissues.     

Trypanosoma cruzi: incorporation of [3H]-palmitic acid and [3H]-galactose into components shed by trypomastigotes.

Trypomastigotes were metabolically labeled with [3H]-palmitic acid or [3H]-galactose and labeled components were detected in the culture medium. Thin layer chromatography of the shed material showed several lipids in the [3H]-palmitic acid labeled sample while the sugar was mainly incorporated into macromolecules. The material incorporated with the lipidic precursor was fractionated by DEAE-Sephadex (acetate form) and the amount of radioactivity was ten times higher in the acidic lipids than in the neutral lipids. When acidic lipids were further separated by Unisil, 73% of the radioactivity was recovered in the less polar fraction. Different patterns were obtained on comparison of the shed components with the lipids remaining in the parasite.     

CBF3基因过量表达的拟南芥细胞质膜组分的变化

通过提取过量表达CBF3基因和对照的拟南芥[Arabidopsis thaliana L.Heyn.(Columbia)]茎叶的质膜,分离并分析其脂类成分和蛋白质含量,从中探讨CBF3对膜脂成分的影响及与抗冷适应的关系。研究结果表明,过量表达CBF3植株的质膜膜脂总量和膜蛋白总量分别是对照的227%和190%,磷脂为105%,与冷适应诱导的效果相似。因此CBF3表达的变化可能对冷适应过程中质膜组成的改变起重要作用。     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

Improved separation and quantification of neutral and polar lipid classes by HPLC–ELSD using a monolithic silica phase: Application to exceptional marine lipids

An improved HPLC method is presented, which allows separation and quantification of a broad range of lipid classes of marine zooplankton with special regard to neutral lipids. Marine zooplankton species often produce high amounts of exceptional lipids, especially at high latitudes, in order to cope with the harsh environmental conditions and strong seasonality in food supply. Major neutral lipid classes are wax esters, triacylglycerols, diacylglycerol ethers, free fatty alcohols and sterols. Neutral and polar lipids were separated and identified on a monolithic silica column (Chromolith^®Performance-Si) using high performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). The method resolves a broad spectrum of lipids, varying in polarity from squalene to lysophosphatidylcholine in a single run. The total run time was 35 min including column re-equilibration. The calibration was made at levels of 0.1–60 μg lipid/injection, but a 10–15-fold greater amount can be injected if single lipid classes need to be separated, e.g. for further determination of individual fatty acids. The method was applied to representative Arctic zooplankton species (copepods, pteropods, euphausiids and ctenophores) that are known to biosynthesize in particular neutral lipids like diacylglycerol ethers and free fatty alcohols.     

Effects of Iron Deficiency on Chloroplast Lipids

The lipids of plastids from three different nodes of Hubbardsquash plants were investigated. A comparison was made of theplastid lipids of plants grown on a complete nutrien with theplastid lipids of plants grown on an iron-deficient nutrientsolution. The iron-deficient leaves were not chlorotic at the time ofharvest. The separated lipids were quantitated by determiningphosphorus, sugar, and fatty acid content. Plastids from olderleaves contained more lipid phosphorus per lipid sugar thanplastids from younger leaves. The plastids treated with an iron-deficientnutrient seemed to have relatively less glycerylphosphoryl glycerol-lipidsthan did the complete plastids. However, few major differencesin the fatty acid composition existed between the iron-deficientand complete grown plastids. It was concluded that the rateof turnover of the plastid lipids may depend upon the individualspecies and the environment to which the plants are subjected.Further changes in the plastid lipids of the iron-dificientplants, as opposed to complete nutrient treated, occurred priorto the appearance of marked visible symptoms of chlorosis.     

Phospholipids and lipid droplets

Lipid droplets are ubiquitous cellular organelles that allow cells to store large amounts of neutral lipids for membrane synthesis and energy supply in times of starvation. Compared to other cellular organelles, lipid droplets are structurally unique as they are made of a hydrophobic core of neutral lipids and are separated to the cytosol only by a surrounding phospholipid monolayer. This phospholipid monolayer consists of over a hundred different phospholipid molecular species of which phosphatidylcholine is the most abundant lipid class. However, lipid droplets lack some indispensable activities of the phosphatidylcholine biogenic pathways suggesting that they partially depend on other organelles for phosphatidylcholine synthesis.     

Polycations. 19. The synthesis of symmetrical dicationic lipids with internal dimethylazonia functionalities separated by a spacer unit and pendant chains

Herein is reported the preparation of several series of symmetrical polyammonium salts that serve as cationic lipids or precursors thereof, and are structurally based on several series of parent diamines where dimethylazonia functionalities are present, separated by a central structural unit, and pendant terminal chains. The resultant materials are of significant interest for a variety of purposes, such as serving as antihydrophobic species and as transfectins, the details of which are provided in separate reports. Attempts to effect selective alkylation to provide the corresponding unsymmetrical cationic lipids were without success, always leading to relatively useless mixtures of products.