Quantitative retention of membrane lipids in the freeze-fracture replica

SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.     

Comparison of two methods for the separation of polar from nonpolar lipids

The countercurrent extraction method of Galanos and Kapoulas for the separation of phospholipids from triglycerides was compared with dialysis in petroleum ether in an apparatus modified from that of Eberhagen and Betzing. The efficiencies of the methods are similar, but dialysis, which is slower, can yield triglycerides that are entirely free from phosphorus. The behavior of free fatty acids and monoglycerides is also described.     

Two-dimensional chromatography on silica gel-loaded paper for the microanalysis of polar lipids

Two-dimensional chromatography on commercially available silica gel-loaded paper for the microanalysis of polar lipids from various tissues is described. All common phospholipids and their lyso derivatives can be reproducibly separated. As many as 22 lipid components were separated on a single chromatogram. Improved methods for staining lipids and for determining phosphorus in the chromatographic spots are reported.     

A phosphoethanolamine-modified glycosyl diradylglycerol in the polar lipids of Clostridium tetani

The polar lipids of the anaerobic bacterium Clostridium tetani, the causative agent of tetanus, have been examined by two-dimensional thin layer chromatography, ESI mass spectrometry, and NMR spectroscopy. Plasmalogen and di- and tetra-acylated species of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and N-acetylglucosaminyl diradylglycerol were the major lipids present in most strains examined except for strain ATCC 10779, the parent of strain E88, the first C. tetani strain to have its genome sequenced. This strain contained the same di- and tetra-acylated species but did not contain plasmalogens. All strains contained a novel derivative of N-acetylglucosaminyl diradylglycerol in which a phosphoethanolamine unit is attached to the 6’-position of the sugar, as judged by selective ³¹P-decoupled, ¹H-detected NMR difference spectroscopy. The N-acetylglucosamine (GlcNAc) residue is presumably linked to the 3-positon of the diradylglycerol moiety, and it has the β-anomeric configuration. Very little plasmalogen component was detected by mass spectrometry in the precursors phosphatidic acid and phosphatidylserine, consistent with the idea that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid assembly in anaerobic clostridia.     

Tetraether type polar lipids increase after logarithmic growth phase of Methanobacterium thermoautotrophicum in compensation for the decrease of diether lipids

Abstract The ratios of tetraether to diether type lipids in the total lipid during cell growth in batch cultures of Methanobacterium thermoautotrophicum ΔH (DSM 1053) were examined. The proportion of tetraether type lipids to the total lipid was about 80% during the log phase, and at the onset of the transient phase it began to rise up to about 93%. It was kept almost constant at that level throughout the stationary phase. The polar lipid composition changed with the age of the cell culture. The proportions of all the diether type polar lipids were lower and the levels of all tetraether type polar lipids were higher in the stationary phase than in the log phase. On the other hand, the composition of polar head groups, irrespective of the core lipids, was nearly constant in both growth phases measured so far despite the change in core lipid composition.     

An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids

A simple small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids from bacterial cells was developed. Extraction with a biphasic mixture of petroleum ether (b.p. 60–80°C) and methanolic saline gave an upper phase containing isoprenoid quinones. The lower phase, containing the partially extracted organisms, was processed according to the Bligh and Dyer extraction method to give a polar lipid extract. As examples of the procedure, the isoprenoid quinones and polar lipids of Bacillus subtilis, Mycobacterium avium, Pseudomonas diminuta and Streptomyces griseus were extracted and analyzed.     

The effect of the polar moiety of lipids on the ion permeability of bilayer membranes

Summary Bilayer membranes were prepared with the negatively charged lipids phosphatidylglycerol and diphosphatidylglycerol, the positively charged lipid lysyl phosphatidylglycerol, the zwitterionic lipid phosphatidylethanolamine, and an uncharged glycolipid, diglucosyldiglyceride, all isolated from gram-positive bacteria. Bilayer membranes of all these lipids manifested specific resistances of 10⁷ to 10⁹ cm² and capacitances of 0.3 to 0.4 F cm^–2. The membrane potentials of these bilayers were measured as a function of the sodium chloride, potassium chloride, and hydrogen chloride transmembrane concentration gradients (0.01 to 0.10m) and were found to be linear with the logarithm of the salt activity gradients. Membranes made from lysyl phosphatidylglycerol (one net positive charge) were almost completely chloride selective, whereas membranes from phosphatidylglycerol and diphosphatidylglycerol (one and two net negative charges, respectively) were highly cation selective. Membranes prepared with either diglucosyldiglyceride or phosphatidylethanolamine showed only slight cation selectivity. These findings indicate that the charge on the polar head group of membrane lipids plays an important role in controlling the ion-selective permeability of the bilayer.     

The influence of carotenoids on the oxidative stability and phase behaviour of plant polar lipids

Carotenoids and total neutral lipids from thylakoids of Nerium oleander were evaluated as antioxidants in liposomes prepared from soybean polar lipids. The extent of lipid oxidation was assessed from the formation of malondialdehyde and conjugated dienes after exposure of the liposomes to free radicals generated by ⁶⁰Co gamma radiolysis. The carotenoids incorporated into the bilayers were isolated from clones of oleander grown at 20° or 45°, growth conditions which are known to result in a difference in the thermal properties of the membrane lipids. The effect of carotenoids on the temperature of the phase transition of thylakoid polar lipids was also examined. The results showed that, in comparison with the effectiveness of a reference antioxidant, α-tocopherol, the carotenoids and total neutral lipids from thylakoids of oleander did not protect the soybean polar lipids from oxidation, nor did they influence the temperature of the phase transition of thylakoid polar lipids.     

Freshwater diatoms as a source of lipids for biofuels

Until recently, biodiesel production has been derived from terrestrial plants such as soybean and canola, leading to competition between biodiesel production and agricultural production for source materials. Microalgae have the potential to synthesize 30 times more oil per hectare than terrestrial plants without competing for agricultural land. We examined four genera (Cyclotella, Aulacoseira, Fragilaria, Synedra) of common freshwater diatoms (Bacillariophyceae) for growth and lipid content in defined medium (sD11) that replicates hypereutrophic conditions in lakes and wastewater treatment plant effluents and optimized the medium for silicon content. Cyclotella and Aulacoseira produced the highest levels of total lipids, 60 and 43 μg total lipids/ml, respectively. Both diatoms are rich in fatty acids C14, C16, C16:1, C16:2,7,10, and C22:5n3. Of the diatoms examined, Cyclotella reached the highest population density (>2.5 × 10⁶ cells/ml) in stationary phase when many of the cells appeared to be filled entirely with oil. Silicon enrichment studies indicated that for optimal utilization of phosphorus and nitrogen by diatoms growing in wastewater effluent, the amount of silicon present or added to the effluent should be 17.5 times the mass of phosphorus in the effluent. With high growth rates, high lipid contents, and rapid settling rates, Cyclotella and Aulacoseira are candidates for biodiesel production.     

Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (±1°C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45°C, the transition was at 7°C while for plants from the same clone, grown at 20°C, it was at −2°C. For mung bean and tomato the transition was between 9 and 12°C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.     

A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids

Lipid extraction using a monophasic chloroform/methanol/water mixture, coupled with functional group selective derivatization and direct infusion nano-ESI-high-resolution/accurate MS, is shown to facilitate the simultaneous analysis of both highly polar and nonpolar lipids from a single retina lipid extract, including low abundance highly polar ganglioside lipids, nonpolar sphingolipids, and abundant glycerophospholipids. Quantitative comparison showed that the monophasic lipid extraction method yielded similar lipid distributions to those obtained from established “gold standard” biphasic lipid extraction methods known to enrich for either highly polar gangliosides or nonpolar lipids, respectively, with only modest relative ion suppression effects. This improved lipid extraction and analysis strategy therefore enables detailed lipidome analyses of lipid species across a broad range of polarities and abundances, from minimal amounts of biological samples and without need for multiple lipid class-specific extractions or chromatographic separation prior to analysis.     

The metabolism of neutral and polar lipids in eleuthero-embryos and starving larvae of the African catfish Clarias gariepinw

Lipid class analysis was carried out on developing eggs, eleuthero-embryos (yolk sac larvae) and starving larvae of the freshwater species Clarias gariepinus , using thin layer chromatography. Samples were taken at fixed intervals from a large pool of fertilized eggs obtained through induced reproduction of several parent fish. The total lipid content of fertilized eggs fluctuated around 22% of the dry weight and decreased from 21% at hatching to about 12–5% at yolk absorption. In starving larvae, the amount of total lipid per individual remained relatively constant. Polar lipids [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] together accounted for 73·6 to 80% of total lipid. PC was by far the most abundant lipid class during the entire experimental period (70–75% of total lipid). PC was catabolized proportionally to total lipid, demonstrating its role as the main energy supplier. All yolk PE was converted to body tissue. The neutral Hpids consisted of triglycerides (TAG), cholesterol and cholesteryl esters (respectively 12·5, 10 and 3% of total lipid in newly fertilized eggs). All TAG were depleted before complete yolk absorption.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

Nuclear retention of RNA as a mechanism for localization.

Two mutant RNAs, one derived from tRNA(imet), the second from U1 snRNA, that are defective in export from the nucleus to the cytoplasm have been studied. In both cases, the RNAs are shown to be transport competent but prevented from leaving the nucleus by interaction with saturable binding sites. This contradicts previous hypotheses to explain the behavior of the tRNA mutant, and highlights a general problem in using mutant RNAs to study nuclear export. In the case of these mutants, it is argued that nuclear retention is likely to be artifactual. However, the additional example of U6 snRNA is described. In this case, nuclear retention appears to be a physiological mechanism by which intranuclear localization is achieved. Evidence that the site of interaction with the La protein in U6 snRNA is important for its nuclear retention is presented.