Multiple Interactions of Glucose with the Extra-Membranous Loops of GLUT1 Aid Transport.

Molecular dynamics simulations amounting to ≈8 µs demonstrate that the glucose transporter GLUT1 undergoes structural fluctuations mediated by the fluidity of the lipid bilayer and the proximity to glucose. The fluctuations of GLUT1 increase as the glucose concentration is raised. These fluctuations are more pronounced when the lipid bilayer is in the fluid compared to the gel phase. Glucose interactions are confined to the extra-membranous residues when the lipid is in the gel phase but diffuses into the transmembrane regions in the fluid phase. Proximity of glucose to GLUT1 causes asynchronous expansions of key bottlenecks at the internal and external openings of the central pore. This is accomplished only by small conformational changes at the single residue level that lower the resistance to glucose movements, thereby permitting unsteered glucose and water movements along the entire length of the pore. When glucose is near salt bridges located at the external and internal openings of the central pore, the distance separating the polar amino acid residues guarding these apertures tends to increase in both fluid and gel phases. It is evident that the multiplicity of glucose interactions, obtained with high concentrations, amplifies the structural fluctuations in GLUT1. The findings that most of the salt bridges and the bottlenecks appear to be operated by glucose proximity suggest that the main triggers to activation of transport are located within the solvent accessible linker regions in the extramembranous zones.

Two Coexisting Membrane Structures Are Defined by Lateral and Transbilayer Interactions between Sphingomyelin and Cholesterol.

The structure of fully hydrated bilayers composed of equimolar proportions of palmitoylsphingomyelin (PSM) and cholesterol has been examined by synchrotron X-ray powder diffraction and atomistic molecular dynamics (MD) simulations. Two coexisting bilayer structures, which are distinguished by the transbilayer phosphate-phosphate distance of coupled PSM molecules, are observed by diffraction at 37 °C. The MD simulations reveal that PSM molecules in the thicker membrane are characterized by more ordered, more extended, and less interdigitated hydrocarbon tails compared to those in the thinner membrane. Intermolecular hydrogen bonds further distinguish the two bilayer structures, and we observe the disruption of a sphingomyelin intermolecular hydrogen bond network induced by the proximity of cholesterol. Through an unsupervised clustering of interatomic distances, we show for the first time that the asymmetry of phospholipids is important in driving their interactions with cholesterol. We identify four distinct modes of interaction, two of which lead to the dehydration of cholesterol. These two modes of interaction provide the first description of precise physical mechanisms underlying the umbrella model, which itself explains how phospholipids may shield cholesterol from water. The most dehydrating mode of interaction is particular to the N-acylated fatty acid moiety of PSM and thus may explain the long-held observation that cholesterol preferentially mixes with sphingomyelins over glycerophospholipids.

Production of L-valine from metabolically engineered Corynebacterium glutamicum.

L-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism; therefore, it is widely added in the products of food, medicine, and feed. L-Valine is predominantly produced through microbial fermentation, and the production efficiency largely depends on the quality of microorganisms. In recent years, continuing efforts have been made in revealing the mechanisms and regulation of L-valine biosynthesis in Corynebacterium glutamicum, the most utilitarian bacterium for amino acid production. Metabolic engineering based on the metabolic biosynthesis and regulation of L-valine provides an effective alternative to the traditional breeding for strain development. Industrially competitive L-valine-producing C. glutamicum strains have been constructed by genetically defined metabolic engineering. This article reviews the global metabolic and regulatory networks responsible for L-valine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in C. glutamicum strain engineering.

Membrane Phase-Dependent Occlusion of Intramolecular GLUT1 Cavities Demonstrated by Simulations.

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.

Sphingolipid symmetry governs membrane lipid raft structure.

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37°C in mixtures containing up to 20mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.

Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers.

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20-27 Šthick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Stabilization of distearoylphosphatidylcholine lamellar phases in propylene glycol using cholesterol.

Phospholipid vesicles (liposomes) formed in pharmaceutically acceptable nonaqueous polar solvents such as propylene glycol are of interest in drug delivery because of their ability to improve the bioavailability of drugs with poor aqueous solubility. We have demonstrated a stabilizing effect of cholesterol on lamellar phases formed by dispersion of distearoylphosphatidylcholine (DSPC) in water/propylene glycol (PG) solutions with glycol concentrations ranging from 0 to 100%. The stability of the dispersions was assessed by determining the effect of propylene glycol concentration on structural parameters of the lamellar phases using a complementary combination of X-ray and neutron scattering techniques at 25 °C and in the case of X-ray scattering at 65 °C. Significantly, although stable lamellar phases (and liposomes) were formed in all PG solutions at 25 °C, the association of the glycol with the liposomes' lamellar structures led to the formation of interdigitated phases, which were not thermostable at 65 °C. With the addition of equimolar quantities of cholesterol to the dispersions of DSPC, stable lamellar dispersions (and indeed liposomes) were formed in all propylene glycol solutions at 25 °C, with the significant lateral phase separation of the bilayer components only detectable in propylene glycol concentrations above 60% (w/w). We propose that the stability of lamellar phases of the cholesterol-containing liposomes formed in propylene glycol concentrations of up to 60% (w/w) represent potentially very valuable drug delivery vehicles for a variety of routes of administration.

Structure of sphingomyelin bilayers and complexes with cholesterol forming membrane rafts.

Sphingomyelin and cholesterol are of interest to biologists because they interact to form condensed structures said to be responsible for a variety of functions that membranes perform. Synchrotron X-ray diffraction methods have been used to investigate the structure of bilayers of D-erythro palmitoyl-sphingomyelin and complexes formed by palmitoyl- and egg-sphingomyelin with cholesterol in aqueous multibilayer dispersions. D-erythro palmitoyl sphingomyelin bilayers exist in two conformers that are distinguished by their lamellar repeat spacing, bilayer thickness, and polar group hydration. The distinction is attributed to hydrogen bonding to water or to intermolecular hydrogen bonds that are disrupted by the formation of ripple structure. The coexisting bilayer structures of pure palmitoyl sphingomyelin are observed in the presence of cholesterol-rich bilayers that are characterized by different bilayer parameters. The presence of cholesterol preferentially affects the conformer of D-erythro sphingomyelin with thicker, more hydrated bilayers. Coexisting bilayers of sphingomyelin and complexes with cholesterol are in register and remain coupled at temperatures at least up to 50 °C. Cholesterol forms a complex of 1.8 mols of sphingomyelin per cholesterol at 37 °C that coexists with bilayers of pure sphingomyelin up to 50 °C. Redistribution of the two lipids takes place on cooling below the fluid- to gel-phase transition temperature, resulting in the withdrawal of sphingomyelin into gel phase and the formation of coexisting bilayers of equimolar proportions of the two lipids. Cholesterol-rich bilayers fit a stripe model at temperatures less than 37 °C characterized by alternating rows of sphingomyelin and cholesterol molecules. A quasicrystalline array models the arrangement at higher temperatures in which each cholesterol molecule is surrounded by seven hydrocarbon chains, each of which is in contact with two cholesterol molecules. The thickness of bilayer complexes of sphingomyelin and cholesterol is less than that of coexisting bilayers of pure sphingomyelin. The implications for protein sorting theories based on bilayer thickness are discussed.

Microbial metabolic engineering for L-threonine production.

L-threonine, one of the three major amino acids produced throughout the world, has a wide application in industry, as an additive or as a precursor for the biosynthesis of other chemicals. It is predominantly produced through microbial fermentation the efficiency of which largely depends on the quality of strains. Metabolic engineering based on a cogent understanding of the metabolic pathways of L-threonine biosynthesis and regulation provides an effective alternative to the traditional breeding for strain development. Continuing efforts have been made in revealing the mechanisms and regulation of L-threonine producing strains, as well as in metabolic engineering of suitable organisms whereby genetically-defined, industrially competitive L-threonine producing strains have been successfully constructed. This review focuses on the global metabolic and regulatory networks responsible for L-threonine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in strain engineering.

Genetic modification and bioprocess optimization for S-Adenosyl-L-methionine biosynthesis.

S-Adenosyl-L-methionine is an important bioactive sulfur-containing amino acid. Large scale preparation of the amino acid is of great significance. S-Adenosyl-L-methionine can be synthesized from L-methionine and adenosine triphosphate in a reaction catalyzed by methionine adenosyltransferase. In order to enhance S-adenosyl-L-methionine biosynthesis by industrial microbial strains, various strategies have been employed to optimize the process. Genetic manipulation has largely focused on enhancement of expression and activity of methionine adenosyltransferase. This has included its overexpression in Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli, molecular evolution, and fine-tuning of expression by promoter engineering. Furthermore, knocking in of Vitreoscilla hemoglobin and knocking out of cystathionine-ß-synthase have also been effective strategies. Besides genetic modification, novel bioprocess strategies have also been conducted to improve S-adenosyl-L-methionine synthesis and inhibit its conversion. This has involved the optimization of feeding modes of methanol, glycerol and L-methionine substrates. Taken together considerable improvements have been achieved in S-adenosyl-L-methionine accumulation at both flask and fermenter scales. This review provides a contemporary account of these developments and identifies potential methods for further improvements in the efficiency of S-adenosyl-L-methionine biosynthesis.

The structure of complexes between phosphatidylethanolamine and glucosylceramide: a matrix for membrane rafts.

Interaction between membrane lipids creates lateral domains within which essential membrane processes like trans-membrane signaling, differentiation etc. take place. Attention has focused on liquid-ordered phases formed by sphingomyelin and cholesterol but formation of ordered domains on the cytoplasmic membrane surfaces has largely been neglected. Synchrotron X-ray powder diffraction methods were used to investigate the interaction between two components of the cytoplasmic leaflet of the plasma membrane, phosphatidylethanolamine and glucosylceramide. Multilamellar dispersions of binary mixtures of different molecular species of phosphatidylethanolamine and glucosylceramide were examined. Stoichiometric complexes are formed when the phosphatidylethanolamine has at least one unsaturated fatty acid. The stoichiometry of the complexes was 2.0 fluid phospholipids per glucosylceramide with C22/24 N-acyl chains and 1.8 with C-12 chains. Saturated molecular species of phosphatidylethanolamines were immiscible with glucosylceramide. The complexes formed with unsaturated phosphatidylethanolamines and glucosylceramide are stable above physiological temperatures. A putative role of these matrices in membrane rafts is considered.

Endotoxins: lipopolysaccharides of gram-negative bacteria.

Endotoxin refers lipopolysaccharide that constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria. Lipopolysaccharide is comprised of a hydrophilic polysaccharide and a hydrophobic component known as lipid A which is responsible for the major bioactivity of endotoxin. Lipopolysaccharide can be recognized by immune cells as a pathogen-associated molecule through Toll-like receptor 4. Most enzymes and genes related to the biosynthesis and export of lipopolysaccharide have been identified in Escherichia coli, and they are shared by most Gram-negative bacteria based on available genetic information. However, the detailed structure of lipopolysaccharide differs from one bacterium to another, suggesting that additional enzymes that can modify the basic structure of lipopolysaccharide exist in bacteria, especially some pathogens. These structural modifications of lipopolysaccharide are sometimes tightly regulated. They are not required for survival but closely related to the virulence of bacteria. In this chapter we will focus on the mechanism of biosynthesis and export of lipopolysaccharide in bacteria.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome.

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase.

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2 degrees /min between 25 and 85 degrees C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 degrees C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 degrees C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 degrees C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 degrees C with pure phospholipid in gel phase and 42:58 mole ratio at 65 degrees C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.

The liquid-ordered phase in membranes.

A range of physiological processes has been imputed to lateral domain formation in biological membranes. However the molecular mechanisms of these functions and the details of how domain structures mediate these processes remain largely speculative. That domains exist in biomembranes and can be modeled in relatively simple lipid systems has contributed to our understanding of the principles governing phase behaviour in membranes. A presentation of these principles is the subject of this review. The condensing effect of sterols on phospholipids spread as monomolecular films at the air-water interface is described in terms of the dependence of the effect on sterol and phospholipid structure. The thermodynamics of sphingomyelin-cholesterol interactions are considered from calorimetric, densitometry and equilibrium cholesterol exchange measurements. Biophysical characterisation of the structure of liquid-ordered phase and its relationship with liquid-disordered phase is described from spectroscopic and X-ray scattering studies. Finally, the properties of liquid-ordered phase in the context of membrane physiology and permeability barrier properties are considered.

Hydrocarbon chains dominate coupling and phase coexistence in bilayers of natural phosphatidylcholines and sphingomyelins.

The structure and thermotropic phase behaviour of aqueous dispersions of egg phosphatidylcholine, egg sphingomyelin, bovine brain sphingomyelin and binary mixtures of phosphatidylcholine and sphingomyelins have been examined by synchrotron X-ray diffraction methods. Small-angle lamellar Bragg peaks and wide-angle X-ray scattering bands have been subjected to peak fitting procedures to identify coexisting gel and fluid as well as fluid-fluid bilayer structures. Molecular species of egg phosphatidylcholine exhibit fluid-fluid immiscibility throughout heating scans from 20 degrees to 50 degrees C. Egg and brain sphingomyelins exhibit gel-fluid bilayer coexistence at temperatures below the main phase transition temperature and fluid-fluid phase coexistence at higher temperatures. Binary mixtures of equimolar proportions of egg phosphatidylcholine and either of the sphingomyelins show gel-fluid phase coexistence at temperatures below the gel phase transition temperature of the respective sphingomyelin. Binary mixtures containing egg sphingomyelin show fluid-fluid immiscibility at all temperatures of the heating scans whereas the fluid phase of mixtures comprising brain sphingomyelin are apparently miscible at all temperatures. An analysis of binary mixtures containing egg sphingomyelin and egg phosphatidylcholine in molar ratios 50:50, 67:33 and 83:17 at 50 degrees C to identify the composition of the lamellar phases indicated that the two phospholipids are immiscible in bilayers in the fluid phase. The results are discussed in terms of the role of intermolecular hydrogen bonds and hydrocarbon chain composition of sphingomyelins in maintaining coupling across fluid bilayers.

Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol.

The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20 degrees and 50 degrees Celsius using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20 degrees and 50 degrees Celsius. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as liquid-ordered phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 degrees Celsius the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disordered hydrocarbon in dispersions of pure sphingomyelin and as liquid-ordered phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel phases of egg and brain sphingomyelins at 20 degrees Celsius. The gel phases coexist with liquid-ordered phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened phase transition endotherm originates from dilution of the cholesterol-rich liquid-ordered phase by mobilization of sphingomyelin from the melting gel phase.

Membrane microdomains: role of ceramides in the maintenance of their structure and functions.

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C(12)-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L(o) domains but induces the formation of a gel-like phase. The activation of phospholipase A(2) by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

The structure of detergent-resistant membrane vesicles from rat brain cells.

The size and the bilayer thickness of detergent-resistant membranes isolated from rat brain neuronal membranes using Triton X-100 or Brij 96 in buffers with or without the cations, K+/Mg2+ at a temperature of either 4 degrees C or 37 degrees C were determined by dynamic light scattering and small-angle neutron scattering. Regardless of the precise conditions used, isolated membrane preparations consisted of vesicles of approximately 100 to 200 nm diameter as determined by dynamic light scattering methods, equating to an area of the lipid based membrane microdomain size of 200 to 400 nm diameter. By means of small angle neutron scattering it was established that the average thickness of the bilayers of the complete population of detergent-resistant membranes was similar to that of the parental membrane at between 4.6 and 5.0 nm. Detergent-resistant membranes prepared using buffers containing K+/Mg2+ uniquely formed unilamellar vesicles while membranes prepared in the absence of K+/Mg2+ formed a mixture of uni- and oligolamellar structures indicating that the arrangement of the membrane differs from that observed in the presence of cations. Furthermore, the detergent-resistant membranes prepared at 37 degrees C were slightly thicker than those prepared at 4 degrees C, consistent with the presence of a greater proportion of lipids with longer, more saturated fatty acid chains associated with the Lo (liquid-ordered) phase. It was concluded that the preparation of detergent-resistant membranes at 37 degrees C using buffer containing cations abundant in the cytoplasm might more accurately reflect the composition of lipid rafts present in the plasma membrane under physiological conditions.

Lipidomics: practical aspects and applications.

Lipidomics is the characterization of the molecular species of lipids in biological samples. The polar lipids that comprise the bilayer matrix of the constituent cell membranes of living tissues are highly complex and number many hundreds of distinct lipid species. These differ in the nature of the polar group representing the different classes of lipid. Each class consists of a range of molecular species depending on the length, position of attachment and number of unsaturated double bonds in the associated fatty acids. The origin of this complexity is described and the biochemical processes responsible for homeostasis of the lipid composition of each morphologically-distinct membrane is considered. The practical steps that have been developed for the isolation of membranes and the lipids there from, their storage, separation, detection and identification by liquid chromatography coupled to mass spectrometry are described. Application of lipidomic analyses and examples where clinical screening for lipidoses in collaboration with mass spectrometry facilities are considered from the user point of view.