Showing papers on "Membrane lipids published in 1986"

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells

Abstract: Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.

Membrane lipid phase as catalyst for peptide-receptor interactions

Abstract: Catalysis of ligand-receptor interactions is proposed as an important function of the lipid phase of the cell membrane. The catalytic mechanism is deduced from observed specific interactions of amphiphilic peptides with artificial lipid bilayers. In our model a direct ligand-receptor reaction is replaced by multiple sequential steps including surface accumulation of charged ligands, ligand-membrane interactions, and ultimately binding to the receptor itself. By dividing the total free energy of binding among several steps, the energy per step, including the intrinsic receptor interaction energy, is kept to moderate values. The model thereby yields simple explanations for the large apparent association constants, the high association and dissociation rates, and the heterogeneity of binding sites so frequently found with pharmacological and biochemical ligand-receptor interactions. Furthermore, the measured apparent association constant is a function of the whole system rather than just the receptor. The same, fully functional receptor may show different binding characteristics in different surroundings, such as in another tissue or in a reconstituted system.

Biochemical function and ecological significance of novel bacterial lipids in deep-sea procaryotes.

Abstract: The fatty acid composition of the membrane lipids in 11 deep-sea bacterial isolates was determined. The fatty acids observed were typical of marine vibrios except for the presence of large amounts of long-chain polyunsaturated fatty acids (PUFAs). These long-chain PUFAs were previously thought to be absent in procaryotes, with the notable exception of a single marine Flexibacter sp. In three barophilic strains tested at 2°C, there was a general increase in the relative amount of PUFAs as pressure was increased from a low growth pressure towards the optimal growth pressure. In Vibrio marinus MP-1, a psychrophilic strain, PUFAs were found to increase as a function of decreasing temperature at constant atmospheric pressure. These results suggest the involvement of PUFAs in the maintenance of optimal membrane fluidity and function over environmentally relevant temperatures and pressures. Furthermore, since these lipids are essential nutrients for higher taxa and are found in large amounts in the lipids of deep-sea vertebrates and invertebrates, an important, specific role for deep-sea bacteria in abyssal food webs is implicated.

Molecular Genetics of Membrane Phospholipid Synthesis

Abstract: I have attempted to illustrate the genetic and biochemical complexity of membrane-lipid synthesis by focusing, primarily, on E. coli. The use of molecular genetics to probe membrane lipids is relatively new. Many important questions of phospholipid biochemistry remain unanswered. In the coming years our growing knowledge of the molecular genetics of phospholipids must be applied to the solution of the following problems: How does a cell regulate its total phospholipid content in relationship to macromolecules, especially membrane proteins, cell wall components, and nucleic acids? Why do E. coli and Caulobacter behave differently in this respect? How does a cell regulate its characteristic ratios of polar headgroups and fatty acyl chains? Why does overproduction of phosphatidylserine synthase have no effect on phospholipid composition? How is lipid topography established, both in terms of intramembrane movement (flip-flop) and intermembrane movement? Are there transport systems (flippases) for short-chain diacylglycerophospholipids in E. coli, as in mammalian microsomes, and can flippase mutants be isolated? What are the functions of the many individual phospholipid species? Does E. coli have a functional equivalent of the mammalian phosphatidylinositol cycle? A complete set of phospholipid mutants, together with phenotypic suppressors, should help to answer these questions by allowing selective perturbations in vivo and physiological studies of associated phenotypes. In addition, molecular cloning is already providing access to large quantities of the lipid gene products, opening the door to biophysical and chemical studies of lipid-protein interactions. A unique feature of genetics, as applied to complex biochemical or physiological systems, is the high frequency of unanticipated discoveries that accompany the characterization of new mutants. In our work, this is best illustrated by the analysis of phosphatidylglycerol-deficient mutants of E. coli, which provided the clue (i.e. lipid X) that permitted the elucidation of lipid A biosynthesis. The interconnection of metabolic pathways and important control mechanisms are often revealed by the study of mutants. In the case of E. coli it is best to consider the many lipids and proteins of the envelope as a whole. Considering how few mutant alleles are available for the lipid genes of E. coli, it will be important to create many more genetic lesions in order to gain a full understanding of regulation and function.

Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp.

Abstract: The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultative strains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids. Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains. Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively high content of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B. subtilis.

Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse.

Abstract: We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.

Enzymes of Lipid Metabolism II

Abstract: Lipolytic Enzymes of the Gastrointestinal Tract- Kinetic Assay of Human Gastric Lipase on Short and Long Chain Triacylglycerol Emulsions- Inhibition of Pancreatic and Microbial Lipases by Proteins: Kinetic and Binding Studies- Tumor-Promoting Phorbol Diesters are Substrates for and Modulators of Diacylglycerol Lipase- Lipolytic Activities Operative at the Outer Surface of Rat Fat Cells- On the Mechanism of Endogenous Lipolysis in Rat Heart: A Role of Lysosomes ?- The Enzymes of Phosphatidylcholine Biosynthesis- Characterization of Choline and Ethanolamine Kinase Activities in Plasmodium-Infected Erythrocytes- Physiological Responses of Intestinal CTP: Phosphocholine Cytidylyltransferase and its Interaction with Lipophilic Drugs- Degradation of Membrane Phosphoglycerides by the Reversal of Phosphotransferase Reactions- Treatment of Rat Brain Microsomal Vesicles with Octyl-?-D-Glucopyranoside: A study on Ethanolamine Base-Exchange after Reaggregation- Effect of Modification of Membrane Phospholipid Composition on Phospholipid Methylation in Aggregating Cell Culture- Acetyltransferases and Transacylases Relative Rates in 1-Alkyl-or 1-Acyl-Phosphatidylcholine Synthesis by Rat Platelet Homogenates- Modulation of Deacylation-Reacylation Pathway of Phospholipid Metabolism and Turnover During Reperfusion of Ischemic Myocardium- Monoacylglycerol Acyltransferase: Stereospecificity and Evidence that the Hepatic and Intestinal Activities are Tissue-Specific Isoenzymes- The Interaction of Pancreatic Phospholipase A2 with Negatively Charged Substrates-Application: The Transformation of Soluble Phospholipase A2 into a Highly Penetrating "Membrane-Bound" Form- Mechanism of Interaction of Phospholipase A2 with Phospholipid Substrates and Activators- Comparison of the Activation of Soluble and Immobilized Phospholipase A2- Some Properties of Membrane-Bound Phospholipases A2- Endogenous Suppression of Neutral-Active and Calcium-Dependent Phospholipase A2 Activity in Human Polymorphonuclear Leukocytes- Properties of Pancreatic Phospholipases A1 and Intestinal Phospholipase A2 from Guinea Pig: Their Complementary Role in the Intestinal Absorption of Phospholipids- Partial Characterization of Cytosolic Phospholipase A1 of Rat Heart- Purification and Properties of Phospholipase A from the Outer Membrane of Overproducing Escherichia Coli K-12- Some Properties of Lysoplasmalogenase and Alkenylhydrolase from Rat Liver Microsomes- Purification of Platelet-Activating Factor Acetylhydrolase- Characterization of an Acetylhydrolase Isolated from Rat Alveolar Macrophages in Comparison with the Enzyme Present In Vivo in Lung Alveoli- Preliminary Study for High Performance Purification of a Hydrophobic Protein: A Biological Fluid Acetylhydrolase- Biosynthesis and Pharmacology of PAF-Acether (Platelet-Activating Factor)- The Peroxisomal Enzymes of Glycerolipid Metabolism- Deficiencies in Ether Glycerolipids and Their Biosynthesis in Inherited Peroxisomal Disorders- Impaired Maturation of Peroxisomal ?-Oxidation Enzymes in Fibroblasts from Patients with the Zellweger Syndrome and Infantile Refsum Disease- New Techniques in Glycosyltransferase Research- Biosynthesis of Gangliosides and Blood Group Glycolipids using Solubilized Glycosyltransferases- Solubilization of Lipid-Glycosyltransferases from Mitochondrial Outer Membranes- UDP-Glucose Sterol ?-Glucosyl Transferase, a Plant Sterol Conjugating Enzyme- Purification and Properties of Acid Sphingomyelinase from Human Urine- Heterogeneity of Human Sphingomyelinase: Relatedness of the Major Polypeptides- Immunological Studies on Acidic Sphingomyelinase- Immunological Studies on Lysosomal Sphingomyelinase: Immunization Procedures, Properties of Polyclonal and Monoclonal Antibodies Obtained and Effect of Triton X-100 on Binding of Enzyme Activity- ?-Glucocerebrosidase: Affinity Purification and Characterization of its Active Site with N-Alkyl Derivatives of L-Deoxynojirimycin- Human Acid ?-Glucosidase: Primary Structure of the Active Site- Specificity of Human Glucosylceramide ?-Glucosidase Towards Structurally Modified Glucosylceramides in a Liposomal Assay-System- Activator Proteins (Protein Cofactors) for the Catabolism of Glycosphingolipids- Glycosphingolipid Activator Proteins- Studies of SAP-1 and SAP-2 in Cultured Skin Fibroblasts- The Specificity of Cerebroside Sulfatase Activator- Two Heat-Stable Low-Molecular-Mass Proteins Stimulating the Enzymic Sphingomyelin Degradation Isolated from Human Gaucher and Normal Spleen- The Effects of Acidic Lipids and Heat-Stable Factor on the Physical-Chemical and Kinetic Properties of Glucocerebrosidase- A New Glucosylceramidase Activator in Human Placenta- Molecular Organization of Glycosphingolipids in Phosphatidylcholine Bilayers and Biological Membranes- Metabolic Incorporation of a New Fluorescent Anthracene Fatty Acid into the Membrane Lipids of Procaryotic and Eucaryotic Cells for Studying the Dynamic and Topology of Membranes- TMA-DPH as Specific Plasma Membrane Fluidity Probe for Intact Cells and Its Limitation- Phospholipid Bilayer Assembly: Facilitated Transmembrane Movement of Phosphatidylcholine- The Asymmetrical Distribution and Biosynthesis of Molecular Species of Phospholipids in Chick Brain Microsomes- Cholesterol Pools in Mycoplasma Membranes: Modifications in Phospholipid Composition Affect the Kinetics of Cholesterol Exchange with Lipid Vesicles- Structure and Function of the Nonspecific Lipid Transfer Protein (Sterol Carrier Protein 2)- A Model for Studying Membrane Fatty Acid Transport: Acyl-Coenzyme A Synthesis in Human Erythrocyte Ghosts- Transport of Fluorescent Fatty Acids into Cultured Cells: Analysis by the Fluorescence Activated Cell Sorter- Transport and Metabolism of Fatty Acids in Cultured Heart Muscle Cells from Neonatal Rats- Hormonal Regulation of Exogenous Fatty-Acid Incorporation into Lipids in Cultured Hamster Fibroblasts- Fatty Acid Composition and Metabolism of Tumor Cells Rendered Resistant to the Anticancer Drug Doxorubicin- HMGR (3-Hydroxy, 3-Methylglutaryl-CoA Reductase) Activity of Cultured Rat Brain Cells: Sensitivity to n-3 and n-6 Polyunsaturated Fatty Acids (PUFAs) from Cod-Liver and Sunflower Oils- Development and Maturation of Lipoprotein Lipase in Cultured Adipose Cells- Synthesis and Properties of Fluorescent Analogs of Cytidine Diphosphate-Diacylglycerol and Phosphatidylinositol- Biosynthesis and Intracellular Transport of Phospholipids in Yeast- Phospholipid Catabolism and Phospholipid Turnover in Cultured Cells- Aging of Rat Heart Myocytes and Fibroblasts: Relationship Between Lipid Composition, Membrane Organization and Biological Properties- Extracellular Fluid Viscosity: Its Role in the Regulation of Membrane Lipid Metabolism and Cellular Secretion- Control of Surface Sphingomyelinase Activity in Cultured Neuroblastoma Cells- Metabolism of Exogenous Gangliosides in Cultured Fibroblasts and Cerebellar Cells- Intracellular Aspects of Ganglioside Metabolism and Transport- Metabolic Aspects of Gangliosides involved in the Maturation of Cultured Nerve Cells- Regulation of Glycosphingolipid Anabolism in Fibroblasts by Ionophores, Plasma Membrane ATP-ASE Inhibiton, and Growth Factors- Nuclear Triiodothyronine Receptors and Mechanisms of Triiodothyronine and Insulin Action on the Synthesis of Cerebroside Sulfotransferase by Cultures of Cells Dissociated from Brains of Embryonic Mice- Utilization of Exogenous Ceramide for the Synthesis of Sphingolipids in Cultured SV40-Transformed Rat Schwann Cells- Genetic Regulation of Phospholipid Synthesis in Yeast- The Regulation of Arachidonic Acid Release in Parturition- Relationship Between Arachidonic Acid Biosynthesis and its Level in Rat Tissues- Regulatory Properties of Carnitine Palmitoyltransferase in the Mitochondrial Membrane of Liver- Structure and Regulation of the sn-Glycerol-3-Phosphate Acyltransferase of E Coli- Mammalian Phospholipase D and Related Activities- Metabolism of Inositol-Glycerophospholipids in Relation to Transmembrane Signalling and Calcium Mobilization- Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism- Effect of Ischemia on Activities of Polyphosphoinositide Phosphodiesterase and Diacylglycerol Kinase in Gerbil Brain Synaptosomes- Receptor-Mediated Degradation of Choline Plasmalogens and Glycerophospho]ipid Methylation: A New Hypothesis- Transport and Processing of Lysosomal Enzymes- Genetic Heterogeneity of Gaucher Disease: Enzymatic and Immunologic Studies- Molecular Forms, Biosynthesis and Maturation of Glucocerebrosidase, a Membrane-Associated Lysosomal Enzyme Deficient in Gaucher Disease- Forms of Glucocerebrosidase Present in Tissues and Urine- The ?-Galactosidase-sialidase Complex: Studies on Normal and Mutant Human Fibroblasts- Intracellular Binding and Transport of Lysosomal Enzymes in Human and Bovine Tissues- Molecular Heterogeneity in O-Variant GM2 Gangliosidosis- Preliminary Characterization of a Ganglioside Sialidase in Normal and Mucolipidosis IV Fibroblasts- Sphingomyelinase and Niemann-Pick Disease- Fluorescent Derivatives of Sphingomyelin: Synthesis, Use as Substrates for Sphingomyelinase and for Diagnosis of Niemann-Pick Disease in Cultured Cells- Lipases, Cholesterylesterases and Carboxylesterases in Lymphoid Cell Lines: Substrate Specificity and Relation to Wolman's, Cholesteryl Ester Storage Diseases and Lipid Storage Myopathy- Use of Fluorescent Fatty Acids for Labelling Acid Lipase-Deficient Cells- Morphological and Biochemical Studies of Induced and Pathological Lipidosis in Cultured Muscle Cells- On the Composition of Autofluorescent Accumulation Products: Ceroid and Lipofuscin- Participants- Contributors

Phase equilibria of membrane lipids from Acholeplasma laidlawii: importance of a single lipid forming nonlamellar phases.

Abstract: A basis for the reorganization of the bilayer structure in biological membranes is the different aggregate structures formed by lipids in water. The phase equilibria of all individual lipids and several in vivo polar lipid mixtures from acyl chain modified membranes of Acholeplasma laidlawii were investigated with different NMR techniques. All dioleoyl (DO) polar lipids, except monoglucosyldiglyceride (MGDG), form lamellar liquid crystalline (L,) phases only. The phase diagram of DOMGDG reveals reversed cubic (I,,), reversed hexagonal (HII), and L, phases. In mixtures of DOMGDG and dioleoyldiglycosyldiglyceride (DODGDG), the formation of an 111 (or HI,) phase is enhanced by DOMGDG and low hydration or high temperatures. For in vivo mixtures of all polar DO lipids, a transition from an L, to an 111 phase is promoted by low hydration or high temperatures (50 "C). The phospholipids are incorporated in this 111 phase. Likewise, 111 and HII phases are formed at similar temperatures in a series of in vivo mixtures with different extents of acyl chain unsaturation. However, their melting temperatures (T,) vary in an expected manner. All cubic and hexagonal phases, except the 111 phase with DOMGDG, exist in equilibrium with excess water. The maximum hydration of MGDG and DGDG is similar and increases with acyl chain unsaturation but is substantially lower than that for, e.g., phosphatidylcholine. The translational diffusion of the lipids in the cubic phases is rapid, implying bicontinuous structures. However, their appearances in freeze-fracture electron microscope pictures are different. The 111 phase of DOMGDG belongs to the Za3d space group. It is concluded that the formation of nonlamellar phases by A. laidlawii lipids depends critically upon the MGDG concentration.

Peroxisome Proliferation and Lipid Peroxidation in Rat Liver

Abstract: Male F344 rats were fed a diet containing the peroxisome proliferators 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropionic acid [ciprofibrate (0.025%)] or [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid [Wy-14643 (0.1%)] for up to 14 months to determine whether hepatic peroxisome proliferation caused by these agents results in the induction of membrane lipid peroxidation in the liver. Peroxidative damage of membrane lipids from whole liver, postnuclear, heavy-particle, microsomal, and nuclear membranes was evaluated by determining the extent of formation of conjugated dienes (ultraviolet absorption, 233 nm). Increased generation of diene conjugates was noted in whole-liver, postnuclear, and heavy-particle membrane lipids of rats fed peroxisome proliferators for 6 months or longer when compared to controls. An additional, more intense absorption profile in the ultraviolet absorption range of ∼276 nm was noted in the membrane lipids derived from whole liver, postnuclear, and heavy particle pellets, but not in the nuclear and microsomal membrane lipids of livers with peroxisome proliferation. Although the exact chemical nature of this Δ276nm peak is not clear, it is attributed to the formation of ketone dienes and/or conjugated trienes. The excess lipid peroxidation correlates with the previous observation of accumulation of abundant quantities of lipofuscin in hepatocytes of rats chronically exposed to peroxisome proliferators. The generation of conjugated dienes and ketone dienes and/or trienes together with increased levels of H2O2 generation by peroxisomal enzymes, and decreased levels of hepatic glutathione peroxidase, glutathione reductase, and glutathione-S-transferases, enzymes responsible for the defense against H2O2 damage, suggest the occurrence of membrane lipid peroxidation and oxidative stress in livers of rats treated with carcinogenic peroxisome proliferators.

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

Abstract: A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

Metabolic Changes of Membrane Lipid Composition in Acholeplasma Laidlawii by Hydrocarbons, Alcohols, and Detergents: Arguments for Effects on Lipid Packing

Abstract: The packing of lipids into different aggregates, such as spheres, rods, or bilayers, is dependent on the hydrophobic volume, the hydrocarbon-water interfacial area, and the hydrocarbon chain length of the participating molecules, according to the self-assembly theory [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200]. The origin of the participating molecules should be of no importance with respect to their abilities to affect the above-mentioned parameters. In this investigation, Acholeplasma laidlawii, with a defined acyl chain composition of the membrane lipids, has been grown in the presence of three different classes of foreign molecules, known to partition into model and biological membranes. This results in an extensive metabolic alteration in the lipid polar head group composition, which is expressed as changes in the molar ratio between the lipids monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), forming reversed hexagonal and lamellar phases in excess water, respectively. The formation of nonlamellar phases by A. laidlawii lipids depends critically upon the MGDG concentration [Lindblom, G., Brentel, I., Sjolund, M., Wikander, G., & Wieslander, A. (1986) Biochemistry (preceding paper in this issue)]. The foreign molecules tested belong to the following groups: nonpolar organic solvents, alcohols, and detergents. Their effects on the gel to liquid crystalline phase transition temperature (Tm), on the order parameter of the acyl chains, and on the phase equilibria between lamellar and nonlamellar liquid crystalline phases in lipid-water model systems are known in several instances.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

Abstract: The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.

Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

Abstract: In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

Abstract: SUMMARY The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cclls dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their cholorophyll content.

The lateral diffusion of lipid probes in the surface membrane of Schistosoma mansoni.

Abstract: The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L.

Abstract: Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO3-insensitive ATPase. The ATPase has high specificities both for substrate and Mg2+ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl−. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.

Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-β-d-Glucan Synthase and Callose Synthesis

Abstract: The activity of 1,3-beta-d-glucan synthase assayed in the presence of digitonin in a microsomal preparation from suspension-cultured cells of Glycine max can be fully inhibited by unsaturated fatty acids, trienoic acids being most effective. Lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B can also fully inhibit the enzyme. Inhibition is observed both when the enzyme is activated by Ca(2+) or by trypsinization. At low amounts some of the substances can also cause stimulation. These effects all may result from a displacement of certain endogenous phospholipids necessary for optimal activity of the 1,3-beta-d-glucan synthase.In the absence of digitonin the enzyme activity is greatly stimulated by lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B within a certain concentration range, presumably by rendering the microsomal vesicles permeable to the substrate and Ca(2+). Dibucaine does not cause such an effect.Acylcarnitine and Echinocandin B at low concentrations can induce callose synthesis in vivo; this effect is enhanced by chitosan. At higher concentrations the two substances and polyunsaturated fatty acids cause severe electrolyte leakage. The effects are discussed in regard to the induction of callose synthesis by enforced Ca(2+) influx, and its modulation by membrane lipids.

Isolation and partial characterization of the plasma membrane from human spermatozoa.

Abstract: Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM) Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation A single membrane population was resolved at the 10 M sucrose interface Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase) Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 083 Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM

Long-chain diols: a new class of membrane lipids from a thermophilic bacterium.

Abstract: Glycerol-derived membrane lipids are essentially absent in the thermophilic bacterium Thermomicrobium roseum. A series of straight chain and internally methyl-branched 1,2-diols of carbon numbers C(18) to C(23) were found to replace glycerolipids in this bacterium. Fatty acids were present but were ester-linked to the diols or amide-linked to polar heads groups and not to glycerol. This thermophile has evolved the integration of diols as a novel approach for the construction of its cytoplasmic membrane.

Membrane alterations in cancer cells: the role of oxy radicals

Abstract: Membranes isolated from tumor cells present profound alterations in their composition, structural organization, and functional properties. In this study we have reported some of these alterations in microsomal and plasma membranes of hepatomas with different growth rate and degree of differentiation. The chemical parameters studied were the phospholipid-to-protein, the cholesterol-to-protein, and the cholesterol-to-phospholipid ratios and the fatty acid composition of the phospholipids. The physical parameters were the molecular order (static) and the fluidity (dynamic), determined, respectively, as the order parameter [P2] and the correlation time tau R of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The functional property investigated was the ability of the membranes to undergo superoxide-induced lipid peroxidation, determined as byproduct (malondialdehyde and lipid hydroperoxides) formation and as changes in the fatty acid acyl residues. Changes in the physical state of the membrane, induced by oxy radicals, were also monitored during lipid peroxidation. A study of the antioxidant activity of the tumor cell, in terms of oxy radical enzymatic defenses (superoxide dismutase, glutathione peroxidase and catalase) was also performed. The main results obtained are the following: hepatoma membranes possess a lower phospholipid content and a lower degree of fatty acid unsaturation; on the other hand, the cholesterol-to-phospholipid ratio is increased; the physical state appears characterized by an increased rigidity (increased molecular order of the lipids and decreased fluidity); the membrane peroxidizability is markedly depressed and its order parameter, in contrast to liver membranes, does not increase with exposure to the action of O2- radicals; and the oxy radical enzymatic defense mechanisms are decreased. All these alterations increase with increasing growth rate and dedifferentiation of the tumor. Considering all of the data, we are inclined to think that tumor membranes are altered structurally and functionally in part as the result of an oxy radical-induced damage that takes place in vivo under conditions of increased oxygen toxicity.

Alteration of red cell membrane organization in sickle cell anaemia.

Abstract: Bee venom phospholipase A2 and the fluorescent probe merocyanine 540 were used to examine plasma membrane phospholipid organization in the spicules released by deoxygenation and reoxygenation of sickle red cells, as well as in reversibly and irreversibly sickled erythrocytes. Digestion of phosphatidyl ethanolamine in spicules was comparable to that of phosphatidyl choline, and these structures were stained by the fluorescent probe. Both assays suggest that membrane lipid asymmetry is disrupted in spicules. The residual cells, from which the spicules were derived, retain the normal asymmetry in phospholipid distribution between the outer and inner leaflets of the plasma membrane bilayer. Comparable experiments with cell fractions enriched in irreversibly sickled cells revealed a partial enhancement of phosphatidyl ethanolamine digestion, confirming the similar experiments of Lubin et al (1981). Staining of these cells with merocyanine 540, however, did not reveal a subfraction of stainable cells, indicating that this increase in phosphatidyl ethanolamine digestion is not due to the presence of a small fraction of cells which have completely lost their membrane asymmetry.