Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Membrane Lipids of Mycoplasma hominis

Essentially all of the lipids of Mycoplasma hominis (200 mug/mg of cell protein) were found to be located in the cell membrane. Over one-half were neutral lipids incorporated from the growth medium and consisting of 43% free cholesterol, 19% esterified cholesterol, 23% triglycerides, 10% free fatty acids, and small amounts of di- and monoglycerides. The polar lipids accounting for about 40% of the total were synthesized by the organisms. Phosphatidylglycerol was the predominant lipid of this fraction. The minor components, tentatively identified as lysophosphatidylglycerol and phosphatidic acid, seem to represent breakdown products of phosphatidylglycerol. No glycolipids were detected. Being unable to synthesize long-chain fatty acids, M. hominis utilized the fatty acids of the growth medium for polar lipid synthesis, preferentially the saturated ones, so that the polar lipids had highly saturated hydrocarbon chains. It is proposed that the large take up of unsaturated neutral lipids and cholesterol from the medium offsets the marked condensing effect of the saturated polar lipids, although electron paramagnetic resonance spectrometry of spin-labeled fatty acids incorporated into the M. hominis membrane indicated that the lipid region is still more rigid than that of the Acholeplasma laidlawii membrane.     

Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis.

Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches. The patches appeared in cells quenched from either 4 or 37 degrees C. Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine. The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M. gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium. Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+. Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M. gallisepticum membranes was almost identical to that of an aqueous dispersion of M. gallisepticum membrane lipids. Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions. The possibility that the osmotic resistance of M. gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 degrees C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

The effect of variations in growth temperature, fatty acid composition and cholesterol content on the lipid polar head-group composition of Acholeplasma laidlawii B membranes

We have systematically investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the membrane lipid polar headgroup composition of Acholeplasma laidlawii B. Two important lipid compositional parameters have been determined from such an analysis. The first parameter studied was the ratio of the two major neutral glycolipids of this organism, monoglucosyldiacylglycerol (MGDG) and diglucosyldiacylglycerol (DGDG). As the former lipid prefers to exist in a reversed hexagonal phase at higher temperatures, with unsaturated fatty acyl chains or in the presence of cholesterol, the ratio of these two lipids reflects the phase state preference of the total A. laidlawii membrane lipids. Although we find that the MGDG/DGDG ratio is reduced in response to an increase in fatty acid unsaturation, increases in growth temperature or cholesterol content reduce this ratio only in cells enriched in a saturated but not an unsaturated fatty acid. The second parameter studied was the ratio of these neutral glycolipids to the only phosphatide in the A. laidlawii membrane, phosphatidylglycerol (PG); this parameter reflects the relative balance of uncharged and charged lipids in the membrane of this organism. We find that the MGDG + DGDG/PG ratio is lowest in cells enriched in the saturated fatty acid even though these cells already have the highest lipid bilayer surface charge density. Moreover, this ratio is not consistently related to growth temperature or changes in cholesterol levels, as expected. We therefore conclude that A. laidlawii strain B, apparently unlike strain A, does not possess coherent regulatory mechanisms for maintaining either the phase preference or the surface charge density of its membrane lipid constant in response to variations in growth temperature, fatty acid composition or cholesterol content.     

Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.     

Membrane lipids of Mycoplasma orale: lipid composition and synthesis of phospholipids.

Lipid composition of Mycoplasma orale was examined and compared with that of horse serum added to the growth medium. Ratios of cholesterol/cholesterol ester and sphingomyelin/phosphatidylcholine were much higher in M. orale than in the horse serum, indicating the organism incorporates selectively cholesterol and sphingomyelin. A distinct difference between the lipids from the two sources was that in phospholipids of M. orale almost all (greater than 95%) of the fatty acyl residues were saturated whereas nearly half of the residues were unsaturated in horse serum phospholipids. Approximately one third of M. orale phospholipids was phosphatidylglycerol, which was synthesized by the organism as was demonstrated by 32P-labeling experiment. Its acyl residues consisted mainly of C16:0 and were efficiently labeled with 14C-palmitate but not with 14C-acetate. These results clearly indicate the de novo synthesis of phosphatidylglycerol by M. orale is through acylation with exogenous saturated fatty acids. On the other hand, all the phosphatidylcholine and sphingomyelin of M. orale were derived from the medium. The 14C-labeling experiment demonstrates that no fatty acid synthesis takes place nor exogenous fatty acid can be incorporated so efficiently as phosphatidylglycerol, suggesting that extremely high proportion of saturated fatty acyl residues in these phospholipids is the consequence of saturation directed to the acyl chains of the incorporated phospholipids.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Ageing-related changes in Mycoplasma canadense membranes

Fluidity and composition of cell membranes during progression of Mycoplasma canadense cultures grown in a serum-free medium was assessed. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at 25°C of intact cells and liposomes in the exponential and stationary phases of growth was compared. A decrease in fluidity and an increase in the ratio of saturated to unsaturated fatty acids was detected in cell membranes on ageing. Nevertheless, membrane density remained unaltered although the molar ratio of cholesterol to phospholipids decreased. It is proposed that the increase in lipid order is primarily due to the increase in the ratio of saturated to unsaturated membrane fatty acids, being the diminished molar ratio of cholesterol to phospholipids involved in the reduced unsaturated fatty acid uptake.     

Ageing-related changes in Mycoplasma canadense membranes.

Fluidity and composition of cell membranes during progression of Mycoplasma canadense cultures grown in a serum-free medium was assessed. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at 25 degrees C of intact cells and liposomes in the exponential and stationary phases of growth was compared. A decrease in fluidity and an increase in the ratio of saturated to unsaturated fatty acids was detected in cell membranes on aging. Nevertheless, membrane density remained unaltered although the molar ratio of cholesterol to phospholipids decreased. It is proposed that the increase in lipid order is primarily due to the increase in the ratio of saturated to unsaturated membrane fatty acids, being the diminished molar ratio of cholesterol to phospholipids involved in the reduced unsaturated fatty acid uptake.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Stable Cell Surface Expression of GPI‐Anchored Proteins,but not Intracellular Transport,Depends on their Fatty Acid Structure

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI‐APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI‐APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI‐AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi‐to‐plasma membrane transport or selective endocytosis via GPI‐enriched early endosomal compartment (GEEC)/ clathrin‐independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI‐APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI‐APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI‐APs particularly under potential membrane lipid perturbation.      

Cholesterol distribution and movement in the Mycoplasma gallisepticum cell membrane.

The time course and extent of transfer of [14C]-cholesterol from resting Mycoplasma gallisepticum cells or membrane preparations to high-density lipoproteins were studied. More than 90% of the total cholesterol in isolated, unsealed membrane preparations was exchanged in a single kinetic process. In intact cells, however, cholesterol exists in two different environments. Cholesterol in one environment, representing approximately 50% of the total unesterified cholesterol, is readily exchanged with the cholesterol of high-density lipoproteins, with a half-time of about 4 h at 37 degrees C. The rate of exchange of [14C]cholesterol from the other environment was exceedingly slow, with a half-time of about 18 days. The fraction of the total cholesterol in the readily exchangeable cholesterol pool in intact cells increased somewhat upon aging of the culture. Electron spin resonance spectra of nitroxide-labeled stearic acids incorporated into membranes of M. gallisepticum cells indicated increased rigidity at the late exponential phase of growth. These results suggest that cholesterol is present in approximately equal concentrations on both surfaces of the M. gallisepticum membrane and that in resting cells the rate of movement of cholesterol molecules from the inner to outer halves of the lipid bilayer is exceedingly slow or nonexistent.