Phase properties of senescing plant membranes. Role of the neutral lipids

Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes.     

Neutral Lipids Rigidify Unsaturated Acyl Chains in Senescing Membranes

Senescence in bean cotyledons is accompanied by a progressiveincrease in the proportion of gel phase lipid in cellular membranesthat can be attributed to qualitative changes in the neutrallipids. The resulting mixture of lipid phases leads to impairedmembrane function. Insight into the molecular basis for thisphenomenon has been gleaned from studies of the effects of theseneutral lipids on the phase properties of pure phospholipidmembranes. Induction of the gel phase, detectable as a risein the liquid-crystalline to gel phase transition temperature,was observed when neutral lipid from senescent membranes wasintroduced into liposomes of unsaturated phospholipids. Thetransition temperature for phosphatidylcholine rose from –6.5° C in control liposomes to 51 ° C when 25% (w/w) neutrallipid was present. A similar rise was obtained for dioleoylphosphatidylcholine.However, for dipalmitoylphosphatidylcholine, which is fullysaturated, the rise in transition temperature upon additionof neutral lipids was only 3 ° C. Thus the neutral lipidsin senescent membranes appear to selectively rigidify unsaturatedacyl chains. At least three types of compounds known to alterthe phase properties of lipid bilayers were detectable in theneutral lipid fraction, suggesting that rigidification reflectsthe concerted action of several neutral lipid components onunsaturated phospholipid. Key words: Senescence, phospholipid, lipid phase properties     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.     

Lipid composition and molecular organization in plasma membrane-enriched fractions from senescing cotyledons

Plasma membrane fractions isolated from cotyledons of Phaseolus vulgaris L. cv. Kinghorn at various stages of senescence showed no significant change in fatty acid saturation with advancing senescence. However, the steroliphospholipid ratio increased by about 400% as senescence intensified. The lipid phase transition temperature of the membranes, which was measured by wide-angle x-ray diffraction, also rose from a point well below the growing temperature for young tissue to about 50°C for membrane from extensively senescent 9-day-old tissue. This means that by day 4 of germination there was a mixture of liquid-crystalline and gel phase phospholipid in the membrane matrices. Crystallinity attributable to sterol-sterol interaction was also apparent in the diffraction patterns for senescent membranes. The co-existence of gel and liquid-crystalline phase phospholipid in the aging membranes as well as the crystalline sterol aggregates presumably render the storage cells of cotyledons leaky and may thus facilitate the translocation of hydrolyzed food reserves into the vascular network.     

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.     

Fluorine-19 nuclear magnetic resonance studies of lipid phase transitions in model and biological membranes.

Fluorinated fatty acids of the general formula CH3(CH2)13-mCF2(CH2)m-2COOH are informative spectroscopic probes of the gel to liquid-crystalline phase transitions in phospholipid dispersions and in biological membranes. We present theoretical considerations to suggest that the 19F nuclear magnetic resonance line shapes are very different for frozen and fluid lipid regions. Our studies confirm this expectation for mixed phospholipid multilamellar dispersions containing a trace of difluoromyristate. The method correctly measures the onset and completion temperatures of the transition in the well-studied dimyristoylphosphaditylcholine distearoylphosphatidylcholine system and also describes the motional behavior of the solid and fluid phases within the transition. Lipids extracted from Escherichia coli membranes show similar motional phenomena through the transition-temperature range according to 19F nuclear magnetic resonance studies of difluoromyristate biosynthetically incorporated into the K1060B5 strain, an unsaturated fatty acid auxotroph. Intact cells or membrane vesicles show substantially different behavior from extracted lipids, indicating that membrane proteins significantly perturb the phase transition. Evidence presented in this paper also shows that the 19F resonance from Escherichia coli phospholipids is sensitive to various intramembrane interactions. There is a general decrease in restriction of motion due to neutral lipids and an opposite effect due to the architecture of the native membrane. Neither effect is temperature sensitive. However, there are interactions in the intact membrane, affecting the 19F resonance, that are temperature dependent both due to the phase-transition process and due to processes occurring at high temperatures.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of a bacterial biosurfactant trehalose lipid with phosphatidylserine membranes

Trehalose lipids are biosurfactants produced by rhodococci that, in addition to their well known potential industrial and environmental uses, are gaining interest in their use as therapeutic agents. The study of the interaction of biosurfactants with membranes is important in order to understand the molecular mechanism of their biological actions. In this work we look into the interactions of a bacterial trehalose lipid produced by Rhodococcus sp. with dimyristoylphosphatidylserine membranes by using differential scanning calorimetry, X-ray diffraction and infrared spectroscopy. Differential scanning calorimetry and X-ray diffraction show that trehalose lipid broadens and shifts the phospholipid gel to liquid-crystalline phase transition to lower temperatures, does not modify the macroscopic bilayer organization and presents good miscibility both in the gel and the liquid-crystalline phases. Infrared experiments show that trehalose lipid increases the fluidity of the phosphatidylserine acyl chains, changed the local environment of the polar head group, and decreased the hydration of the interfacial region of the bilayer. Trehalose lipid was also able to affect the thermotropic transition of dimyristoylphosphatidyserine in the presence of calcium. These results support the idea that trehalose lipid incorporates into the phosphatidylserine bilayers and produces structural perturbations which might affect the function of the membrane.     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermal behavior and lipid composition of cauliflower plasma membranes in relation to ATPase activity and chilling sensitivity

A plasma-membrane fraction rich in ion-stimulated ATPase activity was isolated from cauliflower (Brassica oleracea L.) buds. The activity of the ATPase was dependent on Mg(2+) and stimulated 4-fold by K(+). The lipids of the membrane fraction contained 57% by weight of phospholipid, 16% glycolipid including sterol glycosides, and 27% neutral lipids. Sterols and sterol esters comprised 9% by weight of the total lipid fraction, and the m ratio of total sterol to phospholipid was 0.5. Fatty acid unsaturation of the membrane lipids was 75%. Arrhenius plots of the Mg(2+) and Mg(2+) + K(+) stimulated ATPase activity were biphasic with an increase in activation energy occurring below about 12 degrees C, a response typical of some membrane-associated enzymes of chilling-sensitive plants. No thermal transitions were detected in the membranes or membrane lipids between 0 and 30 degrees C using differential scanning calorimetry and electron spin resonance spectroscopy. This type of thermal behavior is typical of membranes of chilling-resistant plants. It was concluded that the low temperature increase in activation energy of the ion-stimulated, membrane-associated ATPase is an intrinsic property of the enzyme system and not the result of a transition in the bulk membrane lipid.     

Lipid composition of subcellular particles of human blood platelets

Human platelets can be fractionated into three main subcellular components: granules, membranes, and a soluble fraction. In this study we determined the phospholipid and neutral lipid content of the granules and membranes. Quantitative relationships between lipids and protein were examined. The fatty acid and aldehyde composition of individual phospholipids and neutral lipids was also determined. Whole platelets had a lower lipid to protein ratio than did the subcellular particles, but the basic lipid composition of the granules, membranes, and platelets was similar. The phospholipid composition of platelets and subcellular fractions was found to differ only in that granules had a lower percentage of lecithin. Each of the phospholipid classes displayed a distinctive fatty acid pattern which was the same in all fractions and in whole platelets. The major neutral lipid was free cholesterol. Cholesteryl esters, triglycerides, and free fatty acids were minor components. The molar ratio of cholesterol to phospholipid in the platelet membranes was lower than that of brain myelin and erythrocyte ghosts. Some differences in fatty acid composition of the neutral lipids of platelet fractions were found. A special lipid composition or constituent that would correlate with platelet function has not been found.     

Phase Transitions in Coarse-Grained Lipid Bilayers Containing Cholesterol by Molecular Dynamics Simulations

Coarse-grained simulations of model membranes containing mixtures of phospholipid and cholesterol molecules at different concentrations and temperatures have been performed. A random mixing without tendencies for segregation or formation of domains was observed on spatial scales corresponding to a few thousand lipids and timescales up to several microseconds. The gel-to-liquid crystalline phase transition is successively weakened with increasing amounts of cholesterol without disappearing completely even at a concentration of cholesterol as high as 60%. The phase transition temperature increases slightly depending on the cholesterol concentration. The gel phase system undergoes a transition with increasing amounts of cholesterol from a solid-ordered phase into a liquid-ordered one. In the solid phase, the amplitude of the oscillations in the radial distribution function decays algebraically with a prefactor that goes to zero at the solid-liquid transition.     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

Domains in binary SOPC/POPE lipid mixtures studied by pulsed field gradient 1H MAS NMR

We studied domain formation in mixtures of the monounsaturated lipids SOPC and POPE as a function of temperature and composition by NMR. Magic angle spinning at kHz frequencies restored resolution of (1)H NMR lipid resonances in the fluid phase, whereas the linewidth of gel-phase lipids remained rather broad and spinning frequency dependent. In regions of fluid- and gel-phase coexistence, spectra are a superposition of resonances from fluid and gel domains, as indicated by the existence of isosbestic points. Quantitative determination of the amount of lipid in the coexisting phases is straightforward and permitted construction of a binary phase diagram. Lateral rates of lipid diffusion were determined by (1)H MAS NMR with pulsed field gradients. At the onset of the phase transition near 25 degrees C apparent diffusion rates became diffusion time dependent, indicating that lipid movement is obstructed by the formation of gel-phase domains. A percolation threshold at which diffusion of fluid-phase lipid becomes confined to micrometer-size domains was observed when approximately 40% of total lipid had entered the gel phase. The results indicate that common phosphatidylethanolamines may trigger domain formation in membranes within a physiologically relevant temperature range. This novel NMR approach may aid the study of lipid rafts.     

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Effects of a bacterial trehalose lipid on phosphatidylglycerol membranes

Bacterial trehalose lipids are biosurfactants with potential application in the biomedical/healthcare industry due to their interesting biological properties. Given the amphiphilic nature of trehalose lipids, the understanding of the molecular mechanism of their biological action requires that the interaction between biosurfactant and membranes is known. In this study we examine the interactions between a trehalose lipid from Rhodococcus sp. and dimyristoylphosphatidylglycerol membranes by means of differential scanning calorimetry, X-ray diffraction, infrared spectroscopy and fluorescence polarization. We report that there are extensive interactions between trehalose lipid and dimyristoylphosphatidylglycerol involving the perturbation of the thermotropic gel to liquid-crystalline phase transition of the phospholipid, the increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer, and the modulation of the order of the phospholipid bilayer. The observations are interpreted in terms of structural perturbations affecting the function of the membrane that might underline the biological actions of the trehalose lipid.     

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.     

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.