Physical State Of Membrane Lipids Research Articles

Membrane effects of zinc oxide nanorods and nanoparticles in human lymphocytes

The study of the physical state of membrane lipids after the exposure of lymphocytes to ZnO NRs revealed a decrease in the lipid microviscosity of the hydrophilic membrane area and its increase in the hydrophobic area (external monolayer). The effects of ZnO NPs on membrane lipid components were not significant. Investigations of the membrane proteins conformation state revealed a rise of the protein NH 2 -groups level on the membrane surface only after the lymphocytes exposure to ZnO NRs. The level of SH-groups of the membrane protein after the action of ZnO NPs increased, otherwise the interaction of ZnO NRs with cells leads to the oxidation of thiol groups. According to the results of SEM, the geometric size of ZnO NPs did not exceed 30-100 nm, the diameter of ZnO NRs was 70-150 nm and the length - more than 500 nm. So the obtained results of toxicological tests can be partly explained by the possibility of ZnO NPs to enter into the cell but the most probable mechanisms for the NRs interaction with the cell are an electrostatic interaction or membrane puncturing.

28. Using Synchroton infrared microspectroscopy to better understand the freezing-resistance of lactic acid bacteria

Freezing is commonly applied to preserve the functionalities of concentrates of lactic acid bacteria (LAB). However, it is still a critical step in the production of LAB concentrates, as it affects both the viability and acidifying activity upon thawing. Several environmental factors influence the resistance to freezing of LAB (strain, medium composition, temperature, etc.) but the mechanisms of cell injury during freezing need to be elucidated. Cell dehydration taking place during freezing is an important event responsible for alterations of the physical state of membrane lipids and proteins, which, in turn, affect lipid organization and membrane fluidity. FTIR (Fourier Transformed InfraRed) spectroscopy and imaging technology appear to be powerful tools for investigating the biophysical response of LAB and for identifying some key issues in the understanding of the main mechanisms of bacteria degradation during freezing and thawing. In situ and in conditions close to industrial ones, FTIR spectroscopy has recently made it possible to investigate the modifications of the physical state of bacterial lipids of whole cell populations of Lactobacillus delbrueckii subsp. bulgaricus CFL1 (freezing sensitive strain) during freezing and thawing for two different physiological states, corresponding to different behaviours in terms of resistance to freezing and thawing processes. Fresh cells presenting the better resistance to freezing exhibited the lower lipid phase transition temperature, thus remaining in a liquid crystalline state for a longer time during the freezing process (Gautier et al. 2012). This study performed on bacterial suspensions and in real time during freeze–thaw processes, highlighted the difficulties for identifying and quantifying protein conformational changes because of the overlapping of the water band with the protein band. The objective of the present work was to investigate the changes in the secondary structure of proteins of some individual cells of Lb. bulgaricus CFL1 for these two extreme conditions, by using the brilliant Synchrotron infrared beamline SMIS (SOLEIL proposal number 20100789). High reflective index hemispheres (ZnSe) were used in order to get high spatial resolution (2–3 μm 2 ) making possible the quantification of the population heterogeneity and mapping of biophysical and chemical responses of small groups of cells. Concentrated suspensions of Lb. bulgaricus CFL1 were produced using standard procedure of fermentation in MRS broth or mild whey medium at 42 °C in the biology laboratory of SOLEIL Synchrotron. The bacteria were harvested at the beginning of the stationary phase growth, formulated (addition of protective medium), frozen by immersion in liquid nitrogen and stored at −20 and −80 °C. After thawing and washing, cells were dried onto ZnSe hemispheres. ATR (attenuated total reflectance) analysis on the amide I region of dehydrated bacterial suspensions was carried out to examine changes in the protein secondary structures. The impact of the physiological state (viability and acidifying activity) on cell biophysical behaviour and freezing-resistance was investigated by analysing samples before and after each storage conditions. Better resistance to freezing, characterized by a higher acidifying activity and cell survival, was observed for cells presenting a higher proportion of proteins in α-helix conformation. Furthermore, a study of the impact of freezing on the protein secondary structures is in progress.

Growth Temperature and Salinity Impact Fatty Acid Composition and Degree of Unsaturation in Peanut‐Nodulating Rhizobia

Growth and survival of bacteria depend on homeostasis of membrane lipids, and the capacity to adjust lipid composition to adapt to various environmental stresses. Membrane fluidity is regulated in part by the ratio of unsaturated to saturated fatty acids present in membrane lipids. Here, we studied the effects of high growth temperature and salinity (NaCl) stress, separately or in combination, on fatty acids composition and de novo synthesis in two peanut-nodulating Bradyrhizobium strains (fast-growing TAL1000 and slow-growing SEMIA6144). Both strains contained the fatty acids palmitic, stearic, and cis-vaccenic + oleic. TAL1000 also contained eicosatrienoic acid and cyclopropane fatty acid. The most striking change, in both strains, was a decreased percentage of cis-vaccenic + oleic (≥ 80% for TAL1000), and an associated increase in saturated fatty acids, under high growth temperature or combined conditions. Cyclopropane fatty acid was significantly increased in TAL1000 under the above conditions. De novo synthesis of fatty acids was shifted to the synthesis of a higher proportion of saturated fatty acids under all tested conditions, but to a lesser degree for SEMIA6144 compared to TAL1000. The major adaptive response of these rhizobial strains to increased temperature and salinity was an altered degree of fatty acid unsaturation, to maintain the normal physical state of membrane lipids.

Functional in vitro assembly of the integral membrane bacterial thermosensor DesK

The Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coli in vitro transcription–translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 °C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.

The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp PCC 6803.

The stress imposed on living organisms by hyperosmotic conditions and low temperature appears to be perceived via changes in the physical state of membrane lipids. We compared genome-wide patterns of transcription between wild-type Synechocystis sp. PCC 6803 and cells with a mutation in the histidine kinase Hik33 using a DNA microarray. Our results indicated that Hik33 regulated the expression of both osmostress-inducible and cold-inducible genes. The respective genes that were regulated by Hik33 under hyperosmotic and low-temperature conditions were, for the most part, different from one another. However, Hik33 also regulated the expression of a set of genes whose expression was induced both by osmotic stress and by cold stress. These results indicate that Hik33 is involved in responses to osmotic stress and low-temperature stress but that the mechanisms of the responses differ.

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran

Utilization of a fluorescence dye, 1,3-diphenylisobenzofuran (DPBF) as a detector of superoxide anion radical (O ⋅− 2) was examined. The fluorescence intensity of DPBF incorporated in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) is effectively quenched by incubation with xanthine/xanthine oxidase system. On the other hand, xanthine or xanthine oxidase alone did not induce quenching of the DPBF fluorescence in the liposomes. Xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes was almost completely protected by the addition of superoxide dismutase (SOD, 1 U/ml), but not by heat-denatured SOD (10 min boiling) at the same concentration. On the other hand, catalase (1 U/ml), and hydroxyl radical and singlet oxygen scavengers (10 mM sodium benzoate, 300 mM mannitol, 1 mM tryptophan and 1 mM sodium azide) did not protect xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes. The concentration dependence profiles of xanthine oxidase on the DPBF fluorescence quenching and O ⋅− 2 generation showed that there is a good correlation between these parameters. Under the present experimental conditions, approximately 7 μM H 2O 2/30 min were produced, but the addition of H 2O 2 (1 mM) to DPBF-labeled liposomes did not quench the dye fluorescence in the liposomes. Temperature dependence profiles of the DPBF fluorescence quenching induced by xanthine/xanthine oxidase treatment and the excimer fluorescence formation of pyrene molecules embedded in the liposomal membrane suggested that the quenching efficiency of the DPBF fluorescence is largely dependent on their lipid dynamics. Based on these results, we proposed the possibility that DPBF fluorescence quenching method is able to be used as a simple method for detecting O ⋅− 2 inside the membrane lipid layer and that DPBF fluorescence quenching by O ⋅− 2 is controlled by the physical state of membrane lipids.

Inverse relationship between membrane lipid fluidity and activity of Na+/H+ exchangers, NHE1 and NHE3, in transfected fibroblasts.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Modulation of Lipid Unsaturation and Membrane Fluid State in Mammalian Cells by Stable Transformation with the Δ9-Desaturase Gene ofSaccharomyces cerevisiae

The composition and physical state of membrane lipids determine the dynamic nature of membranes, which in turn, could directly be linked to the activity of various membrane-associated cellular functions. To better understand the molecular basis of different membrane-related phenomena we established a novel strategy to alter unsaturation of mammalian cell membranes with an identical genetic background. We transfected L929 mouse fibroblastoid cells with DNA constructs containing the Δ9-fatty acid desaturase gene (Ole1) ofS. cerevisiaeunder the control of desaturase promoters derived either from wild type or mutant strains of the dimorphic fungusH. capsulatum.

Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death.

Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1'-dihexadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiIC[16]) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC(16) stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3x10(-9) cm2/second) than on the midpiece and principal piece (11.8x10(-9) cm2/second). Recovery (R) is >90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20 degrees C and 37 degrees C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R<25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.

Seasonal variations of physical and biochemical membrane properties in trout erythrocytes ( Salmo irideus)

Erythrocyte membranes from Salmo irideus trout were analysed month by month, focusing on (1) Na +,K +-ATPase activity; (2) cholesterol content; (3) generalised polarization of laurdan, reflecting membrane lipid polarity and (4) 1,6-diphenyl-1,3,5-hexatriene steady-state fluorescence anisotropy, related to “membrane fluidity.” Properties (2) and (3) showed clear maxima during winter, property (1) during summer, whereas property (4) was not much influenced by season. Clearly, Na +,K +-ATPase activity of trout erythrocytes is influenced by the physical state of membrane lipids on a monthly scale.

Adaptational Changes of Fatty Acid Composition and the Physical State of Membrane Lipids Following the Change of Growth Temperature in Yersinia enterocolitica

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

Water permeation in Madin-Darby canine kidney cells is modulated by membrane fluidity

Simultaneous determinations of water and antipyrine permeations in monolayers of Madin-Darby canine kidney (MDCK) cells grown on a permeant support were done to study the relationships between water transport and membrane fluidity in these epithelial cells. The changes in permeation of the lipophilic non-electrolyte antipyrine were used to probe the modifications in membrane fluidity. In controls, the apparent diffusional permeability coefficient for water ( P Dw) was three times higher than the antipyrine's one, P DAp (4.2 · 10 −5 vs. 1.4 · 10 −5 cm s −1). Addition of vasopressin or dibutyryl cyclic AMP to the monolayers induced a biphasic increase in antipyrine permeation with peak values at t = 2min, 3–4-fold that of controls. Variations in water permeation were of similar amplitude and obeyed the same time course, leaving the water to antipyrine permeation ratios unchanged. Compound H7, an inhibitor of protein kinases, blunted the increase in permeation for both antipyrine and water. Finally, addition of the fluidizing agent benzyl alcohol to the monolayers resulted in a parallel increase in P DAp and P Dw. These results suggest that the physical state of membrane lipids may control water permeation in MDCK cells.

Effect of K+-depolarization on the physical state of membrane lipids in rat brain synaptosomes

The cardiodepressive effect of PAF has been studied on the electrical and mechanical activities of isolated auricles of guinea pig. Intracellular resting potential, action potential (AP) and isometric contractions elicited by electrical stimulation (0.5 Hz) were measured. PAF (10(-7) M) induced negative inotropic effect, which reached its peak after 5 min with 23.5 +/- 6.6% in respect to prechallenge values (n = 8). After 20 min negative inotropic effect relaxed to 39.6 +/- 8.8%. 1 min after the beginning of washing in Tyrode solution, positive inotropic effect of PAF was evident, that reached its peak (217 +/- 49.5%) after 2 min, decayed after 5-10 min to normal values. PAF did not modify the resting membrane potential, produced a decrease in the amplitude and Vmax of the upstroke AP, shortened the AP duration. Ca-AP and contractions, elicited in partially depolarized myocardium were decreased by PAF (10(-7) M). PAF-produce the change of the AP and the negative effect on auricle contractile force was inhibited in muscles pretreated with 3mM 4 aminopyridine. Histamine (10(-4) M) was also capable of neutralizing the depressant effect of PAF. The obtained results suggested that PAF effects on the membrane of cardiac cells could be related to a change in Ca and K conductance.

Vitamin E, membrane order, and antioxidant behavior in lung microsomes and reconstituted lipid vesicles

Vitamin E, a dietary antioxidant, is known to inhibit peroxidation of membrane lipids and to protect the lungs of vitamin E-deficient animals and to a lesser extent vitamin E-sufficient animals from oxidant injury. Since the protective interaction between vitamin E and biological membranes may be related to alterations in composition and physical state of membrane lipids, we evaluated the effect of vitamin E deficiency on lung microsomal lipids and membrane fluidity. Both intact microsomes and lipid vesicles prepared from the total lipid extracts of these microsomes were used. The percentage incorporation of vitamin E and cholesterol, membrane fluidity, and lipid peroxidation were measured in microsomes as well as their lipid vesicles. Fluidity was measured by monitoring changes in fluorescence anisotropy for 1,6-diphenyl-1,3,5-hexatriene (DPH). Lipid peroxidation was measured by thiobarbituric acid reaction. There were significant increases in the phospholipid ( p < 0.01), the total cholesterol ( p < 0.05), and the total saturated fatty acids ( p < 0.05) and decreases in total polyunsaturated fatty acid ( p < 0.01) content of vitamin E-deficient microsomes. There were no detectable peroxidative products in freshly isolated microsomes from either vitamin E-sufficient or -deficient lungs. However, lipids from vitamin E-deficient microsomal membranes were more susceptible to free radical initiated peroxidation than lipids from vitamin E-sufficient microsomes. Fluidity in vitamin E-deficient microsomes or in their lipid vesicles was significantly ( p < 0.05) decreased compared to the respective controls. In vitamin E-deficient microsomes or their lipid vesicles, the incorporation rate of vitamin E was two- to three-fold greater than in vesicles of vitamin E-sufficient microsomes or their lipid vesicles. However, the percentage incorporation of cholesterol was identical in both vitamin E-deficient and vitamin E-sufficient microsomes or in their respective lipid vesicles. As a result of vitamin E incorporation, fluidity was significantly decreased ( p < 0.05) in vitamin E-sufficient vesicles and was further decreased ( p < 0.001) in vitamin E-deficient vesicles. Incorporation of cholesterol also decreased fluidity in both vitamin E-deficient and vitamin E-sufficient vesicles but to the same extent ( p < 0.001). Lipid peroxide formation was two-fold greater in the vitamin E-deficient than in the vitamin E-sufficient vesicles. The increased incorporation of vitamin E (but not cholesterol) diminished ( p < 0.001) lipid peroxide formation in both vitamin E-deficient and vitamin E-sufficient vesicles. These results suggest that alterations in the physical state of membrane lipids in vitamin E deficiency favors incorporation of more vitamin E. Although both vitamin E and cholesterol decreased membrane fluidity, only vitamin E protects against membrane lipid peroxidation.

Effect of tocopherol and taurine on membrane fluidity of retinal rod outer segments

The deficiency of taurine and alpha-tocopherol results in disturbances of the structure of retinal rod outer segments. In this study the effect of alpha-tocopherol (vitamin E) and taurine on the fluidity of membranes from frog retinal rod outer segments and of liposomes prepared with lecithin or with lipids from outer segment membranes was examined by steady-state fluorescence polarization of diphenyl-hexatriene (DPH). alpha-Tocopherol increased the DPH anisotropy parameter in both preparations. The vitamin modified the breakpoint temperature of Arrhenius plots of DPH anisotropy, and decreased the activation energy. Taurine failed to modify any of the parameters examined in both outer segment membranes and lecithin liposomes. These results suggest a stabilizer role for tocopherol in rod outer segment membranes. In contrast, the requirement of taurine to maintain outer segment structure seems unrelated to an effect on the physical state of membrane lipids.

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 degrees 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 degrees 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 degrees C for 10 to 17 days and then transferrd to 22 degrees C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3 degrees C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3 degrees 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.

Alterations in the physical state and composition of brush border membrane lipids of rat enterocytes during differentiation

The physical state of the membrane lipid of brush border membranes, prepared from rat small intestinal villus and crypt cells, was examined by steady-state fluorescence polarization using three lipid-soluble fluorophors. Membranes prepared from crypt cells were found to possess a higher lipid fluidity than those of villus cells with each probe. Analysis of the composition of these membranes revealed that those from crypt cells had lower ratios of cholesterol/phospholipid (mol/mol), protein/lipid ( w w ), and saturated fatty acyl chains/unsaturated chains ( w w ). Alterations in the levels of stearic (18:0) and oleic (18:1) acids were responsible for differences in the latter ratio. The results, therefore, demonstrate that alterations in the lipid composition and fluidity of brush border membranes of enterocytes occur during the process of differentiation.

Fatty acyl chain structure, orientational order, and the lipid phase transition in Acholeplasma laidlawii B membranes. A review of recent 19F nuclear magnetic resonance studies.

The orientational order parameters of monofluoropalmitic acids biosynthetically incorporated into membranes of Acholeplasma laidlawii B in the presence of a large excess of a variety of structurally diverse fatty acids have been determined via 19F nuclear magnetic resonance (19F NMR) spectroscopy. It is demonstrated that these monofluoropalmitic acids are relatively nonperturbing membrane probes based upon physical (differential scanning calorimetry), biochemical (membrane lipid analysis), and biological (growth studies) criteria. 19F NMR is shown to convey the same qualitative and quantitative picture of membrane lipid order provided by 2H-NMR techniques and to be sensitive to the structural characteristics of the membrane fatty acyl chains, as well as to the lipid phase transition. Representatives of each naturally occurring class of fatty acyl chain structures, including straight-chain saturated, methyl-branched, monounsaturated, and alicyclic-ring-substituted fatty acids, were studied and the 19F-NMR order parameters were correlated with the lipid phase transitions (determined calorimetrically). The lipid phase transition was the prime determinant of overall orientational order regardless of fatty acid structure. Effects upon orientational order attributable to specific structural substituents were discernible, but were secondary to the effects of the lipid phase transition. In the gel state, relative overall order was directly proportional to the temperature of the particular lipid phase transition. Not only the overall order, but also the order profile across the membrane was sensitive to the presence of particular structural substituents. In particular, in the gel state specific fatty acyl structures demonstrated a characteristic disordering effect in the membrane order profile. These various observations can be merged to provide a unified picture of the manner in which fatty acyl chain chemistry modulates the physical state of membrane lipids.

The effect of anaesthetics on protein conformation in membranes as studied by the spin-labelling technique

The effect of general and local anaesthetics was studied on the membrane components (proteins and lipids) of frog sciatic nerve using the spin-labelling technique. A rapid and effective method was developed to incorporate fatty acid labels into the lipid regions and to attach the maleimide spin labels to the thiol sites of the proteins of the membrane. On the basis of the rotational correlation time of the attached maleimide spin labels, at least three different protein domains were identified. One part of the labels has a preferred orientation with respect to the axis of the nerve fibre. The effect of halothane and of local anaesthetics such as lidocaine and tetracaine, which influence primarily the lipid regions of the membrane, is efficiently transferred to the spin-labelled membrane proteins via strong lipid-protein interaction. The results support the concept that the architecture and the physiological activity of the membrane-bound proteins are sensitive to changes in the physical state of membrane lipids.