Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca²⁺ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca²⁺ concentration. The high values of the order parameters found in intact platelets (S_{DPH, 36.c}=0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDP, in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous microviscosity values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (DPH, 36°C =–0–5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipidprotein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25° to 36°C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36°-40°C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca²⁺ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (2%) of the order parameter of CE-DPH for Ca²⁺ concentrations in the millimolar range.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[(4-trimethyl-amino)phenyl]-6-phenyl-1,3,5-hexatriene - CE-DPH (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene - 16AP 16-(9-anthroyloxy)-palmitic acid; c-PnA, CIS-parinaric acid; t-PnA, trans-parinaric acid - PER perylene - POPOP p-bis[2(5-phenyl-oxazolyl)benzene] - ESR electron spin resonance Offprint requests to: A. U. Acuña     

Lateral heterogeneity in human platelet plasma membrane and lipids from the time-resolved fluorescence of trans-parinaric acid

We have investigated the complex behaviour of the time resolved fluorescence intensity and anisotropy of trans-parinaric acid, incorporated into fragments of the plasma membrane of human platelets and in multibilayers of lipids extracted from that membrane. It is shown that the observation of anisotropies that increase at long times can be satisfactorily interpreted by assuming two populations of the fluorescence probe with distinct lifetimes, rotational relaxation times and order parameters. The heterogeneous probe distribution was correlated with a similar heterogeneity in the lipid composition of the bilayer, modulated by temperature. Below 35°C an important fraction of the lipids of the plasma membrane are apparently in the form of solid-like domains (20% at 20°C). However, in the physiological temperature range that solid/fluid heterogeneity is almost negligible. Since these effects were also observed in multibilayers of lipids from the platelet membrane, the formation of solid-like clusters appears to arise from lipid-lipid interactions only, and most probably involving cholesterol. These results support the previous finding of a lateral phase separation for temperatures less than 37°C described by Gordon et al. (1983) in a spin-probe study of the platelet plasma membrane.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - DPPC dipalmitoyl-phosphatidylcholine - tPnA trans-parinaric acid Offprint requests to: A. U. Acuña     

Changes in the lipid physical state in human erythrocyte membranes subjected lead exposure in vitro

The effect of lead acetate on the physical state of membrane lipids in human erythrocytes in vitro was studied using the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and spin probes 16-doxyl-stearate and iminoxyl palmitic acid. It was shown that 2-10 microM lead acetate causes an increase in both intensity and polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, indicating changes in the microviscosity of the lipid bilayer of erythrocyte membranes. Judging from the parameters of EPR spectra of 16-doxyl stearate and iminoxyl palmitic acid incorporated into erythrocyte membranes, 2-10 microM lead acetate increases the heterogeneity of the lipid bilayer in surface and deep hydrophobic layers of the erythrocyte membrane.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

Partition coefficients of fluorescent probes with phospholipid membranes

A method for determination of membrane partition coefficients of five fluorescent membrane probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic acid), 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH propionic acid), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide (4-di-10-ASP), was developed utilizing the fluorescence enhancement of a constant probe concentration by titration with excess phospholipid liposomes. The partition coefficients of DPH, DPH carboxylic acid, DPH propionic acid, TMA-DPH and 4-di-10-ASP into dipalmitoylphosphatidylcholine membranes were determined to be 1.3.10(6), 1.0.10(6), 6.5.10(5), 2.4.10(5) and 2.8.10(6) respectively. Knowledge of the partition coefficients may help select a lipid concentration for membrane studies that necessitate a probe's dominant incorporation into membranes.     

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.     

Perturbing probes and the study of lipid-protein bilayer membranes

Recent experimental and theoretical developments concerning perturbing probes are outlined. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene and nitroxide electron paramagnetic resonance spin labels attached to lipid hydrocarbon chains are taken as the most widely used examples of such probes. The reliability of these probes as indicators of the statics and dynamics of unlabelled lipid hydrocarbon chains is discussed, and the use of such probes in giving information about protein size, protein oligomerization and protein lateral distribution is outlined. Examples are given of studies to determine protein packing in lipid bilayers membranes.     

Changes of membrane fluidity in chemotactic peptide-stimulated polymorphonuclear leukocytes

Although the phenomenon of stimulus-response coupling in polymorphonuclear leukocytes involves a series of membrane events the influence of stimulation on membrane fluidity is to clarify. In our experiments we have used 1-(4-trimethylaminophenyl) 6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization technique to evaluate membrane fluidity in living polymorphonuclear leukocytes after stimulation with N-formyl-methyonil-leucyl-phenylalanine peptide which has a well defined membrane receptor on the plasma membrane. We report that polymorphonuclear leukocytes stimulation increases 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene polarization, only when colcemid, a microtubule disrupting drug, is added to polymorphonuclear leukocytes. This can be viewed as an indirect evidence that microtubules are involved in the control of polymorphonuclear leukocytes membrane fluidity. On the contrary no changes have been observed with 1,6-diphenyl-1,3,5-hexatriene. This study indicates the potential use of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene to evaluate the involvement of plasma membrane physical state during intact cell activity.     

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

Various models for the analysis of time-dependent fluorescence anisotropy measurements were evaluated. The discussion was based on the analysis of pulsed experiments with 1,6-diphenyl-1,3,5-hexatriene embedded in small unilamellar vesicles of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine and in dimyristoylphosphatidylcholine/alpha-lactalbumin complexes. It was shown that a recently proposed model (Van der Meer, W., H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx, H. Schröder, 1984, Biophys. J., 46:515-523) described the data better than did the earlier suggested cone model (Kinosita K., Jr., S. Kawato, and A. Ikegami, 1977, Biophys. J., 20:289-305). This permitted the use of the new model for the estimation of the second- and fourth-rank order parameters on nonoriented systems. The results indicated that a fraction of the probes was oriented perpendicularly to the preferred direction of the lipids. An increase of the rotational correlation times of the fluorescent probe and a higher order of its environment were detected after the interaction of alpha-lactalbumin with the dimyristoylphosphatidylcholine vesicles at acidic pH at 24.2 degrees C.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

Incorporation of sterol into chloroplast thylakoid membranes and its effect on fluidity and function

The sterol, cholesteryl hemisuccinate, has been incorporated into isolated thylakoid membranes of pea and lettuce chloroplasts in order to modify the fluidity of the lipid matrix. Changes in fluidity have been monitored using fluorescence polarization of the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene and the electron-spin-resonance, spin-label probe, 5-doxyl stearate. Both methods indicate that incorporation of increasing levels of sterol reduces the fluidity of the thylakoid lipid matrix. At room temperature the thylakoid lipid matrix is relatively fluid and the effect of increasing the viscosity is to inhibit partially the maximum rate of steady-state electron flow and reduce the dark rate of reduction of flash-oxidised cytochrome f. The results are discussed in terms of lipid fluidity influencing the rate of lateral diffusion of reduced plastoquinone from photosystem II to photosystem I.     

Ionizing Radiation Alters the Properties of Sodium Channels in Rat Brain Synaptosomes

The effect of ionizing radiation on neuronal membrane function was assessed by measurement of neurotoxin-stimulated 22Na+ uptake by rat brain synaptosomes. High-energy electrons and gamma photons were equally effective in reducing the maximal uptake of 22Na+ with no significant change in the affinity of veratridine for its binding site in the channel. Ionizing radiation reduced the veratridine-stimulated uptake at the earliest times measured (3 and 5 s), when the rate of uptake was greatest. Batrachotoxin-stimulated 22Na+ uptake was less sensitive to inhibition by radiation. The binding of [3H]saxitoxin to its receptor in the sodium channel was unaffected by exposure to ionizing radiation. The effect of ionizing radiation on the lipid order of rat brain synaptic plasma membranes was measured by the fluorescence polarization of the molecular probes 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene. A dose of radiation that reduced the veratridine-stimulated uptake of 22Na+ had no effect on the fluorescence polarization of either probe. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive sodium channels in rat brain synaptosomes. This effect of radiation is not dependent on changes in the order of membrane lipids.     

Interaction of the herbicide atrazine with model membranes. I: Physico-chemical studies on dipalmitoyl phosphatidylcholine liposomes

Atrazine (2-chloro-4 ethylamino-6-(isopropylamino)-s-triazine) is one of the most widely used herbicides. Fourier transform infrared spectroscopy, differential scanning calorimetry and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were used to study the interaction of atrazine with dipalmitoyl phosphatidylcholine liposomes used as a model for biological membranes. The results show that atrazine does not perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide localizes near the glycerol backbone of the lipid.     

The use of the fluorescent probe α-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides

α-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of α-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature.The rotational mobility of α-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50°C, a region where no phase transitions were detectable. The rotational mobility of α-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2–3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

Interaction of alpha-tocopherol with model human high-density lipoproteins.

The effects of alpha-tocopherol on the properties of model high-density lipoproteins (HDLs), composed of human apolipoprotein A-I and dimyristoylphosphatidylcholine, were investigated by physicochemical methods. The intrinsic fluorescence of alpha-tocopherol and its effects on the polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, which probes the hydrocarbon region of the lipids, and 4-heptadecyl-7-hydroxycoumarin, which is a probe of lipid surfaces, suggest that alpha-tocopherol is located at the lipid-water interface. Relative to cholesterol, alpha-tocopherol in lipid surfaces is virtually inert physicochemically. Incorporation of alpha-tocopherol into HDLs induces only a modest increase in particle size, no change in the transition temperature, and little change in lipid polarity and lipid-lipid interactions. Moreover, alpha-tocopherol has only a negligible effect on the kinetic parameters of the lipophilic enzyme lecithin:cholesterol acyltransferase, which binds to phosphatidylcholine surfaces and forms cholesteryl esters. However, alpha-tocopherol has a dramatic inhibitory effect on the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine, a process that occurs through the insertion of the protein into preformed defects in the lipid surface. It is proposed that alpha-tocopherol inhibits the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine by inserting into defects within the lipid surface, thereby reducing the size and/or number of sites for insertion of apolipoprotein A-I.