Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.     

Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26 degrees C (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (r infinity), and lifetime (tau) revealed that DPH reported a phase transition because of changes in r infinity, whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (greater than 25 ns) and short (less than 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55 degrees C) as the membrane passed through the phase transition, with 50% of the change occurring at 27 degrees C (c-PnA) or 33 degrees C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Charged anesthetics selectively alter plasma membrane order

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.     

The lateral fluidity of erythrocyte membranes. Temperature and pressure dependence

The pressure and temperature dependence of the lateral and rotational fluidity of erythrocyte membranes was investigated by inserting the excimeric membrane probe 1'-pyrenedodecanoic acid (PDA) into the membranes of intact cells and measuring the probe excimer formation rate and the steady-state polarization of the monomer at pressures up to 2000 atm (2 kbar). At that pressure the lateral diffusivity of PDA was found to decrease by a factor of 10 and its emission anisotropy by a factor of 5 at 22 degrees C. At atmospheric pressure, the local lateral diffusion coefficient of PDA at 2 and 33 degrees C is 1.5 and 4.3 x 10(-8) cm2 s-1, respectively. The activation energy for probe translation was found to decrease from 6 to 3 kcal M-1 in going from atmospheric pressure to 2 kbar, while the entropy decreased by approx. 15 cal M-1 K-1, indicating greater lipid order at the high pressure. The experimental data are consistent with a 'free-area' model for the membrane, analogous to the free-volume model for nonassociated liquids. The lateral diffusivity of PDA was found to be proportional to the free membrane area and linear extrapolation to zero diffusivity indicates that at atmospheric pressure, the fractional free area of the erythrocyte membrane is 6%.     

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

DPH标记法研究杀虫剂对二化螟线粒体膜流动性的影响

以DPH为荧光探剂,采用荧光偏振法研究了几种常用农药对二化螟Chilo supperssalis(Walekr)线粒体膜流动性的影响。结果表明,DPH是一种有效的荧光探剂,可以用来研究线粒体膜脂的流动性。不同种类的农药对二化螟线粒体膜的流动性都有一定的影响,但是以三氟氯氰菊酯、高效氯氰菊酯和硫丹影响较大,甲胺磷、三唑磷和克百威影响较小。三氟氯氰菊酯和高效氯氰菊酯可使膜的流动性下降,而硫丹、甲胺磷、三唑磷和克百威则使膜的流动性增强。对膜影响较大的三氟氯氰菊酯和硫丹对膜流动性的影响,还存在一定的剂量-效应关系。另外,膜的流动性受温度的影响很大,在温度分别为17、27、37℃的条件下,在药剂浓度为1×10-4mol/L时,甲胺磷在3个温度下对膜的流动性影响都很小,在误差范围内几乎没有影响;硫丹不同温度下都使膜的流动性增强,而三氟氯氰菊酯则使膜的流动性降低。     

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.     

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures

Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6-35 degrees C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38 MPa, a phase transition temperature of 12 +/- 2 degrees C was observed, which increased to 17 +/- 4 degrees C at 43.7 MPa, 21+/- 7 degrees C at 61.8 MPa, and 24 +/- 9 degrees C at an osmotic pressure of 133.1 MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.     

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of archaebacterial bipolar tetraether liposomes by perylene fluorescence

Membrane packing and dynamics of bipolar tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied by perylene fluorescence. At a probe-to-PLFE lipid ratio of 1:400, we have detected an unusual fluorescence intensity increase with increasing temperature, while the fluorescence lifetime changed little. As the ratio was decreased, the intensity anomaly was diminished. At 1:3200 and 1:6400, the anomaly disappeared. A remarkable perylene intensity anomaly was also observed in bilayers composed of saturated monopolar diester phosphatidylcholines at their main phase transition temperatures. These results suggest that the intensity anomaly may be due to probe aggregation caused by tight membrane packing. At the same probe-to-lipid ratio (1:400), however, 1, 2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1, 2-diphytanoyl-sn-glycero-3-phosphoglycerol (DPhPG) liposomes did not exhibit any intensity anomaly with increasing temperature. This suggests that DPhPC and DPhPG liposomes are more loosely packed than PLFE liposomes; thus the branched methyl groups are not the contributing factor of the tight membrane packing found in PLFE liposomes. Using a multiexcitation method, we have also determined the average (R), in-plane (R(ip)), and out-of-plane (R(op)) rotational rates of perylene in PLFE liposomes at various temperatures (20-65 degrees C). R and R(ip), determined at two different probe-to-lipid ratios (1:400 and 1:3200), both undergo an abrupt increase when the temperature is elevated to approximately 48 degrees C. These data suggest that PLFE liposomes are rigid and tightly packed at low temperatures, but they begin to possess appreciable "membrane fluidity" at temperatures close to the minimum growth temperature ( approximately 50 degrees C) of thermoacidophilic archaebacteria.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Combined cold, acid, ethanol shocks in Oenococcus oeni: effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 degrees C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 degrees C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 degrees C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

Order measurements in plasma membranes from Duchenne dystrophy fibroblasts

Plasma membranes have been isolated using different methods from Duchenne dystrophy and control human skin fibroblasts. Fluorescence techniques were utilized to resolve the rotational properties and the degree of hindered rotation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene in the membranes. Under specific conditions of fibroblast processing and membrane fractionation, plasma membranes from Duchenne fibroblasts showed significantly less order (0.0125 greater than P less than 0.0025) and less hindrance to probe rotation than membranes from control fibroblasts. The order differences did not seem to be the result of heterogeneity in the membrane environment sampled by the probe. The frequency dependence of the fluorescence lifetime for diphenylhexatriene indicated no measurable contribution by a short lifetime component. Analysis of diphenylhexatriene rotation in the plasma membranes using the 'wobbling-in-cone' theory suggested that both the angle of probe rotation (theta c) and the rotational rate (Dw) were important parameters in understanding the variations between Duchenne and control membranes at 16, 22 and 30 degrees C. Electron spin resonance studies with 5'-doxylstearic acid at 25 degrees C confirmed our fluorescence results. The segmental motion exhibited by the spin label revealed less order in the Duchenne membranes.     

Rotational relaxation times of 1,6-diphenyl-1,3,5-hexatriene in phospholipids isolated from LM cell membranes. Effects of phospholipid polar head-group and fatty acid composition.

Phospholipids were isolated from mitochondrial, microsomal, and plasma membranes of LM cells and fractionated into individual phospholipid classes on silicic acid columns. The fatty acid composition and the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each phospholipid class. Sphingomyelin was the only phospholipid isolated from LM cell membranes that showed a phase transition within the temperature range investigated, 5-40 degrees C. The rotational relaxation times for DPH were lowest in phosphatidylcholine in all the membrane fractions. Phosphatidylcholine isolated from the three membrane fractions of choline-supplemented cells had similar rotational relaxation times and phosphatidylcholine isolated from microsomal membranes of linoleate-supplemented cells had lower rotational relaxation times. The results indicate that the differences in the rotational relaxation times of DPH between mitochondrial, microsomal, and plasma membrane phospholipids could be explained primarily by differences in the polar head-group composition, while differences in the fatty acid composition had only a minor effect. This provides a basis for understanding how the different lipid components in these cells contribute to membrane fluidity.