Adaptation of biological membranes to temperature. The lack of homeoviscous adaptation in the sarcoplasmic reticulum

Temperature adaptation of biological membranes was examined by comparing the fragmented sarcoplasmic reticulum preparation of goldfish acclimated to different temperatures. Membrane fluidity was estimated using the fluorescence polarization technique. There was considerable variation between preparations, but no consistent differences in fluidity were observed between 5- and 25°C-acclimated goldfish, fish species adapted over an evolutionary period to arctic or desert temperatures, and rat. The fatty acid composition of the sarcoplasmic reticulum preparations of differently acclimated goldfish showed differences in the proportion of mono- and polyunsaturated fatty acids while the proportion of saturated fatty acids remained relatively constant. However, the fatty acid composition of sarcoplasmic reticulum phosphoglycerides became more unsaturated in the order rat, desert pupfish, arctic sculpin, which correlates with their respective environmental or body temperature. It is concluded that differences in membrane components other than fatty acids are important in determining membrane dynamic structure. The inability to demonstrate homeoviscous adaptation in sarcoplasmic reticulum is supported by other evidence suggesting that functions of the sarcoplasmic reticulum that are measured in vitro are not affected by such modifications of their phosphoglyceride fatty acid composition as occur during thermal acclimation.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

Polyunsaturated fatty acyl residues of galactolipids are involved in the control of bilayer/non-bilayer lipid transitions in higher plant chloroplasts

Total polar lipid extracts of chloroplasts isolated from broad beans (Vicia faba) tend to form non-bilayer structures when dispersed in dilute salt solutions. Monoglactosyldiacylglycerol is shown to play a dominant role in this process. The tendency of this lipid to form non-bilayer structures when dispersed alone in water was found to depend upon the degree of unsaturation of its associated fatty acyl chains. Highly unsaturated lipids (average number of double bonds per lipid molecule greater than about 5.0) form inverted hexagonal (Hex_II) structures in water at 20°C, whilst more saturated lipids (average number of double bonds per lipid molecule less than about 4.5) form lamellar sheets. Wide-angle X-ray diffraction and differential scanning calorimetry measurements indicate that these lamellae consist of gel-phase lipid that can adopt either of two structures depending on the thermal history of the sample. Freeze-fracture studies performed on total polar lipid extracts that have been hydrogenated using Adams' catalyst, and reconstituted extracts in which monogalactosyldiacylglycerol has been selectively hydrogenated, show that the degree of unsaturation of this lipid is a key factor in determining whether or not non-bilayer structures are formed in such extracts. Increasing the extent of saturation of the acyl residues of monogalactosyldiacylglycerol reduces the tendency to form non-bilayer structures. Similar effects are observed on lowering the temperature of the dispersions. Fluorescence polarisation measurements using 1,6-diphenyl-1,3,5-hexatriene indicate that the disappearance of non-bilayer structures is accompanied by a marked decrease in the fluidity of the lipid matrix. The possible significance of these observations is discussed in terms of the thermal adaptation and chilling sensitivity of plant membranes.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.     

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495–501). A light fraction F₁ was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (F_II and F_IV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of F_II and F_IV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Limited rotational motion of amphiphilic flavins in dipalmitoylphosphatidylcholine vesicles

The rotational motion of amphiphilic flavins in dipalmitoyl phospholipid bilayers was investigated with fluorescence anisotropy decay measurements. At temperatures between 10 and 50°C the rotation proved to be anisotropic, which indicated composite motion of both the aliphatic side-chain and the isoalloxazine moiety of the octadecyllumiflavin derivatives. Above the phase transition temperature (crystalline→liquid-crystalline state) the depolarization is complete within the average flavin fluorescence lifetime, implicating unrestricted motion and resulting in a non-ordered microenvironment. In the gel or crystalline state the flavin motion can best be characterized as a limited rotation or librational motion. The fluorescence decay of the flavins is heterogeneous at temperatures between 10 and 50°C, which is explained by assuming nanosecond relaxation of the polar phosphatidyl head-groups around the excited flavin. The lack of a significant cholesterol effect suggests that the isoalloxazine is located at the interphase of the bilayer and not in the hydrocarbon region. The microstructure is fluid-like, not in agreement with a preferred static localization of the flavins in the bilayer.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Organization and dynamics in micellar structural transition monitored by pyrene fluorescence

Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be induced in charged micelles at a given temperature by increasing ionic strength of the medium. We have monitored the change in organization and dynamics associated with sphere-to-rod transition of SDS micelles utilizing pyrene fluorescence. We report here, utilizing changes in the ratio of pyrene vibronic peak intensities (I₁/I₃), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be ∼32. Interestingly, the apparent micellar dielectric constant exhibits a reduction with increasing NaCl concentration. The dielectric constant in rod-shaped micelles of SDS (in presence of 0.5 M NaCl) turns out to be ∼22. To the best of our knowledge, these results constitute one of the early reports on polarity estimates in rod-shaped micelles. In addition, pyrene excimer/monomer ratio shows increase in SDS micelles with increasing NaCl concentration. We interpret this increase due to an increase in average number of pyrene molecules per micelle associated with the sphere-to-rod structural transition. These results could be significant in micellar drug solubilization and delivery, and in membrane morphology changes.     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast

We report sphingolipid-related reorganization of gel-like microdomains in the plasma membrane of living Saccharomyces cerevisiae using trans-Parinaric acid (t-PnA) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Compared to control, the gel-like domains were significantly reduced in the membrane of a sphingolipid-deficient lcb1-100 mutant. The same reduction resulted from sphingolipid depletion by myriocin. The phenotype could be reverted when a myriocin-induced block in sphingolipid biosynthesis was bypassed by exogenous dihydrosphingosine. Lipid order of less-ordered membrane regions decreased with sphingolipid depletion as well, as documented by DPH fluorescence anisotropy. The data indicate that organization of lateral microdomains is an essential physiological role of these structural lipids.     

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than $\text{2 · 10}{}^{\mathrm{?3}}$ wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and $\text{poly}\text{(N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.     

Difference in plasma membrane structure between two sublines of ehrlich-lettrè ascites tumor cells

The plasma membranes of the glycogen-free and the glycogen-containing subline of Ehrlich-Lettrè ascites cells were purified and compared with respect to their enzyme activity, chemical, lipid and protein composition, and membrane fluidity. Both membrane fractions differed in a number of parameters which are discussed as differences in the expression of malignant transformation of the two sublines. 1. The 5′-nucleotidase activity was 3–5-times higher and the sialic acid content 3-times lower in the glycogen-containing than in the glycogen-free subline. 2. Differences were also observed with respect to the phospholipid composition, that is in the relative proportions of mainly phosphatidylcholine, -inositol and -serine. 3. The fatty acid spectrum of the two sublines differed in the C-18 series and in the percentage of polyunsaturated acids, which was about 6% lower in the glycogen-containing line. 4. Measurements of fluorescence polarization (P) using 1,6-diphenyl-1,3,5-hextriene as probe generally gave higher P values, indicating a decreased membrane fluidity for the plasma membranes of the glycogen-containing subline both below and above the transition temperature at 33°C. 5. Polyacrylamide gel electrophoresis revealed different protein patterns mainly in the molecular weight range of around 90 000 and in the range between 31 000 and 14 000.