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.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes

Nanosecond decays of the fluorescence anisotropy, $\text{r}$, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.The relative contributions of the fast and the infinitely slow decaying component to the steady-state value, $\text{r}$, of the fluorescence anisotropy were very similar for artifical and biological membranes.Angles, θ, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with ‘microviscosities’, 〈η〉. An increase in 〈η〉 from 1.5 to 5.2 P in our systems was accompanied by a decrease in θ from 49° to 30° while the decrease in the mean motional relaxation times, $\text{φ}{}_{\mathrm{<mtext>f</mtext>}}$, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in ‘microviscosities’ of cholesterol-containing membranes ($\text{r}\text{> 0.15}$) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

Tests of the carrier model for ion transport by nonactin and trinactin

The fluxes of K⁺ and NH₄⁺ carried by nonactin and trinactin across thin lipid membranes have been measured as functions of ion activity, electric potential and time. In agreement with the predictions of a version of the carrier model in common use, the shape of the initial current-voltage relation is independent of the activity of the electrolyte, $\text{a}{}_{\mathrm{<mtext>i</mtext>}}$ while the ratio of the initial conductance, $\text{G}{}_0$, to the steady-state conductance, $\text{G}{}_{\mathrm{\infty }}$, increases according to $\text{G}{}_0\text{/G}{}_{\mathrm{\infty }}\text{=}\text{const}{}_1\text{+ const}{}_2\text{× a}{}_{\mathrm{<mtext>i</mtext>}}$. For trinactin the data presented allow the estimation of the rate constants of the carrier process (in the limit of zero potential) in a manner which does not assume any particular variation with potential for the constants. Using empirically determined functions of potential, a complete set of values is also available for nonactin. The curve fitting which is necessary is described in the following paper. The data presently available for valinomycin are sufficient neither to test the model nor to determine a complete set of constants.     

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$ efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$. (iii) When melittin tetramer is stabilized, it decreases $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ by 10–15 deg. C. The transition broadens, and is finally abolished for $\text{R}{}_{\mathrm{<mtext>i</mtext>}}\text{? 2}$. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, fluorescence and Raman show that melittin decreases the amount of CH₂ groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, while from Raman the positional intermolecular order decreases without significant change in the CH₂ groups ‘trans’/‘gauche’ ratio.     

Major intrinsic polypeptide of lens membrane: Biochemical and immunological characterization of the major cyanogen bromide fragment

A protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide from bovine lenses yields a major fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 15 000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide is buried within the lipid bilayer.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Influence of sterols on ion transport through lipid bilayer membranes

Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb⁺-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in $\text{n}$-hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant ${}_{\mathrm{<mtext>i</mtext>}}$ of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$. The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb⁺ transport only cholesterol had a strong influence on transport properties. The rate constants of association ($\text{k}{}_{\mathrm{<mtext>R</mtext>}}$) as well as the rate constants of translocation of the complex ($\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$) and of the free carrier ($\text{k}{}_{\mathrm{<mtext>S</mtext>}}$) were reduced by incorporation of cholesterol up to eight-fold. The decrease of $\text{k}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$ are possibly caused by a decrease of membrane fluidity, whereas the decrease of $\text{k}{}_{\mathrm{<mtext>R</mtext>}}$ may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer.     

Transport kinetics of dipicrylamine through lipid bilayer membranes. Effects of membrane structure

Charge-pulse current-relaxation studies have been performed with lipid bilayer membranes in the presence of the hydrophobic ion dipicrylamine. From the analysis of the relaxation times and amplitudes the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ of dipicrylamine as well as the partition coefficient β between membrane surface and water could be evaluated. In a first series of experiments membranes made from monoolein or dioleoylphosphatidylcholine in a number of different $\text{n-}\text{alkane}$ solvents were studied, as well as virtually solvent-free bilayer membranes made from monolayers. The thickness $\text{d}$ of the hydrocarbon layer of these membranes varied between 5.0 and 2.5 nm. While β was almost insensitive to variations in $\text{d}$, a strong decrease of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ with increasing membrane thickness was found; the observed dependence of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ on $\text{d}$ approximately agreed with the theoretically expected influence of membrane thickness on the height of the dielectric barrier. No specific differences between Mueller-Rudin films and solvent-free (Montal-Mueller) membranes other than differences in thickness were found. In a further series of experiments the chemical structure of the lipid was systematically varied (number and position of double bonds in the hydrocarbon chain, nature of the polar head group). The translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was much larger in phosphatidylethanolamine membranes than in phosphatidylcholine membranes. A strong increase of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was found when the number of double bonds in the hydrocarbon chain was increased from one to three. These changes were discussed in terms of membrane fluidity and dielectric barrier height. Much higher values of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ were observed in lipids with ester linkage between hydrocarbon chain and glycerol backbone, as compared with the corresponding ether analogs. This finding is qualitatively consistent with determinations of dipolar potentials in monolayers of ester and ether lipids. When cholesterol is added to phosphatidylcholine membranes, the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ increases up to five-fold, while the partition coefficient β remains virtually constant. The variation of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ in this case can be largely accounted for by a decrease in membrane thickness and a concomitant reduction in dielectric barrier height. In membranes made from the negatively charged lipid phosphatidylserine the partition coefficient of dipicrylamine strongly increased with ionic strength, as expected from the Gouy-Chapman theory of the surface potential.     

Transversal mobility in bilayer membrane vesicles: Use of pyrene lecithin as optical probe

Pyrene lecithin, a new excimer-forming lipid molecule, has been synthesized to examine the transversal mobility of probe molecules in lecithin bilayer vesicles. The rate of the lipid exchange is obtained by following the excimer yield as a function of time after mixing of fluorescence doped and undoped vesicles. A rapid exchange ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 11}\text{s}$) is followed by a slow transfer ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8}\text{h}$). Above the lipid phase transition the fast transfer can be attributed to an exchange of lipid molecules from the outer layer of one vesicle to the outer layer of another one. The slow exchange is interpreted in terms of the ‘flip-flop’ process between the two layers of a single bilayer vesicle.Using pyrene and pyrene decanoic acid as probe molecules only the fast transfer through the water phase is observed ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4}\text{s}$ for pyrene and $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7}\text{s}$ for pyrene decanoic acid). This indicates that molecules like fatty acids or apolar membrane constituents must equilibrate very rapidly in a single bilayer vesicle.The water solubility or the critical micelle concentration of the probe molecules is determined and related to the transfer rates. An exchange process through the water phase via a monomeric state can be excluded.     

The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity

The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=?0.94, P<0.0005}$) and a positive correlation for peritoneal lymphocytes ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=+0.97,P<0.0005}$) and spleen cells ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=+0.76,P<0.005}$) existed.     

Protein-catalyzed phospholipid exchange in bilayer vesicles determined by flow cytometry and electron microscopy

The protein-induced lipid transfer between phosphatidylcholine vesicles was investigated. Measurements of the degree of polarization at single vesicles were made by flow cytometry using diphenylhexatriene as the optical probe. Vesicles differing in phase transition temperature could be distinguished by their degree of polarization at a temperature where one population was in the fluid ($\text{T > T}{}_{\mathrm{<mtext>t</mtext>}}$) and the other one in the quasi-crystalline ($\text{T < T}{}_{\mathrm{<mtext>t</mtext>}}$) state. Besides vesicles containing exchanged lipids we also observed fractions of unaffected vesicles. The lipid exchange was visualized directly by freeze-fracture electron microscopy. The characteristic ‘ripple’ structure of phosphatidylcholine vesicles disappeared upon exchange with lipid in the fluid state.     

Deuterated phospholipids as raman spectroscopic probes of membrane structure: Phase diagrams for the dipalmitoyl phosphatidylcholine(and its d62 derivative)-dipalmitoyl phosphatidylethanolamine system

Raman spectroscopic techniques have been used to construct phase diagrams for the binary phospholipid systems, DPPC-d₆₂/DPPE and DPPC/DPPE (DPPC, dipalmitoyl phosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine). For the former, the half-width of the C-²H stretching modes of the deuterated component near 2100 cm^?1 serves as an indicator of phospholipid fluidity. The phase behavior is described semi-quantitatively using regular solution theory with the following non-ideality parameters:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.75}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=1.05}\text{kcal/mol}$

The use of deuterated phospholipids as one component of a binary mixture permits direct evaluation of the conformation of a particular component in the mixture throughout the phase separation region. The approach is demonstrated with the help of a simple model correlating the half-width of the symmetric C-²H stretching mode with the fraction of DPPC-d₆₂ hydrocarbon chains in the liquid crystalline state.The effect of chain perdeuteration on the phase behavior of DPPC with DPPE is evaluated by comparison of the phase diagram of the DPPC-d₆₂/DPPE system with that of DPPC-DPPE. The latter has been constructed previously from both probe and calorimetric techniques, and is created from the Raman spectroscopic data using the $\text{I(}\text{1130}\text{1100}\text{)}$ ratio to characterize the transgauche population ratio in non-deuterated hydrocarbon chains. A reasonable fit to the phase behavior is obtained using:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.85}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=0.90}\text{kcal/mol}$

The similarities of the non-ideality parameters in the two phase diagrams indicate that the effect of perdeuteration on the phase behavior of DPPC is not extensive. The use of deuterated phospholipids as essentially unperturbed components of a model membrane system is justified.     

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point ($\text{p}\text{I}$), apparent molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP_132–135^4–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ but different $\text{p}\text{I}$ were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.     

Quantum-chemical and empirical calculations on Phospholipids VIII. The electrostatic potential from isolated molecules up to layer systems

Using 1-6-12 empirical functions with a solvent-averaged electrostatic contribution $\text{q}{}_{\mathrm{I}}\text{q}{}_{\mathrm{j}}\text{ε(r}{}_{\mathrm{Ij}}\text{)}\text{× r}{}_{\mathrm{Ij}}$ and electrostatic potentials from CNDO-type wavefunctions, the development of specific interactions of ions visualized by the molecular electrostatic potential of PO₄-group containing molecules was studied. Going from single molecules to monolayers made up of 37 head groups of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) for quantum-chemical calculations, or of 23 head groups for empirical calculations we found decreasing potential minima. Only the inclusion of the screening effect of water, simulated by a distance dependent dielectric constant, ε(r), gives an explanation of stereospecific interactions of model membranes with ions. This finding can be compared with results of simulation calculations on water structure above a PE head group layer.     

Effect of para-chloromercuribenzenesulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules

The apparent Arrhenius energy of activation ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}$) of the water osmotic permeability ($\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$) of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ (kcal/mol) was $\text{3.2 ± 1.4}$ (controls) and $\text{9.2 ± 2.2}$ (pCMBS), while $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ decreased with pCMBS to $\text{0.26 ± 0.17}$ of its control value. Mersalyl also decreased $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}$ are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.     

Chloroplast membrane damage during freezing: the lipid phase

In order to study the effect of freeze damage to chloroplast membranes microviscosity of spinach thylakoids was probed by stearic acid spin labels. Changes in ESR parameters have been determined either as a function of temperature or during freezing at ?15 °C as a function of time. An empirical parameter $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ (ratio of height of a low field line component h₊ over height of the central line h₀) proved to be very sensitive to minute changes in membrane structure.In cryoprotected chloroplast membranes Arrhenius plot breaks indicative of phase changes are observed at +15 and ?10 °C. Breaks in the Arrhenius plots were not observed in vesicles prepared from chloroplast lipids by sonication. Instead, a melting zone was indicated below ?30 °C.Freeze damage of thylakoids during storage at ?15 °C is reflected in an increase of $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ and a decrease in central line width W₀. At +20 °C, differences between the ESR parameters of active as compared to freeze-damaged membranes could be detected, if the osmolarity of the suspending medium exceeded 200 mosm. The observed changes in line shapes are interpreted as an increase in mobility and/or orientation of the lipids following the swelling of thylakoids. They do not indicate a disorganization of the lipid phase. Sedimentation experiments indicated that the freeze-damaged swollen membranes still exhibited osmotic responses. It is suggested that freezing which is known to dissociate proteins from the membranes altered the charge distribution of the membranes leading first to membrane swelling and finally, by the opening of hydrophilic channels, to membrane collapse.     

Fluorescence polarization study of human erythrocyte membranes with 1-phenyl-3-(2-naphthyl)-2-pyrazoline as orientational probe

The emission and polarization spectra of 1-phenyl-3-(2-naphthyl)-2-pyrazoline (PNP) in various environments were studied. Compared to the widely used orientational membrane probe 1,6-diphenylhexatriene (DPH), PNP is five times less photolabile and since its fluorescence emission maximum is at longer wavelengths $\text{(γ}{}_{\max }\text{≈ 445}\text{nm}\text{)}$, it is more suitable for use with intact erythrocytes. The limiting fluorescence anisotropy of PNP is 0.385. In erythrocyte ghosts, the steady-state emission anisotropy of PNP is a decreasing function of wavelength and its temperature dependence parallels that of DPH, dropping from 0.298 at 2°C to 0.185 at 38°C when averaged between 420 and 470 nm.