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.     

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.     

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.     

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.     

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.     

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.     

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

Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-1,3,5-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 r, of the fluorescence anisotropy were very similar for artifical and biological membranes. Angles, theta, 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', (eta). An increase in (eta) from 1.5 to 5.2 P in our systems was accompanied by a decrease in theta from 49 degrees to 30 degrees while the decrease in the mean motional relaxation times, phi f, 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 (r greater than 0.15) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method

A method is described to measure the oxygen diffusion-concentration product, $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$, at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time $\text{T}{}_1$ of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to $\text{T}{}_1$ of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying $\text{T}{}_1$ are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$ at the main phase transition was observed.     

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.     

Correlation of the perceived texture of random coil polysaccharide solutions with objective parameters

A wide range of concentrated random coil polysaccharide solutions have been assessed for textural attributes by a trained sensory panel. The only textural terms invoked to describe these model systems were ‘thickness’ and ‘stickiness’, which were shown to be highly correlated, and essentially identical numerically, using a ratio scaling technique. Viscosity (η) measurements over a wide range of shear rates (γ) for all these samples gave flow curves (log η versus log γ) of the same form. Differences in flow behaviour between samples could then be characterised completely by two parameters, the maximum viscosity at low shear rates (η₀), and the shear rate ($\text{γ}\text{?}{}_{\mathrm{0·1}}$) at which $\text{η =}\text{solη}{}_0\text{10}$. A simple linear relationship was demonstrated between these two parameters and perceived thickness (T) or stickiness (S), irrespective of polysaccharide type. For Newtonian liquids, log T (or log S) varied linearly with log η. Hence the effective ‘in-mouth’ thickness of random coil polysaccharide solutions, in normal viscosity units, may be predicted directly from η₀ and $\text{γ}\text{?}{}_{\mathrm{0·1}}$ by the simple relationship: $\text{log}\text{η}{}_{\mathrm{N}}\text{= 1·13}\text{log}\text{η}{}_0\text{+ 0·45}\text{log}\text{γ}\text{?}{}_{\mathrm{0·1}}\text{? 1·72}$ where η_N is the viscosity of a Newtonian solution which would be perceived as identical in thickness (and stickiness) to the polysaccharide solution.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

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.     

Melittin-phospholipid interaction studied by employing the single tryptophan residue as an intrinsic fluorescent probe

The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, $\text{k}{}_{\mathrm{<mtext>q</mtext>}}\text{= 1.4·10}{}^9\text{M}{}^{\mathrm{?1}}\text{·}\text{s}{}^{\mathrm{?1}}$, by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1·10⁹ and 0.7·10⁹ M^?1·s^?1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case in aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its α-helical content (suggested by published CD data). The magnitude of the $\text{k}{}_{\mathrm{<mtext>q</mtext>}}$ values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer.     

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.     

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 $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.     

Possible effects of the detachment of stromal lamellae from granal stacks on salt-induced changes in spillover. A study by sonication of chloroplasts

Salt-induced chlorophyll fluorescence and spillover changes in control and briefly sonicated chloroplasts have been studied under conditions where Photosystem II traps are closed. In a low-salt medium containing 10 mM KCl, control envelope-free chloroplasts exhibited good spillover, as measured by low chlorophyll fluorescence yield at room temperature, a high ratio of the fluorescence peaks $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K, and increased Photosystem I activity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and Photosystem II light. In contrast, when stacked chloroplasts were briefly sonicated and subsequently diluted into a low-salt medium, a high fluorescence yield at room temperature and a low ratio of $\text{F}{}_{735}\text{F}{}_{685}$ at 77 K persisted. When unstacked chloroplasts were sonicated and then diluted into a high-salt medium, the room temperature fluorescence yield remained low. The results are interpreted in terms of a model relating the changes in chlorophyll fluoresecence with the lateral diffusion of Photosystem I and Photosystem II chlorophyll-protein complexes in the plane of the thylakoid membrane creating randomized or segregated domains, depending on the degree of electrostatic screening of surface charges (Barber, J. (1980) FEBS Lett. 188, 1–10). It is argued that brief sonication of stacked chloroplasts separates stromal membranes from granal stacks, thus limiting the inter-mixing of the photosystems via lateral diffusion even when the ionic composition of the medium is varied. Consequently energy transfer from Photosystem II to Photosystem I is relatively poor and chlorophyll fluorescence from Photosystem II is enhanced. The loss of the salt effect on sonicated unstacked membranes can also be accommodated by the model. In this case it seems that the generation of small membrane fragments does not allow the normal salt-induced phase separation of the pigment-protein complexes to occur.