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The interaction between glycophorin and a spin-labeled cholesterol analogue has been investigated by EPR spectroscopy. In vesicles which were reconstituted by the freeze and thaw technique, direct evidence was obtained for a reorganisation of the membrane at low protein content (protein/lipid ratio less than 1:300). From the spin exchange interaction we were able to show a protein-induced clustering of the steroid in fluid and in gel state membranes. Tryptic cleavage of the complete N-terminus of glycophorin vanishes the effect. Whereas the removal of the sialic acid residues by neuraminidase digest had no influence on the EPR spectra. The interaction seems to be cholestane spin label specific since it was not observed with an androstane spin-label.

The interactions of the antibiotic polymyxin B1 and its enzymatic cleavage product polymyxin B1 nonapeptide with phosphatidic acid monolayers and with bilayer membranes were investigated. Temperature-dependent pressure-area analysis of the monolayer reveals a linear increase of the lipid mean molecular area in the liquid condensed state for polymyxin concentrations between 10(-8) and 4 x 10(-7) M. Depending on the surface pressure, the area increase amounts to 30-70 A2. A linear dependence was also observed in the liquid expanded state but saturation is reached already at 10(-7) M polymyxin. The adsorption of polymyxin to phosphatidic acid bilayers is also linear and of a Langmuir type. Saturation is reached at a 1:4 polymyxin/lipid molar ratio. Polymyxin induces a phase separation in phosphatidic acid monolayers which was concluded from the thermotropic phase transition curves. In agreement with earlier bilayer experiments a second lowered phase transition appears in the presence of polymyxin. These fluidized domains again exhibit a linear polymyxin uptake comparable to the one of the liquid expanded monolayer at a temperature, where the undisturbed lipid is still in the condensed state. Polymyxin nonapeptide also causes an expansion of phosphatidic acid monolayers but only by maximally 10 A2. The thermotropic phase transition of the monolayer is reduced and considerably broadened by the nonapeptide. In phosphatidic acid bilayers we observed a decrease of the lipid phase transition temperature by 24 degrees C. The lateral chain packing is considerably disturbed by the peptide part of polymyxin.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.

Endothelial cells were isolated with high viability (greater than 93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of gamma-glutamyl transpeptidase. The gamma-glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70 +/- 10 h. In the absence of collagen, gamma-glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the gamma-glutamyl transpeptidase to 30% of the initial value.

The excimer formation technique has been applied to investigate the mechanism of the lateral diffusion in the crystalline P beta' phase of dipalmitoylphosphatidylcholine vesicles. This became possible at low pyrene concentrations. From the shape of phase-transition curves, from the effect of pressure up to 150 bar and from the perturbation induced by cholesterol, we conclude that the free volume model could also be applied to the P beta' phase. However, the diffusion is thought to occur in defect structures that are considered to form fluid pathways between domains of crystalline lipid. Partition coefficients of amphipaths provide a basis for testing for the role of defects. The amphipath chlorpromazine partitions into fluid membranes with a partition coefficient, kp, of 3200, into the crystalline phase with kp = 200 but into the P beta' phase with a value of kp = 800. This again gives rise to the assumption that the P beta' phase contains fluid domains that behave like the fluid L alpha phase and make up about 10-20% of the total amount of lipid in the bilayer. Cholesterol is known to interfere especially with defect structures in the P beta' phase. It fills up the gaps, and therefore reduces the partition coefficient to almost zero in the P beta' phase.

The thermotropic properties of aqueous phosphatidylethanolamine dispersions vary with hydration. Measured by EPR-spectroscopy freshly hydrated dimyristoylphosphatidylethanolamine dispersions exhibit a gel to liquid-crystalline phase transition at Tml = 48 degrees C. Dehydration could be induced by prolonged incubation of a hydrated sample at 4 degrees C. The phase transition temperature of the dehydrated phase was determined to be Tmh = 54 degrees C. From the measured phase transition curves we followed the dehydration with time and found a cooperative nucleation process. A 50% dehydration was reached after 5 days. This dehydration process could be prevented by gangliosides: 1.5 mol% of GT1b, 4 mol% of GM1 or 7 mol% of GD1a or GM3 but also 7 mol% of phosphatidic acid were able to stabilize the hydrated phase completely. The effect of gangliosides GM1, GM3, GD1a, GT1b and of the negatively charged phosphatidic acid on the phase behaviour of dimyristoylphosphatidylethanolamine (DMPE) dispersions were investigated. The phase transition temperature of freshly hydrated DMPE samples was successively decreased from 48 to 43 degrees C with increasing amounts of GD1a up to 10 mol% whereby the phase transition was significantly broadened. Gangliosides GM1, GM3 and GT1b as well as phosphatidic acid had minor effects. Dispersions of pure DMPE prepared below the transition temperature Tml form the dehydrated phase again with a melting temperature of Tmh = 54 degrees C. In the presence of 10 mol% GD1a or GT1b this value is reduced to Tml, the phase transition temperature of the hydrated phase. The reduction induced by GM3 is less pronounced. With GM1 or phosphatidic acid the samples remain partially dehydrated and the phase transition curves become biphasic up to 7 mol% ganglioside or phosphatidic acid.

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20 degrees C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.

The lateral mobility of pyrene, pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters. The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the charges of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers. The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of phi = 17 degrees is estimated.

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.

The protein-mediated phospholipid exchange between small unilamellar vesicles was investigated by fluorescence polarization measurements with diphenylhexatriene as optical probe. Thermotropic phase-transition measurements were taken after mixing two vesicle preparations of distinct and different phase-transition temperatures or having different states of charge. From the heights of each phase-transition step, we were able to follow the lipid-exchange process in the presence, as well as in the absence (natural exchange), of so-called transfer protein isolated from beef liver. A strong enhancement of the lipid transfer was observed at the corresponding lipid-phase-transition temperature, which is explained by the presence of fluctuating fluid and ordered domains co-existing at the lipid-phase-transition temperature. A unidirectional lipid transfer of the neutral component was observed between negatively charged phosphatidic acid and neutral phosphatidylcholine vesicles. Fluorescence polarization measurements showed the disappearance of the phosphatidylcholine phase transition, whereas the phosphatidic acid phase transition broadened and its phase transition temperature became lower.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.

Binding of the positively charged drug chlorpromazine to phospholipid monolayers was investigated. A preferential uptake was observed near the phase transition of the corresponding lipid. Cholesterol considerably diminishes the chlorpromazine uptake, again particularly near a lipid phase transition. The binding properties depend on the chlorpromazine concentration in the subphase. A critical concentration is 5 X 10(-5) M, where higher uptake occurs in the liquid condensed than in the liquid expanded state of the monolayer at pressures of about 10 mN/m. Dipalmitoylphosphatidylcholine monolayers spread on a subphase containing chlorpromazine are comparable to monolayers at higher temperature but in the absence of chlorpromazine. These data are in agreement with previous fluorescence and electron paramagnetic resonance experiments on lipid bilayer membranes (Luxnat and Galla 1986).

Seminalplasmin, a strongly basic protein isolated from bull semen, was found to antagonize with high potency and extraordinary specificity the function of calmodulin. Calmodulin antagonism is the result of an interaction between the two proteins, which is mainly determined by electrostatic forces. The stimulation of Ca2+-transporting ATPase and phosphodiesterase by calmodulin was half-maximally inhibited at approx. 0.1 microM-seminalplasmin. However, the basal activity of calmodulin-dependent enzymes was not significantly altered by seminalplasmin over the concentration range investigated.

The translational diffusion of pyrene, pyrene butyric acid and pyrene decanoic acid has been determined in phosphatidylcholine bilayers of different chain length and under pressure up to 200 bars. In the liquid crystalline phase and at a given temperature the diffusion decreases with increasing chain length. At a constant reduced temperature, Tred (about 10 K above the transition temperature), long chain lipids exhibit the fastest diffusion which is in disagreement with hydrodynamic models but favours free volume models for diffusion in lipid bilayers. The volume of activation, Vact, calculated from the decrease of the diffusion coefficient with pressure, delta lnD/delta P, depends on lipid chain length. Vact decreases with decreasing lipid chain length at a given temperature, T = 65 degrees C, and increases at the reduced temperature. These results are again in agreement with the dependence of the diffusion on lipid chain length and therefore with the free volume model.

In 32 healthy blood donors, 20 patients with histologically verified cancer and 18 patients with non-neoplastic diseases, the fluorescence polarization changes of fluorescein samples incorporated in mononuclear leucotyes were measured after incubation with PHA. The leucocytes of healthy persons, and 16/18 persons with non-neoplastic diseases, responded with a decrease in the degree of fluorescence polarization by about 20% from that in non-PHA-stimulated cells. In 19/20 patients with a variety of malignant tumours, the leucotyes did not respond to PHA stimulation with such a decrease. The exceptions among the patients with neoplastic and non-neoplastic diseases are considered, and may not be "false-negative" or "false-positive" respectively, but indicative of a particular situation in that disease. The biophysical mechanisms underlying the observed changes remain to be investigated.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.

The lateral mobility of the lipid analog N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine and of the integral protein glycophorin in giant dimyristoylphosphatidylcholine vesicles was studied by the photobleaching technique. Above the temperature of the chain-melting transition (Tm = 23 degrees C), the diffusion coefficient, Dp, of the protein [Dp = (4 +/- 2) X 10(-8) cm2/s at 30 degrees C] was within the experimental errors equal to the corresponding values DL of the lipid analog. In the P beta 1 phase the diffusion of lipid and glycophorin was studied as a function of the probe and the protein concentration. (a) At low lipid-probe content (cL less than 5 mmol/mol of total lipid), approximately 20% of the probe diffuses fast (D approximately equal to 10(-8) - 10(-9) cm2/s), while the mobility of the rest is strongly reduced (D less than 10(-10) cm2/s). At a higher concentration (cp approximately 20 mmol), all probe is immobilized (D less than 10(-10) cm2/s). (b) Incorporation of glycophorin up to cp = 0.4 mmol/mol of total lipid leads to a gradual increase of the fraction of mobile lipid probe due to the lateral-phase separation into a pure P beta 1 phase and a fraction of lipid that is fluidized by strong hydrophilic lipid-protein interaction. (c) The diffusion of the glycophorin molecules is characterized by a slow and a fast fraction. The latter increases with increasing protein content, which is again due to the lateral-phase separation caused by the hydrophilic lipid-protein interaction. The results are interpreted in terms of a fast transport along linear defects in the P beta 1 phase, which form quasi-fluid paths for a nearly one dimensional and thus very effective transport. Evidence for this interpretation of the diffusion measurements is provided by freeze-fracture electron microscopy.