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Adriamycin was shown to affect electron transport in the inner mitochondrial membrane in two ways. In a first step, adriamycin inhibits the cytochrome c oxidase activity. In a second step, adriamycin catalyzes the deviation of the electron flow between NADH and cytochrome c towards oxygen. Most likely, highly reactive oxygen species induce polymerization of the membrane components.

A new method is described to demonstrate conformational changes in oligopeptide monolayers. A probe (2- p -toluidinylnaphthalene-6-sulfonate) which fluoresces strongly in an hydrophobic environment, is adsorbed on the oligopeptide monolayer. Fluorescence intensity is measured as a function of the degree of polymerization of the oligopeptides. Between the hexa- and octapeptide, a discontinuity is obtained and interpreted as a transition from a random coiled conformation to a helical conformation. Because a change in the oligopeptide optical properties (circular dichroism) appeared for the same value of the degree of polymerization in organic solvents, the possibility of a surface denaturation can be excluded.

Two thirds or more of adriamycin (ADM) is found in the hydrophilic phase when the drug is dissolved in the two-phase system of Folch. This distribution is changed dramatically by the presence of all the negatively charged phospholipids, which form an electrostatic complex with the drug in the lower lipophilic phase. The molar ratio of ADM to phospholipids in the lower phase is 2 to 1 with cardiolipin, which has 2 phosphate molecules, and 1 to 1 with phosphatidic acid which has only one. ADM is recovered from lipophilic phase by acidification. No complex was obtained with sulfatides or ADM-ADN complex. The relevance of these data to the pathogenesis of ADM induced cardiac mitochondrial lesions is discussed.

A new procedure for conformational analysis is described to define the orientation of chlorophyll a in model membranes. The method allows to predict the configuration of assembled amphiphilic molecules. The minimal conformational energy is calculated as the sum of the contributions, resulting from the Van der Waals interactions, the torsional potentials, the electrostatic interactions and the transfer energy. In the calculated conformation, the porphyrin ring is orientated at an angle of 45° ± 5° to the membrane interface, with the phytol chain inserted into the lipid layer. This orientation of the porphyrin ring is in excellent agreement with the experimental value obtained with model membranes. The method could allow to define the orientation of other components in the thylakoid membrane.

A pre-sequence of 25 amino acids is required for import of yeast cytochrome oxidase subunit IV into mitochondria. Structure and orientation of the 25 amino acids synthesized peptide (p25) in a lipid bilayer were investigated by infrared attenuated total reflection spectroscopy. This method allowed to overcome the difficulties related to the optical turbidity due to the light scattering on membrane fragments which prevents the use of circular dichroism. We demonstrate here that incubation of the peptide with DOPC (dioleoylphosphatidylcholine) and DOPC-CL (dioleoylphosphatidylcholine - cardiolipin) liposomes is accompanied by an increase in alpha-helical content as compared to beta structure. Polarisation measurements indicate that the amphipathic helical segment is inserted parallel to the lipid acyl chains in cardiolipin containing liposomes.

The possible molecular interaction between phorbol esters and a phospholipid was examined in monolayer films at an air-water interface. The surface pressure isotherms indicated that, at close-to-physiological pressure, there existed a repulsive interaction between the phospholipid and biologically active phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-didecanoate (PDD). There was a close parallelism between the relative biological potency of different phorbol esters, as tumour promoters, and the magnitude of their repulsive interaction with the phospholipid. These findings raise the possibility that a biophysical interference of phorbol esters with the phospholipid domain of biological membranes may represent an important determinant of their biological actions.

Summary Fluorescence polarization and differential scanning calorimetry measurements made on dipalmitoyl phosphatidyl choline-stearylamine mixed liposomes show striking changes in the biophysical properties of these vesicles. The liposomes formed have transition temperature largely shifted to higher temperature and evolve to larger structure. These results strongly suggest that the alteration of liposomal structures due to the incorporation of lipid soluble compound like drugs requires physico-chemical studies before any screening with liposomes as pharmacological capsules.

Lipoxin A and lipoxin B (LXA and LXB) are formed from the oxygenation of arachidonic acid by interactions between the 5- and 15-lipoxygenases of human leukocytes. Each compound displays highly stereospecific biological actions. Here, we present a computational description of the following compounds: lipoxin A, (5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; 11-trans-lipoxin A, (5S,6R,15S)-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid; lipoxin B, (5S,14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid; and 8-trans-lipoxin B, (5S,14R,15S)-trihydroxy-6,8,10,12-trans-eicosatetraenoic acid. The analyses considered van der Waals energy, electrostatic interactions, torsional potential, and alterations in electrostatic forces. Additional analyses were carried out with each of the four compounds forming complexes with one calcium ion. Each compound gave very different conformers. Both lipoxin A and lipoxin B can form globular conformations, while their all-trans isomers form rigid extended structures. When complexes with each of these compounds and one calcium ion were examined (i.e., (LXA)2Ca: (11-trans-LXA)2Ca), both LXA and LXB formed several flexible conformations including crumpled, wrapped or extended conformations. In this situation, LXA showed a higher probability than LXB to wrap around one Ca2+. In contrast, the two all-trans isomers always lead to extended conformations. Results from the present study illustrate that changes in the stereochemistry of LXA and LXB lead to unique conformations which may underlie the different biological actions of these compounds. Moreover, they indicate that the conformations of eicosanoids can change while in aqueous or hydrophobic environments (i.e., biomembranes).

Beef heart mitochondria were incubated with ADM and NADH. An adriamycin semiquinone radical was detected using ESR spectroscopy. The semiquinone radical production rate is decreased upon addition of a scavenger (AD 20) in the reaction medium. NMRI mice were treated with AD 20 (70 mg/kg, i.p.) 15 min prior ADM injection (20 mg/kg, i.p.) or with ADM alone. Heart mitochondria were isolated 48 hr later. The enzymatic activities of complex I-III and complex IV of the mitochondrial respiratory chain were strongly depressed in animals receiving ADM alone, whereas these activities were almost completely restored in animals receiving AD 20 and ADM. Fluorescence depolarization measurements indicated that only mice treated with ADM alone presented a decreased fluidity of their cardiac mitochondrial membrane.

Amiodarone is a potent antianginal and antiarrhythmic drug which affects the lipid dynamics. The influence of amiodarone ionization on the lipid transition temperature and enthalpy associated to the liquid crystalline to gel state transition was studied in multilamellar vesicles (MLV) of dipalmitoylphosphatidylcholine (DPPC) by differential scanning measurements (DSC) at different pH. These data were correlated with the calculated number of charged amiodarone molecules inserted into the lipid vesicles. The procedure of calculation requires the knowledge of the intrinsic ionization constant of amiodarone and the area occupied per amiodarone molecule in the close packed state; it can be applied successfully to water insoluble amphiphilic molecules. Only the ionized form of amiodarone molecule destabilizes the lipid matrix organisation whereas no effect was observed with the uncharged form. This destabilizing effect could be explained in terms of a modification of the drug structure induced by its ionization state or in terms of its distribution in the lipid matrix, as an isolated molecule or assembled in clusters depending on its ionization state.

Model membranes have been used to describe the interaction between charged drugs (acridine orange, ethidium bromide) and lipids. A new method of evaluation of the surface charge density is described. It allows, from surface potential measurements, the evaluation of the drug-lipid association constant. The low perturbation of the monolayer dipole orientation after complexation is correlated to the low penetration of the drug into the lipid bilayer of liposomes as shown by a fluorescence titration technique.

It is demonstrated here that adriamycin (ADM), a widely used antimitotic agent, has a similar affinity for its target (DNA) and for a mitochondrial membrane component (cardiolipin). This cardiolipin—adriamycin complex is responsible for the inhibition of cytochrome c oxidase. The activity change of cytochrome oxidase evaluated for several adriamycin derivatives in beef heart mitochondria indicates that the inhibition is not a consequence of a direct drug—enzyme interaction but of a modification of the enzyme lipid environment. The adriamycin—cardiolipin complex segregates in a separate lipid phase and the new enzyme environment does not allow its activation. Cardiolipin (CL) is essential for cytochrome c oxidase activity. This inactivation could explain the adriamycin cardiotoxicity. Indeed, it is significant that the most cardiotoxic adriamycin derivatives form the strongest complexes with cardiolipin. A precise knowledge of this inhibition mechanism is a prerequisite for the synthesis of new non-cardiotoxic compounds.

The mode of orientation of gramicidin A in the β 6.3 conformation in a monolayer of lysophosphatidylcholine was studied on the basis of conformational data. The approach involves localization of the centers of hydrophobicity and hydrophilicity of the gramicidin molecule, and takes into account the dielectric constant discontinuity of the interface. The most probable orientation is that with the C-terminal, tryptophan-rich, end directed towards the hydrophobic medium. In this orientation, four lysophosphatidylcholines surround a gramicidin monomer leading to a complex with an overall cylindrical shape compatible with the bilayer structure found in aqueous dispersions of mixtures of these molecules (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141–144).

Adriamycin, an anthracycline glycoside antibiotic that inhibits the electron flow in mitochondria, also inhibits photosynthetic electron transport (PSI+PSII). The oxygen consumption curves suggest an inhibitory effect of PSII activity at very low adriamycin concentrations. Surface potential and differential scanning calorimetry measurements coupled with the use of tritiated daunomycin demonstrated that adriamycin interacts specifically with negatively charged thylakoid lipids, and induces a clustering of these negatively charged lipids in a neutral lipid matrix. These properties have made it possible to suggest a mechanism for the adriamycin-induced inhibition of mitochondrial enzymes (cytochrome c oxidase, NADH: cytochrome c oxidoreductase). We did not identify precisely the target responsible for the adriamycin effect in the thylakoid membrane, but the preliminary studies reported herein indicate evident similarities between the two inhibition mechanisms.

We have measured the rate of hydrolysis of liposomes made of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A2.

A change of conformation of macromolecules has been investigated at the air-water interface. The model chosen is a copolypeptide of glutamic acid and methyl glutamate. The modification of the conformation of the copolypeptide is induced by changing the pH of the support. The presence of electrostatic charges on the spread macromolecules has as consequence an abrupt change of the surface properties. Measurements of surface potential and surface viscoelasticity demonstrate a discontinuity in the surface behavior for a pH of the subphase of about 7.5 units. This result is interpreted as a transition from the helical conformation to the coiled conformation at the air-water interface.

Interaction between suloctidil (CP 556 S) and lipids (phosphatidylcholine, phosphatidylserine) is studied using a new conformational analysis procedure. This analysis is extended to two compounds related to suloctidil but bearing no protonable group (CP 894 S) or of different hydrophobicity (CP 1136 S). It gives a molecular description of the mode of insertion of the drugs into the lipid layer. The influence of the calculated lipid-drug interaction and area occupied per drug molecule in the lipid layer is tentatively related to the effect on the lipid dynamics. For a given conformer, an effect on the lipid dynamics is expected only if (a) the area occupied per conformer is similar to that of the lipid and/or (b) the drug-lipid interaction energy is equal or superior to that of the lipid-lipid interaction. These predictions are analyzed in terms of the available experimental data.

In this paper we attempt to outline some features which determine the encapsulation of small molecules into lipid vesicles. Spin labels derived from five carboxylic acids of different lengths were synthesized and incorporated in varying amounts into multilamellar and unilamellar vesicles made up of four different phosphatidylcholines. The influence on the release process of the bilayer rigidity and of the hydrophobicity of the entrapped molecule was systematically studied. The hydrophobicity is of critical importance and was estimated by measuring the partition coefficient (P) between octanol and buffer. In multilamellar vesicles, molecules characterized by extreme P values (log P less than -0.3 and log P greater than 5) can be efficiently entrapped. The rate of leakage is related to the P value according to a bell-shaped curve. Moreover, gel state of the bilayer and long acyl chains of the lipids are properties which favor a good entrapment. Small unilamellar vesicles may be formed in the presence of high concentrations of hydrophilic and lipophilic spin labels. However, the formation of unilamellar vesicles produces a significant reduction of the internal volume and of the entrapped water-soluble spin lables. High fractions of lipid-soluble spin labels can be incorporated in unilamellar vesicles but the vesicle stability is diminished.

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.

The effects of an antiarrhythmic and antianginal drug, amiodarone, on the physical state of membrane phospholipids was investigated by means of fluorescence polarization using the apolar probe 1,6 diphenyl-1,3,5-hexatriene incorporated in the hydrocarbon core. Multilamellar vesicles were prepared from neutral phospholipids (egg phosphatidylcholine, synthetic saturated phosphatidylcholine) alone or mixed with cholesterol or various lipids representative of the main lipid classes. Amiodarone reduces the temperature of the gel to liquid-crystalline phase transition and either increases or decreases lipid mobility in the gel or liquid-crystalline phase. In the gel state, the lipid mobility depends on drug concentration, degree of ionization and the length of the lipid acyl chains. In the liquid-crystalline state, the decreased lipid mobility which is concentration-dependent is essentially due to hydrophobic interactions. Amiodarone increases the lipid order parameter to the same extent as cholesterol. The data suggested that amiodarone is a rigid molecule deeply buried in the hydrocarbon core of the lipid and that amiodarone-lipid interactions are mainly hydrophobic.

The interaction of a number of positively charged anti-tumor drugs with cardiolipin-containing model membranes has been investigated using 31P nuclear magnetic resonance (31P-NMR), differential scanning calorimetry (DSC) and monolayer techniques. It appeared that the ellipticines used (i.e., celiptium and 2-N-methylellipticinium), and also ethidium bromide, completely blocked Ca2+-induced HII phase formation in pure cardiolipin liposomes at molar ratios of drug-to-lipid of approx. 1:1. For the anthracyclines adriamycin and 4'-epi-adriamycin, a similar effect was observed, but now a 2:1 ratio was required. 31P-NMR experiments on dioleoylphosphatidylethanolamine/cardiolipin mixed liposomes indicated that the two anthracyclines, but not the other three drugs, were capable of inducing macroscopic phase separation into domains enriched in drug-cardiolipin complexes and domains enriched in the zwitterionic phospholipid species. DSC experiments on dipalmitoylphosphatidylcholine/cardiolipin mixtures led, with the exception of 2-N-methylellipticinium, to the same conclusion. Measurements of surface pressure and surface potential of cardiolipin monolayers at the air/water interface as well as conformational analysis of the various drug-cardiolipin recombinants showed that the elliptciines are deeply embedded in the acyl chain region of the bilayer, while the anthracyclines and ethidium bromide are preferentially localized in the interface. All drugs share an important electrostatic interaction with the negatively charged phosphates of cardiolipin.

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p-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.

Previous studies [Laurent et al., Biochem. Pharmac. 31, 3861 (1982)] have demonstrated that aminoglycoside antibiotics bind to negatively charged phospholipid bilayers and inhibit the activity of lysosomal phospholipases. This inhibition also occurs in vivo in animal and man. It is considered to be an early and significant step in the development of aminoglycoside-induced nephrotoxicity. The binding of 6 aminoglycosides in current clinical use (dibekacin, gentamicin, tobramycin, kanamycin A, amikacin and streptomycin) to phosphatidylinositol has been studied by gel filtration technique and by conformational analysis. Variation of the phosphatidylinositol content from 0 to 27% of total phospholipids causes a cooperative increase in aminoglycoside binding. At fixed phosphatidylinositol concentration, the binding of the different aminoglycosides is related to the number of aminogroups carried by the drug (viz., gentamicin greater than kanamycin A greater than streptomycin) and is largely, but not entirely dependent upon electrostatic interactions. Conformational analysis of the interaction of aminoglycosides with phosphatidylinositol monolayers was made by a step-wise computation approach. We first have taken into account the Vander Waals, torsional and electrostatic energies and we have calculated the hydrophobic and hydrophilic centers of each molecule. Assembly was then computed by successive association of one molecule of drug and up to 4 molecules of phosphatidylinositol. The calculated interaction energies varied from -8.5 kcal/mol (gentamicin) to -4.9 kcal/mol (amikacin) and -3.9 kcal/mol (streptomycin).(ABSTRACT TRUNCATED AT 250 WORDS)

A computational approach is used to predict the mode of organization of lipid molecules. The calculated aggregate states are fully compatible with the lipid phase preference: bilayers for dipalmitoylphosphatidylcholine, micelles for palmitoyllysophosphatidylcholine, and inverted structures for protonated dioleoylphosphatidic acid. The minimal conformational energy of the isolated molecule is calculated as the sum of the contributions resulting from the Van der Waals interactions, the torsional potentials, the electrostatic interactions, and the transfer energy. Assembly of these structures in monolayers yields the most probable modes of organization of lipids. The described method may represent a useful toot to predict the phase preference of lipids and to give a molecular description of their mode of association.

A new approach is presented which allows to describe the binding of different local anesthetics to lipids. Lipids (DL- alpha-dipalmitoylphosphatidylcholine, phosphatidylserine, cardiolipin) are spread at the air-water interface and the anesthetic (procaine, butacaine, tetracaine) injected into the aqueous subphase. The equilibrium constants associated to the interfacial reaction: D+ (subphase) +L- (monolayer) in equilibrium DL (monolayer) (where D+ denotes the anesthetics, L- the lipid anionic site and DL the complex) are calculated from an experimental evaluation of the surface potential of the lipid monolayer. This mode of determination is based essentially on the good correlation between the experimental values of the surface potential and the theoretical predictions from the Gouy-Chapman theory. Fluorescence measurements on liposomes are carried out in order to locate the position of the drug in the lipid layer. This method can be extended to any positively charged drug-anionic lipid interaction.

The effect of adriamycin on cardiolipin-containing model membrane systems have been studied by 31P-NMR, freeze-fracture electron microscopy and binding experiments. Adriamycin effectively inhibits the formation of non-bilayer lipid structures induced by Ca2+ and cytochrome c in cardiolipin-containing liposomes. This drug also strongly inhibits the uptake of Ca2+ by cardiolipin into an organic phase. These results are discussed in relation to the cardiotoxic effect of adriamycin and the possible importance of non-bilayer lipid structures for the functioning of the mitochondrion.

Despite the fact that photosynthetic membranes show the conventional bilayer structure, their major lipid component monogalactosyldiacylglycerol does not form lamellar structure but takes up an hexagonal-II structure when dispersed alone in water and forms inverted lipids micelle structures when dispersed together with other lipid components of the photosynthetic membrane. We present here evidence that the mode of organization of these lipids can be predicted from a conformational approach allowing to describe the configuration of assembled amphiphilic molecules. The minimal conformational energy is calculated as the sum of the contributions resulting from the Van der Waals interactions, the torsional potentials, the electrostatic interaction and the transfer energy. Because of its calculated "cone shaped" structure monogalactosyldiacylglycerol forms inverted lipid structure with the hydrophilic groups pointing inward; for digalactosyldiacylglycerol, an other essential lipid constituent of photosynthetic membrane, its calculated cylindrical shape induces an organization in bilayer structures.

This paper seeks to investigate the molecular organization of fatty acid spin label (4-palmitamido2,2,6,6-tetramethylpiperidine-1-oxyl) and dipalmitoylphosphatidylcholine molecules in monolayer vesicles. The molar ratio of the spin probe was varied from 1 to 22% and the corresponding electron spin resonance spectra were measured at 25 and 48°C. These spectra were analyzed in terms of the dipole-dipole and exchange interactions occurring between the radicals. From a computer simulation, a quantitative analysis of these interactions could be made. Comparison of these data with theoretical models indicate that above the lipid phase transition, i.e., at 48°C, the membrane is homogeneous and the label molecules diffuse randomly in the lecithin bulk phase (D = 3.8 x 10−8 cm2/sec). At 25°C, the membrane model can be viewed as a mosaic structure composed of spin probe clusters (7.9 x 1011 clusters/cm2). The size of the clusters as well as the minimal mean distance between two cluster edges could be calculated as a function of the spin label molar ratio. These results are used to interpret the observations made in the first paper of this series [Chatelain et al., J. Colloid Interface Sci. 72, 287 (1979)] concerning the surface viscosity of the mixed monolayer and the kinetics of the enzymatic activity of phospholipase A2. It can be concluded that above a critical spin label concentration (10–15%) the properties of the entire lecithin phase are influenced by the presence of the spin probe clusters.

In order to determine the role of the membrane charge density on the ionophore-ion (valinomycin-K+, antamanide-Na+) complexation process at the lipid-water interface, antibiotic-palmitic acid mixed monolayers are spread at the air-water interface. By changing the composition of the subphase (pH, nature of the cation) and of the mixed film, it is possible to modify the surface charge density and the cationic environment of the spread ionophore. Surface potential measurements are interpreted in terms of the Gouy-Chapman theory and demonstrate that the surface charge density determines the ionophore binding capacity.

Pancreatic endocrine cells prepared from rat islets were labelled with 1,6-diphenyl-1,3,5-hexatriene and examined in a microviscosimeter. Glucose caused a dose-related decrease in fluorescence polarization. This decrease was observed within 1 min after increasing the concentration of glucose. These findings suggest that glucose affects membrane viscosity in pancreatic endocrine cells. At a glucose concentration of 16.7 mM, the estimated viscosity was 15 ± 3 per cent lower than basal value (2.01 ± 0.12 P).

Multilamellar liposomes made of equimolar mixtures of dimyristoyl and distearoylphosphatidylcholine were hydrolysed by porcine pancreatic phospholipase A 2 . Ph-stat titration, equilibrium gel filtration and differential scanning calorimetry were used to study respectively the enzymic hydrolysis, the enzyme binding and the lipid phase repartition. We demonstrated that the optimal enzyme activity observed in the region where liquid crystalline and gel lipid phases coexist, is due to a drastic increase of the enzyme binding to its substrate. It is suggested that the border region separating the lipid phases could be a privileged site for enzyme insertion. Increase of lateral compressibility due to coexistence of solid and fluid lipid phases will promote the penetration of the hydrophobic interface recognition site (IRS) of phospholipase A 2 into the lipid matrix whereas the active site, distinct from the IRS will attack its substrate independently of the lipid physical state.

Staphylococcus aureus can be protected by unsaturated unesterified fatty acids against the growth inhibitory effects of miconazole and ketoconazole observed at concentrations greater than 10(-6) M and greater than 10(-5) M, respectively. Miconazole's fungicidal activity is partly antagonized by oleic acid. However, the effect of ketoconazole on the viability of Candida albicans was not affected by this fatty acid. Cytochrome oxidase and ATPase activities are more sensitive to miconazole (10(-5) M) than to ketoconazole (greater than 10(-4) M) and also liposomes are more susceptible to lysis induced by miconazole. Using differential scanning calorimetry it is shown that high concentrations of miconazole shift the lipid transition temperature of multilamellar vesicles to lower values without affecting the enthalpy of melting. Ketoconazole induces a broadening of the main transition peak only. It is suggested that miconazole changes the lipid organization without binding to the lipids, whereas ketoconazole is localized in the multilayer without having an important direct effect on the lipid organization. The results indicate that miconazole, and to a lesser extent ketoconazole, at doses that can be reached by topical application only, interfere with a third target (the two others are ergosterol synthesis and fatty acid elongation plus desaturation). It is hypothesized that the induced change in lipid organization may play some role in miconazole's topical antibacterial and fungicidal activity, whereas it does not seem to play a significant role in ketoconazole's activities.

Adriamycin and several derivatives were found to inhibit the last oxidation site of the respiratory chain (cytochrome c oxidase EC.1.9.3.1.) both on mitochondria and on purified reconstituted systems. A new mechanism of membrane enzyme inactivation is proposed to explain the experimental results: adriamycin does not interact directly with cytochrome c oxidase but inactivates it by changing the cardiolipin environment essential for its activity. In presence of adriamycin, cardiolipin is extracted from the lipid surrounding environment of cytochrome c oxidase and segregates in a separate phase inaccessible for the enzyme. We suggest that other cardiolipin dependent enzymes could be inactivated by adriamycin.

The influence of stearylamine upon the behavior of unilamellar liposomes formed from dimyristoyl, dipalmitoyl, and distearoyl phosphatidylcholine was investigated. In the presence of stearylamine, optical density measurements indicated that larger liposomal structures are formed. However, when stearylamine was incorporated in distearoyl phosphatidylcholine, i.e., when a stearoyl chain was present in both components of the liposome, no change could be detected. Differential scanning calorimetry of the different liposomal suspensions revealed that for the different systems used, new liposomal structures were formed. The kinetics of transformation depends strongly on the chemical structure of the lipid and of the amine. The possible change in physicochemical properties of the liposome due to the incorporation of a lipid-soluble compound should be kept in mind in any attempt to use liposomes as pharmacological capsules.

A semi-empirical conformational analysis is used to compute the conformation of (+)-propranolol inserted in dipalmitoylphosphatidylcholine. In a first step, the minimal conformational energy of the isolated molecule at the hydrocarbon-water interface is calculated as the sum of the contributions resulting from the Van der Waals, the torsional, the electrostatic and the transfer energies. Five pairs of conformers of minimal energy are determined. They are compared to data available from other experimental approaches. In a second step, they are assembled with dipalmitoylphosphatidylcholine at the interface. Although propranolol is considered in its protonated form, the electrostatic interaction with dipalmitoylphosphatidylcholine is negligible as compared to the Van der Waals interaction. The area occupied per propranolol molecule is between 0.53 and 0.64 nm2/molecule. In the most probable modes of insertion of propranolol into the lipid layer, the naphthyl moiety of the compound interacts with the lipid acyl chains. The protonated amino group is located in the vicinity of the phosphate residue possibly causing an electrostatic interaction.

A peptide of diphtheria toxin B fragment (residues 147-266) has been shown to induce pore formation in lipid bilayer membranes at low pH. Such an effect was obtained at a much lower extent or not at all at pH = 7. The region localized between residues 225 and 246 is highly hydrophobic (27.3% polarity) and characterized by a high concentration of proline residues. Since proline cis-trans isomerization is highly sensitive to the pH of the medium, we investigated the capability of the cis and trans isomers to penetrate into the lipid matrix. Obviously, the cis-trans isomerization of proline 242 and 245, assumed to be imposed by a low pH, uncovers the hydrophobic region and induces its insertion into a lipid layer of dipalmitoylphosphatidylcholine. The lipid matrix destabilization resulting from this process could promote the penetration into the lipid bilayer of an amphipatic structure (153-178) similar to the transverse lipid associating domains of membrane proteins.

The procedure of surface charge density evaluation using 9-aminoacridine as a fluorescent probe implies that cations do not interact with the negatively charged lipids. From surface potential measurements on lipid monolayers coupled with fluorescence measurements on liposomes, we demonstrate that the cations present in the aqueous phase, including the fluorescent probe itself, interact with the lipidic sites. These association reactions should not be neglected in the evaluation of the surface charge density associated with model membranes.

This paper presents a new approach to study A23187 ionophore in an aqueous environment. The ionophore was spread at the air—water interface. Surface pressure and tritium—hydrogen surface exchange measurements demonstrated the conformational stability of A23187 in the free and complexed forms at the interface. Exchange measurements suggested that the A23187 ionophore adopts an interfacial conformation in which hydrogen bonds are not accessible to the aqueous solvent. Surface potential measurements allowed the calculation of the intrinsic dissociation constant of the ionophore carboxylic group in an aqueous environment (pKi = 5). The ionophore activity sequence for bivalent ions was Ca2+>Mg2+⇓Ba2+ . This sequence is comparable to those obtained by other authors inorganic solution. Surface radioactivity measurements performed with 45Ca2+ in the subphase demonstrated the formation of a neutral complex between two negatively charged ionophore molecules and one Ca2+ ion. This reaction was only observed at pH higher than 5. The association constant of the interfacial ionophore-Ca2+ complex was calculated.

Conductance measurements on planar lipid bilayers demonstrate that CB1, a CNBr peptide of diphtheria toxin fragment B located in its middle region, possesses the unique property to destabilize the lipid bilayer organization. It is suggested that a segment of 25 amino acids in the N-terminal sequence of CB1 could be responsible for this effect. Its very low polarity, its predicted amphipathic helical structure and a helix length corresponding to the thickness of the hydrocarbon region of the lipid bilayer should specifically favor its insertion in the membrane. The existence of such a transverse lipid-associating domain could confer upon the molecule the properties leading to the anchoring of diphtheria toxin in the cytoplasmic membrane.

A general procedure of evaluation of the lipid—drug association constant in monolayer is presented. Lipids are spread at the air—water interface and the drugs injected into the aqueous subphase. Association constant evaluation is based essentially on the good correlation between experimental values of the surface potential and predictions from the Gouy—Chapman theory. Several procedures of calculation are described.

To demonstrate the possibility of cell delivery of water insoluble products, L1210 cells were incubated with liposomes containing Nocodazole® (NCL), an experimental drug totally insoluble in water. Liposomes of various lipid compositions readily incorporated NCL, but only those formed from dipalmitoyl phosphatidylcholine, cholesterol and stearylamine (molar ratio 4:3:1) had a clear affinity for L1210 cells, as shown by fluorescence studies. This affinity is mediated by the degree of both cell and liposome membrane fluidity. Cytoplasmic and intranuclear location of these liposomes containing NCL or drug-free and subcellular changes induced by their presence were demonstrated by ultrastructural studies.

The monolayer approach has been used to study an (Na + +K + )-activated ATPase, free and in its lipidic environment. Surface pressure, surface tritium-hydrogen exchange, and surface viscosity measurements of ATPase-lipid films suggest the following: A lipid-ATPase interaction depends on the nature of the lipid; the globular ATPase molecule induces complete fluidity of the lipid layer; the ATPase orientation in the mixed films depends on the lipid fluidity.

The amphiphilic character of different plasma apolipoproteins was investigated by a combination of established hydrophobicity analysis methods. These methods proved to be powerful in the detection of amphiphilic phospholipid-binding domains. Within this class of lipid-binding domains, lecithin-cholesterol acyltransferase activating and non-activating helices could be differentiated by calculating hydrophobic moments at different angles. We conclude that the hydrophobic characteristics of the different helices determined the mode of lipid binding and the substrate properties of these phospholipid-protein complexes for the lecithin-cholesterol acyltransferase reaction.

The possibility of an α-helix-coil transition of the poly-γ-methyl- l -glutamate spread at the air-water interface has been investigated. The helical conformation of this polypeptide has been shown to exist in the solid film removed from an aqueous support. Starting from a well-defined conformation, the transition is induced by simple methods similar to those used in solution. 1. (i) Effect of the solvent: Addition to the support of dioxane-dichloroacetic acid mixtures of various relative proportions allows the inducement of a transition. 2. (ii) Effect of temperature: An increase of the substrate temperature allows the modification of the interaction forces responsible for the polypeptide structure. The transition phenomenon has been followed by the evolution of the surface pressure. The study at very low surface concentrations enables us to associate with the transition both the surface virial coefficient, which represents specifically the interaction between the spread polypeptide molecules and the molecules of the substrate, and the co-surface which is proportional to the dimension of the macromolecule at the interface.

Resume Par analogie avec une etude realisee sur des solutions bi- et tridimensionnelles d'acetates de polyvinyle branche et lineaire, et montrant la concordance existant entre les variations de proprietes de ces deux types de solutions, l'influence du branchement sur une macromolecule plus rigide a ete examinee, de maniere a determiner la part due a la cohesion du polymere. Les mesures ont ete realisees principalement a l'interface eau-air ou le support manifeste les proprietes d'un solvant pauvre, ce qui permet une differentiation plus significative entre les deux polymeres, des polymethacrylates de methyle branche et lineaire, de meme masse molaire. En solution bidimensionnelle, l'evolution de la pression superficielle en fonction de la temperature, aux faibles concentrations superficielles permet de determiner les deuxiemes coefficients du viriel superficiels, les enthalpies et entropies d'etalement, et de demontrer l'existence d'une interaction plus faible entre le polymere branche et le support. Cette etude est completee par les mesures de pression superficielle et de viscoelasticite de monocouches plus condensees. La differentiation montre la structure plus organisee du polymere branche par rapport au lineaire.

The validity of Silberberg's hypothesis concerning the conformation of branched polypeptides was tested by studies on solutions and on monolayers. The poly(α-amino acids) investigated consisted of poly-L-lysine backbones with side chains of poly(gamma;-benzyl L-gutamate) and poly(β-benzyl L-aspartate). In solution, the conformation was studied in the mixed solvent chloroform–dichloroacetic acid. Optical rotatory dispersion measurements demonstrated the existence of a helix–coil transition which is a function of the length of the side chain. A significant “transition curve” appeared between 9 and 15 residues. From the results, it may be assumed that the poly-L-lysine backbone is extended in a coiled conformation and the side chains are coiled or helical, depending on the number of glutamate residues. At the air–water interface, a discontinuity in the surface-pressure curve appears between 9 and 15 residues per side chain. The similarity with the results obtained in solution appears significant; however, this has to be confirmed with methods more specific for structural modifications.