Interfacial activity of an ion channel-generating protein cytolysin from the sea anemone Stichodactyla helianthus.

The ability of a purified sea anemone (Stichodactyla helianthus) protein cytolysin to interact with a variety of interfaces was investigated by means of the Wilhemy plate method. At the air:water and lipid:water interfaces, the toxin lowered the surface pressure most readily as the aqueous phase pH increased towards the isoelectric point (9.8) of the toxin. The affinity of the toxin for both the phospholipid:water and the oil:water interfaces was much greater than for the air:water interface. Although the toxin had previously been found to avidly bind to sphingomyelin-containing phospholipid dispersions and bilayers, it failed to display any preferential interaction with a sphingomyelin monolayer relative to one of dipalmitoylphosphatidyl-choline under identical conditions, even when the monolayers were maintained at 40 dynes/cm, a pressure considered to produce phospholipid packing densities similar to those observed in cell membranes. Unlike many other membrane-active protein cytolysins, the ability of Stichodactyla cytolysin to penetrate these phospholipid monolayers was not affected by the initial surface pressure over the range 0-32 dynes/cm. However, at 40 dynes/cm initial packing pressure, the surface pressure generated by the cytolysin was similarly reduced in both sphingomyelin and dipalmitoylphosphatidylcholine monolayers. Our results suggest that the protein cytolysin initially binds reversibly to cell and artificial bilayer membranes in a non-specific manner; sphingomyelin domains in the bilayer then provide optimal conditions for insertion into the membrane and subsequent assembly of a stable multimeric complex which functions as an ion channel.

Interfacial tensions and partition coefficients in water/heptane systems containing 2,6-diisopropylphenol, n-alkylphenols and cycloalkanols.

The free energies of absorption, dehydration, and transfer in a heptane-water system for a set of phenolic compounds and a series of cycloalkanols have been investigated. The free energy of adsorption depends only upon the molecular surface area of the hydrophobic fraction of the molecule, whereas the free energy of dehydration is independent of this fraction.

Surface activities of barbital, phenobarbital, and pentobarbital and their interaction energies with phospholipid monolayers.

The adsorption free energies of barbital, phenobarbital, and pentobarbital at the air-water interface were estimated from plots of the surface pressure (pi less than or equal to 5 dynes/cm) against the bulk concentration. Their energies of interaction with dipalmitoylphosphatidylethanolamine and dipalmitoyllecithin monolayers spread at the air-water interface were estimated from the surface pressure increase with increasing concentrations of the subphase-injected barbituric acid derivatives. Adsorption free energies and interaction energies were barbital less than phenobarbital less than pentobarbital, which correlate with their nerve blocking concentration.

Kinetics and mechanisms of monolayer interactions V: surface activities of alkylamides, alkylmonocarboxylic acids, and alkylketones and their interaction energies with phospholipid monolayers.

The adsorption free energies of the C1-C6 alkylamides, C3-C8 alkylketones, and C1-C10 alkylmonocarboxylic acids at the air-water interface, estimated from plots of the surface pressure (less than or equal to 5 dynes/cm) versus the bulk concentration, were linear functions of the total surface area per molecule (square angstroms per molecule), with a slope 46% higher for the alkylamides and 25% lower for the alkylketones than that for the monocarboxylic acids. The interaction energies of alkylamides with dipalmitoyl lecithin and dipalmitoyl phosphatidylethanolamine spread at the air-water interface, estimated from the surface pressure increase with increasing concentrations of the injected C1-C5 compounds, were linear functions of the total surface area per molecule. The diffusion free energies, delta Gdif, of the alkylamides within a phospholipid bilayer, predicted from the permeability equation and their interaction energies with dipalmitoyl lecithin monolayers by assuming the additivity of their free energies of adsorption and dehydration at the solution-bilayer interface, agreed with the literature data.

Surface activities of procaine, lidocaine, and tetracaine and their interaction energies with phospholipid monolayers.

The free energies of adsorption of procaine, lidocaine, and tetracaine at the air--water interface were estimated from plots of surface pressure (pi less than or equal to 5 dynes/cm) against bulk concentration. Their interaction energies with dipalmitoylphosphatidylethanolamine and dipalmitoyllecithin monolayers, previously spread at the air--water interface, were estimated from the increase of surface pressure with increasing concentrations of the subphase-injected anesthetic. Free energies of adsorption and the interaction energies were in the order procaine less than lidocaine less than tetracaine and correlate with relative anesthetic potencies and the blocking of nerve conduction.

Interaction of doxorubicin with phospholipid monolayers.

The energies of interaction of doxorubicin hydrochloride and sodium 1,2,4-trihydroxy-9,10-dioxo-3-anthracenesulfonate with dipalmitoylphosphatidylethanolamine and dipalmitoyllecithin monolayers spread at the air-water interface were estimated from the increase in surface pressure with increasing concentrations of the subphase-injected compound. Their orders of magnitude were consistent with those of the energies of interaction of doxorubicin and acridines with double-stranded DNA, which suggests that the same type of van der Waals forces are operative.

Kinetics and mechanisms of monolayer interactions IV: Surface activity of alkanols and energies of their interaction with dipalmitoyllecithin and dipalmitoylphosphatidylethanolamine.

The free energies of adsorption of the C1 to C14 alkanols at the air--water interface, estimated from plots of the surface pressure (pi less than or equal to 5 dynes/cm) against the bulk concentration, were a linear function of the chain length for 1-alkanols. From C3 to C8, the 2- and 3-isomers showed lower values than the 1-isomers. The energies of interaction of the C1 to C14 alkanols with dipalmitoylphosphatidylethanolamine and dipalmitoyllecithin monolayers, previously spread at the air-water interface, were estimated from the increase of the surface pressure with increasing concentrations of the injected alkanol. The energies of interaction of the C1 to C5 1-alkanols were linear functions of the chain length. The energy of interaction per methylene group of the alkyl chain suggests that the phospholipid monolayers behaved as ultrathin "oil" phases. The 2- and 3-isomers presented marked departures from linearity. The literature data for reflection coefficients in biomembranes and for partition coefficients between olive oil and water, red cell membranes and water, and phospholipid liposomes and water for the C1 to C5 alkanols show similar linearities with the chain length for the 1-isomers and comparable departures for the 2- and 3-isomers.

Kinetics and mechanisms of monolayer interactions. III: models to explain time-dependent effects of injected cetrimonium bromide.

Two kinetic models are derived to explain the time-dependent effects of cetrimonium ions injected beneath an air-aqueous solution interface without any previously adsorbed or spread monolayer at different ionic strengths. The data show excellent fit and consistency with a nulticompartmental kinetic model which postulates a barrier or intermediate compartment between the ionic surfactant in the subphase and the sites on the surface. Such a barrier inhibits free diffusion of the surfactant from the bulk of the solution to these sites. The experimental data are not inconsistent with a model that postulates a time-dependent binding process and the absence of a diffusion-limiting subinterface since, within the limits of the error, the derived rate constants conform to the model prediction that they should be proportional to the surfactant-ion concentration.

Drug-biomolecule interactions: mechanisms and kinetics of interactions of biomolecules at interfaces.

At the air-water interface, the area occupied by the molecules of each component of mixed monomolecular layers of cholesterol with hexadecyl alcohol, hexadecylic acid, or hexadecylamine was independent of the presence of the other component. The values of the free energy of mixing of these lipids were within the range of the entropic factor of the free energy even at high surface pressures. Mixed monolayers of cholesterol and bovine serum albumin showed a similar independence of the area per molecule and of the free energy of mixing values when the concentration of the protein was expressed in terms of amino acid residues per molecule of protein forming the mixed monolayer. Higher values of the free energy of mixing were obtained for mixed monolayers of cholesterol with dipalmitoyl lecithin and dipalmitoyl phosphatidylethanolamine than were expected from an entropic factor. The interaction between monomolecular layers of lipidic biomolecules with bulk subphase components, the energy of activation, interaction kinetics, and effects of added electrolytes were also studied. The implications of these data to a mechanism of action are discussed.

The penetration of sodium into the epithelium of the frog skin.

The aim of this paper is twofold. First, to describe a method for the measurement of the unidirectional flux of Na from the outer bathing solution into epithelium (J(OT)), and second, to describe the use of this method under a variety of experimental conditions in order to obtain some insight into the nature of this flux. The method developed is based on the exposure of a frog skin to a Ringer solution containing (22)Na. The exposure is made so that neighboring points along the surface remain in contact with the (22)Na solution for gradually longer periods, ranging from 0 to 46 sec. Some 8 to 10 samples of the exposed part are used to obtain the time course of the uptake of (22)Na and this time course is used, in turn, to evaluate J(OT). This flux is then studied in skins mounted between two identical Ringer solutions with 115 mM Na (11.25 +/- 0.10 [18] micromole.hr(-2) cm(-2)), and in skins mounted with Ringer with 1 mM Na on the outside and 115 mM Na on the inside (0.43 +/- 0.05 [18] micromole.hr(-1).cm(-2). From the observations that the flux is much larger than the net Na flux across the whole skin, that it is inhibited by K(+), and is unaffected by ouabain, it is concluded that the penetration of Na(+) into the epithelium does not occur by simple diffusion and is not directly dependent on an ouabain-sensitive mechanism. In the course of these experiments it was observed that when the skin was crushed between two chambers the uptake of Na in the neighboring exposed areas was decreased.