Computation of mixed phosphatidylcholine-cholesterol bilayer structures by energy minimization.

The energetically preferred structures of dimyristoylphosphatidylcholine (DMPC)-cholesterol bilayers were determined at a 1:1 mole ratio. Crystallographic symmetry operations were used to generate planar bilayers of cholesterol and DMPC. Energy minimization was carried out with respect to bond rotations, rigid body motions, and the two-dimensional lattice constants. The lowest energy structures had a hydrogen bond between the cholesterol hydroxyl and the carbonyl oxygen of the sn-2 acyl chain, but the largest contribution to the intermolecular energy was from the nonbonded interactions between the flat alpha surface of cholesterol and the acyl chains of DMPC. Two modes of packing in the bilayer were found; in structure A (the global minimum), unlike molecules are nearest neighbors, whereas in structure B (second lowest energy) like-like intermolecular interactions predominate. Crystallographic close packing of the molecules in the bilayer was achieved, as judged from the molecular areas and the bilayer thickness. These energy-minimized structures are consistent with the available experimental data on mixed bilayers of lecithin and cholesterol, and may be used as starting points for molecular dynamics or other calculations on bilayers.

Molecular origin of the internal dipole potential in lipid bilayers: calculation of the electrostatic potential.

The finite difference linearized Poisson-Boltzmann equation was solved for a segment of bilayer for two lipids (phosphatidylcholine dihydrate and phosphatidylethanolamine-acetic acid) in order to obtain the transbilayer electrostatic potential. Atomic coordinates derived from the crystal structures of these lipids were used, and partial changes were assigned to all atoms in the polar parts of the molecules. These calculations confirmed that a dipole potential exists in the uncharged hydrophobic interior of a bilayer. The phosphocholine and phosphoethanolamine groups make negative contributions to the internal potential, and the glycerol acyl esters make positive contributions, but the sum of these terms is negative. The water of hydration in phosphatidylcholine, and the acetic acid which is present in the phosphatidylethanolamine crystal structure, make positive contributions to the internal potential. It is concluded that the water of hydration in fully hydrated lipid bilayers is mainly responsible for the experimentally inferred positive sign of the internal potential.

Multibilayer structure of dimyristoylphosphatidylcholine dihydrate as determined by energy minimization.

Complete energy minimization was carried out on the multibilayer crystal structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine dihydrate (DMPC.2H2O), starting from the X-ray structure determination reported by Pearson and Pascher (1979) Nature 281, 499-501. The asymmetric unit contains two nonidentical DMPC molecules and four water molecules. Minimization removed the acyl chain disorder present in the X-ray structure and caused the carbon planes of the acyl chains to become mutually parallel. Two energy-minimized structures (structures I and II) were found which mainly differed in the hydrogen-bonding arrangement of the waters of hydration. In structure I as in the X-ray structure, one of the water molecules forms a hydrogen-bonded bridge between successive bilayers; but in structure II, all hydrogen bonds are satisfied on the same bilayer. Structure II corresponds to the global energy minimum and is also a suitable structure for single bilayers. The lattice constants and cell volume of the minimized structures are close to the experimental values. The electrostatic force between DMPC bilayers is attractive. The mean hydration energy of the water is -14.2 kcal/mol, which is 2.5 kcal/mol lower than the binding energy of ice.

Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release.

We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted.

Comparison of energy-minimized crystal structures of 2,3-dilauroyl-D-glycerol, 3-palmitoyl-DL-glycerol-1-phosphorylethanolamine and 1,2-dilauroyl-DL-phosphatidylethanolamine:acetic acid.

Computational procedures have been developed by which the total energy of a lipid multibilayer can be calculated and minimized. The energy is expressed as a sum of non-bonded, electrostatic, hydrogen bonded and torsional energy terms and includes intramolecular and intermolecular components. Calculations were carried out on three lipid crystals for which structural data are available from X-ray diffraction analysis. For each crystal, the energy was minimized as a function of all bond rotations, molecular rotations and translations and the lattice constants. The minimized structures differed by only small amounts from the experimental structures, which confirms the validity of the current set of energy functions and parameters for use with lipids. The intermolecular energy of each crystal is analyzed in terms of lateral interactions, interactions between the two monolayers of the same bilayer and interactions between bilayers. The intermolecular non-bonded energy per CH2 or CH3 group in the acyl chains is also given.

Evidence for electrogenic accumulation of mefloquine by malarial parasites.

The uptake of mefloquine and chloroquine by Plasmodium chabaudi-infected mouse erythrocytes was measured in the presence and absence of ionophores and uncoupler in order to distinguish between the pH-dependent and pH-independent absorption of these drugs. Nigericin and CCCP (carbonylcyanide m-chlorophenylhydrazone) were used to relax the proton gradients and electrical potentials across the membranes. It was found that 40-60% of the mefloquine uptake, and 90% of the chloroquine uptake, was pH-dependent, the remainder being due to passive binding to cellular constituents. The distribution ratio of the pH-dependent uptake for mefloquine was about three times greater than for chloroquine. According to the lysosomotropic weak base hypothesis in which the neutral forms of weak bases are assumed to equilibrate across membranes, the mefloquine distribution should be smaller than the chloroquine distribution: since mefloquine is singly charged and chloroquine is doubly charged, the chloroquine distribution ratio should vary as the square of the mefloquine ratio. We interpret the greater uptake ratio of mefloquine to be evidence for the involvement of secondary active transport, with drug uptake being coupled to proton outflow by an antiporter protein. It is proposed that the uptake of mefloquine is electrogenic, with the proton gradient and the electrical potential both contributing to the driving force, but that the proton gradient alone is responsible for the chloroquine uptake.

Temperature dependence and mechanism of local anesthetic effects on mitochondrial adenosinetriphosphatase.

Chloroform-released ATPase prepared from beef heart mitochondria is inhibited by tetracaine and dibucaine over the entire temperature range in which the enzyme is active. The temperature of maximal activity is at 60 degrees C in the absence of anesthetic and is shifted upward by 2-3 degrees C by the addition of 0.3 mM dibucaine. Local anesthetics protect ATPase from irreversible cold inactivation. The kinetics of this protective effect are analyzed by a thermodynamic model in which the associated/dissociated subunit equilibrium is shifted toward the associated state by the preferential binding of anesthetic to the associated state. The accessibility of buried sulhydryl groups to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) is increased by local anesthetics; this is interpreted to mean that the anesthetics increase the conformational flexibility of the protein. It is proposed that the hydrophobic moieties of local anesthetics and related compounds bind to numerous hydrophobic sites or crevices on ATPase; this binding induces a perturbation of the protein conformation, which in turn causes a decrease of enzyme activity. This model is sufficiently general to encompass the diversity of molecules which have similar anesthetic-like effects, and since it relates to common fundamental features of protein structure, it may also be the mechanism of the nonspecific effects of these molecules on other proteins.

Stoichiometry and dissociation constants for interaction of tetracaine with mitochondrial adenosinetriphosphatase as determined by fluorescence.

The stoichiometry and dissociation constants for the interaction of tetracaine with chloroform-released ATPase prepared from beef heart mitochondria were determined from the enhancement of tetracaine fluorescence intensity that occurs upon binding. There is a single class of approximately 60 thermodynamically equivalent binding sites on ATPase for tetracaine; these have a microscopic dissociation constant of 4.9 X 10(-4) M at 25 degrees C under solvent conditions that are similar to those used for enzyme assay. Analysis of enzyme kinetic data according to a partial noncompetitive scheme gave an inhibitor constant for tetracaine of 4.8 X 10(-4) M. The numerical agreement between the dissociation constant and the inhibitor constant shows that the filling of the same class of sites is probably responsible for both the enzyme inhibition and the fluorescence enhancement. The sites are hydrophobic, as evidenced by the blue shift and the magnitude of the fluorescence enhancement that occur upon binding.

Differential scanning calorimetry study of local anesthetic effects on F1ATPase and submitochondrial particles.

The differential scanning calorimetry trace of F1ATPase, prepared from beef heart submitochondrial particles, has a single sharp endothermic transition at 80.5 +/- 1.0 degrees C and a half-height peak width of 2.0 +/- 0.2 degrees. The transition enthalpy is 19 +/- 2 cal/g of protein. Submitochondrial particles (SMP) have a similar peak at 75.1 +/- 0.5 degrees C with a half-height peak width of 1.8 +/- 0.1 degrees and an enthalpy of 5 +/- 1 cal/g of SMP protein. The SMP transition is provisionally identified as being due to membrane-bound F1ATPase. Tetracaine and dibucaine cause these transitions to shift to lower temperatures; addition of 0.3 mM dibucaine gives peaks at 71.7 and 64.9 degrees C for F1ATPase and SMP, respectively, and 1.0 mM tetracaine gives peaks at 70.0 and 60.5 degrees C for F1ATPase and SMP, respectively. These anesthetic concentrations also give appreciable inhibition of enzyme activity at 25 degrees C. We conclude that the local anesthetics induce conformational alterations in the F1ATPase-protein complex which result both in enzyme inhibition and in the lowering of the thermal denaturation transition temperature.

Local anesthetics: a new class of partial inhibitors of mitochondrial ATPase.

The following characteristics are reported for mitochondrial ATPase prepared by the chloroform extraction method: (1) The pH optimum for enzyme activity is at 8.0. (2) The neutral anesthetic benzocaine inhibits the enzyme at all pH values. (3) Reciprocal plots of 1/v versus 1/[ATP] show that inhibition by lidocaine, tetracaine, dibucaine, and chlorpromazine is noncompetitive; slope and intercept replots are hyperbolic, showing that the inhibition is partial rather than complete.

Inhibition of synaptosomal enzymes by local anesthetics.

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na+ + K+)-ATPase (ouabain-sensitive), Mg2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPases, for example 7.9 and 10 mM tetracaine for Mg2+-ATPase and (Na+ + K+)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.

Further studies on F1-ATPase inhibition by local anesthetics.

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.

Cytochrome c oxidase inhibition by anesthetics: thermodynamic analysis.

The thermodynamic parameters that characterize the inhibition of cytochrome c oxidase activity, in rat liver submitochondrial particles, by n-butanol, tetracaine, and dibucaine were obtained. Three equilibria were assumed in order to account for the data: for the interaction of inhibitor with the native state of the enzyme, for the interaction of inhibitor with the thermally (reversibly) denatured state, and for the change between the native and thermally denatured states. Inhibition results from interaction with both the native and denatured states but, because the interaction is stronger with the denatured than with the native state, the native/denatured equilibrium is shifted to the right by the anesthetics. The enthalpies of interaction are -2.3, -4.7, and 3.7 kcal/mol (1 cal = 4.18 J) for the native state and -10, -6, and -14 kcal/mol for the denatured state, for n-butanol, tetracaine, and dibucaine, respectively. These values are much smaller than the previous estimates obtained by using the assumption that anesthetics interact only with the thermally denatured state of enzymes (e.g., -81 kcal/mol for tetracaine inhibition of luciferase). Our results suggest that local anesthetics inhibit enzyme activity by causing a reversible perturbation of protein conformation. The magnitude of the perturbation is much smaller (in energetic terms) than that which accompanies thermal denaturation.

Multiple sites of inhibition of mitochondrial electron transport by local anesthetics.

Local anesthetics and alcohols were found to inhibit mitochondrial electron transport at several points along the chain. THe anesthetics employed were the tertiary amines procaine, tetracaine, dibucaine, and chlorpromazine, and the alcohols were n-butamol, n-pentanol, n-hexanol, and benzyl alcohol. Uncoupled sonic submitochondrial particles from beef heart and rat liver were studied. We report the following: (1) All of the anesthetics were found to inhibit each of the segments of the electron transport chain assayed; these included cytochrome c oxidase, durohydroquinone oxidase, succinate oxidase, NADH oxidase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, and NADH-cytochrome c oxidoreductase. (2) NADH oxidase and NADH-cytochrome c oxidoreductase required the lowest concentration of anesthetic for inhibition, and cytochrome c oxidase required the highest concentrations. (3) We conclude that there are several points along the chain at which inhibition occurs, the most sensitive being in the region of Complex I (NADH dehydrogenase). (4) Beef heart submitochondrial particles are less sensitive to inhibition than are rat liver particles. (5) Low concentrations of several of the anesthetics gave enhancement of electron transport activity, whereas higher concentrations of the same agents caused inhibition. (6) The concentrations of anesthetics (alcohol and tertiary amine) which gave 50% inhibition of NADH oxidase were lower than the reported concentrations required for blockage of frog sciatic nerve.

On the mechanism of action of anesthetics. Direct inhibition of mitochondrial F1-ATPase by n-butanol and tetracaine.

The concentrations of n-butanol and tetracaine required for 50% inhibition of the ATPase activity of F1 particles isolated from bovine heart mitochondria were 160 mM and 1.1 mM, respectively. The results are offered as evidence that the physiological effects of these anesthetics may be due to direct interaction with membrane proteins rather than with the lipids.

Block poly(Ala)-poly(Lys). A water-soluble model for intrinsic membrane proteins?

Block poly(Ala)16-poly(Lys)13.5 was synthesized by the Leuchs anhydride method. This polypeptide is water soluble in a largely monomeric form, but binds rapidly and spontaneously to unilamellar vesicles of dimyristoyl phosphatidylcholine at pH 7.4. The interaction is evidently of a hydrophobic nature since the complex is not disrupted by salt and no similar reaction is given by polylysine. Evidence for the interaction was obtained by ultrafiltration, chromatography on Sepharose 4B, and sedimentation velocity ultracentrifugation. While direct information on the molecular structure of the complex is still lacking, we propose that this amphipathic block copolymer binds to lipids in a similar manner as intrinsic membrane proteins and hence can be used to study the interactions of intrinsic proteins with lipids.

A calorimetric comparison of the interaction of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins.

The interactions of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins have been studied by the method of titration calorimetry. It was found that the initial addition of sodium dodecyl sulfate to cytochrome c caused an endothermic unfolding of the protein, detectable by circular dichroism (CD). This was followed by the exothermic binding of sodium dodecyl sulfate to the protein, without further CD-detectable conformational changes. In contrast, sodium dodecyl sulfate bound directly to the erythrocyte glycoproteins in an exothermic reaction without any accompanying CD-detectable conformation changes. This indicates that the glycoproteins solubilized in aqueous media have exposed hydrophobic regions which can interact directly with this detergent. The enthalpy changes and stoichiometries of binding are reported.