The influence of pharmacologically active substances of different classes at ultra low doses on lipid peroxidation in brain cell membranes and activity of acetylcholinesterase in vivo and in vitro

One of the approaches to the development of new medicines now is the exploration of the effects of low and ultra low doses (ULD) of biologically active substances and preparations traditionally used in rather high dosages. The purpose of our work was to investigate the influence of pharmacologically active substances of various classes at wide range of concentrations, including ultra low, on lipid peroxidation in cell membranes of mice brain and activity of acetylcholinesterase (AChE). The action of synthetic antioxidant (AO) from the group of hindered phenols (inhibitors of free-radical reactions) phenozan, neurotransmitter acetylcholine (ACh), hybrid compound "phenozan + ACh + alkyl radical C-10" (perspective for Alzheimer's disease therapy), tranquilizer from benzodiazepines phenazepam and hydrogen peroxide were investigated. The influence of the investigated substances at ULD (concentrations) on kinetic parameters of the reaction, catalysed with soluble and membrane AChE (Michaelis constant and maximal velocity), and also on lipid peroxidation (LPO) system (level of products and LPO velocity, contents of total lipids, phospholipids, cholesterol) in mice brain cell membranes in vitro and in vivo was revealed. Concentrational and dose curves were of compilated character with the presence of zero effect zones typical for the agents capable to work at ULD. The effects of super low and "usual" doses of investigated substances were commensurable.     

Effect of superlow doses (10(-18)-10-(-14) M) of biologically active substances: general rules, features, and possible mechanisms]

The effects of ultra-low (10(-18)-10(-14) M) doses (ULD) of biologically active substances have been reviewed in terms of common regularities of ULD effects and peculiarities of action of various groups of compounds. The most common and at the same time paradoxical regularities of ULD action are bi- or polymodal patterns of dose dependence, absence or presence of an inverse effect at higher doses, and instability of ULD effect. Possible mechanisms of ULD action including the mechanism based on the adaptation theory are discussed.     

Ensembles of carboxymethyl cyclodextrins on cationic liposomes as highly efficient nanocontainers for the delivery of hydrophobic compounds

The increase of payload is one of the key tasks in creation of nanocontainers for the delivery of bioactive substances (BAS). In this work the adsorption of anionic carboxymethyl cyclodextrins (CMCDs) on the surface of cationic liposomes was studied as mechanism of formation of capacious nanocontainers for the encapsulation and delivery of hydrophobic BAS. The formation and physico-chemical characteristics of complexes were studied by means of laser microelectrophoresis, dynamic light-scattering, conductometry and atomic force microscopy (AFM). As a model, bioactive molecule hydrophobic curcumin was chosen for the investigation. The encapsulation of curcumin was controlled by UV–Vis spectrometry. Interaction of CMCDs/liposomes complexes with model cell membranes was visualized by fluorescent microscopy. Finally, cytotoxicity of nanocontainers was studied by MTT-test. It was estimated that colloid stable complexes with net positive charge could contain up to 2.5÷5 CMCD molecules per one cationic lipid. Incorporation of curcumin in CMCDs does not change the character of interaction of oligosaccharides with liposomal membranes of individual liposome. CMCDs/liposomes complexes adsorb on model cell membranes without significant loss of CMCD molecules. This fact in addition to low cytotoxicity of cationic CMCDs/liposomes complexes demonstrates potential of their application as nanovehicles for the delivery of BAS.     

Functional reconstitution of the integral membrane enzyme, isoprenylcysteine carboxyl methyltransferase, in synthetic bolalipid membrane vesicles

Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (also known as bolalipids) have been prepared to determine (1) the influence of molecular structure on the physical properties of bolalipid membranes and (2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. Three bolalipids were synthesized: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 degrees C; C32phytBAS Tm = 14 degrees C; and C20BAS Tm = 17 degrees C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 25 degrees C, were 1.5 x 10(-8) and 1.8 x 10(-9) cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS/E. coli polar lipid, C20BAS/POPC, C32phytBAS/E. coli polar lipid, and C32phytBAS/POPC. Ste14p activity was lost in vesicles composed of 75-100 mol % C20BAS and 0-100 mol % C32BAS but retained in vesicles with 0-50 mol % C20BAS and 0-100 mol % C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol % C20BAS and 100 mol % C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 A. Because Ste14p activity was not affected by either the gel-to-liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75-100 mol % C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

Potato plants lacking the CDSP32 plastidic thioredoxin exhibit overoxidation of the BAS1 2-cysteine peroxiredoxin and increased lipid Peroxidation in thylakoids under photooxidative stress

The CDSP32 protein (chloroplastic drought-induced stress protein of 32 kD) is a thioredoxin participating in the defense against oxidative damage. We recently have identified in vitro the BAS1 2-Cys peroxiredoxin, a peroxide-detoxifying enzyme, as a target for CDSP32. Here, we report the characterization under stress conditions of transgenic potato (Solanum tuberosum) plants lacking CDSP32 with regard to the BAS1 redox state and the level of lipid peroxidation. Under control conditions, BAS1 is present at similar levels both in wild-type (WT) and transgenic plants. Under drought and methyl viologen treatment, CDSP32-lacking plants display, compared with WT, an increased proportion of BAS1 monomer corresponding to an overoxidized form of the protein. Leaf discs from transgenic plants treated with methyl viologen exhibit earlier degradation of BAS1 than WT plants do. Using several approaches, i.e. a probe emitting fluorescence when reacting with peroxides, high-performance liquid chromatography determination of lipid hydroxy fatty acid content, and measurement of chlorophyll thermoluminescence, we show a higher lipid peroxidation level under methyl viologen treatment in thylakoids from CDSP32-lacking plants compared with WT. These data show that CDSP32 is a critical component in the defense system against lipid peroxidation in photosynthetic membranes, likely as a physiological electron donor to the BAS1 peroxiredoxin.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Effect of dilute solutions of biologically active substances on cell membranes

The article presents data on changes in physicochemical properties of different biological membranes (plasmatic, microsomal, synaptosomes) under the action of biologically active substances, which are different in their chemical structure and the mechanism of action (natural and synthetic antioxidants, thyrotropin - releasing hormone, phorbol esters), in the wide range of concentrations (10^?22?10^?3 M). Dose dependences of the effect of biologically active substances on the activity of membrane-bound enzymes, lipid peroxidation, the structural state of the various regions of the lipid bilayer of membranes have been obtained and analyzed in terms of their formal generality of polymodality, number and position of the maxima, a sign change of the effect. An attempt to explain the mechanism of each of the observed peaks in these curves has been made. The maximum in the range of relatively high “physiological” concentrations (10^?3–10^?7 M) is associated with introduction of biologically active substances into biomembranes. In this study maxima in the range of ultra-low doses (10^?11–10^?16 M) and “apparent” concentrations (10^?18 M), where the presence of biologically active substance molecule in a reaction volume is probabilistic in nature, are explained by physicochemical properties of diluted biologically active substances solutions. This conclusion is based on our data on the changes in IR spectra of aqueous solutions of biologically active substances and the results obtained by academician A.I. Konovalov et al. concerning the physicochemical properties of dilute solutions of biologically active substances (conductivity, surface tension, charge), due to the formation of so-called “nanoassociates” from biologically active substance molecule and numerous number of water molecules. The nanoassociates formation and biological effect disappear if the low concentration solutions are kept in a special shielded permalloy container protecting its contents from external electromagnetic field. Thus, nanoassociates are the material carriers of the unique ability of the ultra-low doses of biologically active substances to exhibit biological effects.     

A study of the effect of low alpha-tocopherol concentrations on the dynamical parameters of microsomal membranes by the method of spin probes

The effect of alpha-tocopherol (alpha-tp) prepared in solvents of different polarity in a wide range of concentrations (10(-4) M - 10(-25) M) on lipid phase structural characteristics of microsomal membranes isolated from mouse liver cells has been investigated in vitro. Structural changes in membranes were detected on a Bruker-200D ESR-spectrometer (Germany) by the method of spin probes. Changes in the rigidity of surface lipid bilayer regions (8 A) and microviscosity of deep membrane layers (20 A) were studied using the stable nitroxyl radicals 5- and 16-doxylstearic acids, correspondingly. As a result, nonlinear multimodal dose dependences were obtained. It was demonstrated that the physiological (10(-4) M - 10(-9) M) and ultralow doses of alpha-tocopherol up to "apparent" concentrations (10(-11) M - 10(-25) M) increased the rigidity of surface lipid bilayer regions and microviscosity in the depth of membrane. Additionally, these doses of alpha-tp induced an increase in the number of thermoinduced structural transitions in deep lipid bilayer regions. The effect at "apparent" concentrations (< 10(-18) M) has only been observed in polar alpha-tocopherol solutions. The results obtained are statistically reliable with a significance level of 95%.     

Radioimmunologic and radioreceptor studies of the pharmacokinetics of the enkephalin analog dalargin in man

Pharmacokinetics of dalargin, an opioid hexapeptide, was investigated on 7 males by two approaches. Dalargin radioimmunoassay was performed using a highly specific antiserum reacting only with the whole molecule. In radioreceptor assay lyophilized rat brain membranes containing opiate receptors were used. 2-6 min after intravenous introduction of 1-10 mg dalargin, immunoreactive dalargin blood concentration was lower than 0.5 ng/ml. The results of radioreceptor assay were presented as a biexponential curve with a fast main phase of activity changes (90%, characteristic time 1.5-5.0 min) and a slow "clearance" phase (10% of the substance, characteristic time 85-200 min). Prolonged presence of receptor-active substances in the blood can be attributed to the products of dalargin degeneration, namely its N-terminal penta- and tetrapeptides.     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.     

Rapid protein anchoring into the membranes of Mammalian cells using oleyl chain and poly(ethylene glycol) derivatives

The cell membrane is an important interface for communication with extracellular events, and incorporation of bioactive substances, such as antibodies and receptors, into the cell membrane may enhance the potential abilities of cells. Gene manipulation, chemical modification of membrane proteins, and cell surface painting using a GPI anchor have been performed to introduce substances into cell membranes. Furthermore, many lipid anchors have also been used to modify lipid membranes such as liposomes. In this study, we have focused on developing an easy and rapid method for anchoring of substances including macromolecular proteins into the membranes of living mammalian cells. We employed a single oleyl chain derivative coupled with hydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitate solubilization in water. This water-soluble derivative was designated Biocompatible Anchor for Membrane (BAM). Some proteins (streptavidin, EGFP and an antibody) were coupled with BAM90 at the distal terminal of PEG and rapidly (within a few minutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3, hybridoma 9E10). However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention time of anchoring was shortened (1-2 h) in culture medium containing 10% FBS. We further prepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80, the EO unit is 80) as a double oleyl chain derivative for comparison with the single oleyl chain derivative, BAM90. The retention time of anchored DOPE-BAM80 was longer than that of BAM90 and more than 8 h in culture media with and without 10% serum. Furthermore, the treatment time of DOPE-BAM80 for anchoring was nearly as short (within a few minutes) as that of BAM90. In addition, both types of BAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity. Therefore, DOPE-BAM80 is useful for protein anchoring to cells. Although the utilization of BAM90 is considered to be limited, it is expected to useful in restricted environments, for example, in tissues such as the cornea, peritoneum, bladder, and various mucosae, which are less exposed to serum. Thus, we suggest the possibility that both types of BAM can be applied to cell surface engineering.     

Effect of electronic homeopathic copy of biohumus fertilizer on tomato sprout development

Electronic homeopathic copies (EHC) are remedies prepared without traditional dilution/potentiation but by means of so-called "imprinting" of initial substance to water (or other carriers) with the help of M. Ray's devices. EHC are interpreted by modern homeopathic medicine as functional analogs of biologically active substances (BAS) in supersmall doses (SSD). The authors have undertaken 3 blind randomised experiments concerning BAS aqueous solution of fertilizer biohumus, its EHC and placebo influence on tomatoes' sprouts development. Filtered and intermixed water have been encapsulated in 1.5-liter polyethylene containers. The solution of fertilizer has been prepared in accordance with the instruction. EHC has been "transferred" from the concentrated fertilizer with the help of Simulator (Metabolics, GB) apparatus. "Deleting" of information have been made for placebo. Seed of tomatoes (kind "Yasniye") have been pre-soaked in preparations and landed in accordingly numbered plastic boxes (97 x 15 x 14 sm) by 40-44 plants in each one. The seedbed was from one well intermixed portion. The plants were top-dressed by preparations (200-300 ml) one time per week. The boxes were exposed in one room (rotation was made twice per week). The plants have been cut up on the 38th day from pre-soaking. The height of a green part and its mass have been measured for each plant. The differences between independent samples (preparations studied) have been estimated. The differences between EHC and placebo have appeared to be reliable, in the first experiment (p < 2 x 10(-4)--mass, p < 10(-9)--height), in the second one (p = 0.014--mass), in the third one (p < 3 x 10(-6)--mass, p = 0.028--height) The results obtained verify a reality of EHC phenomenon. Thus it is quite within reason to suggest an existence of uniform physical (instead of chemical) mechanism of BAS's EHC/SSD structuring and acting on biological objects.     

Insulin-induced changes in mechanical characteristics of lipid bilayers modified by liver plasma membrane fragments

Insulin interaction with BLM with incorporated fragments of rat liver plasma membranes, containing hormone receptors, was studied by determining Young modulus of elasticity of bilayer lipid membranes in direction perpendicular to the surface, E. The presence of membrane proteins in a concentration of 60 micrograms.ml-1 induced a significant decrease in parameter E (to approx. 50%) as compared with values obtained in non-modified membranes during insulin action (concentration interval 10(-11)-10(-9) mol.l-1). The extent of the effect was dependent on the initial phase state of the membrane, on cholesterol content in BLM as well as on membrane proteins concentration in lipid bilayer.     

Permeation of membranes by the neutral form of amino acids and peptides: Relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10⁵ slower than facilitated inward transport across biological membranes. This suggests that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10¹⁰ times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g., for certain signal sequences, toxins and thylakoid proteins) in vivo.Abbreviations LUV large unilamellar vesicle - pH transmembrane pH gradient - PAH polyaromatic hydrocarbon Correspondence to: A.C. Chakrabarti     

Reactive oxygen species and sperm cells

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Lipid peroxidation modifies the picture of membranes from the "Fluid Mosaic Model" to the "Lipid Whisker Model"

The “Fluid Mosaic Model”, described by Singer and Nicolson, explain both how a cell membrane preserves a critical barrier function while it concomitantly facilitates rapid lateral diffusion of proteins and lipids within the planar membrane surface. However, the lipid components of biological plasma membranes are not regularly distributed. They are thought to contain “rafts” - nano-domains enriched in sphingolipids and cholesterol that are distinct from surrounding membranes of unsaturated phospholipids. Cholesterol and fatty acids adjust the transport and diffusion of molecular oxygen in membranes. The presence of cholesterol and saturated phospholipids decreases oxygen permeability across the membrane. Alpha-tocopherol, the main antioxidant in biological membranes, partition into domains that are enriched in polyunsaturated phospholipids increasing the concentration of the vitamin in the place where it is most required. On the basis of these observations, it is possible to assume that non-raft domains enriched in phospholipids containing PUFAs and vitamin E will be more accessible by molecular oxygen than lipid raft domains enriched in sphingolipids and cholesterol. This situation will render some nano-domains more sensitive to lipid peroxidation than others. Phospholipid oxidation products are very likely to alter the properties of biological membranes, because their polarity and shape may differ considerably from the structures of their parent molecules. Addition of a polar oxygen atom to several peroxidized fatty acids reorients the acyl chain whereby it no longer remains buried within the membrane interior, but rather projects into the aqueous environment “Lipid Whisker Model”. This exceptional conformational change facilitates direct physical access of the oxidized fatty acid moiety to cell surface scavenger receptors.     

Characterization of diphtheria toxin-induced lesions in liposomal membranes. An evaluation of the relationship between toxin insertion and "channel" formation

Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the "channel-forming" B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between "channel formation" and fragment A translocation across membranes. However, the nature of the toxin channel is called into question by our observations that channel size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin "channel" is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer.