Mechanism Of Membrane Damage Research Articles

Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis.

Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A(2) variant present in the venom of Bothrops jararacussu, in which the Asp(49) residue is replaced with a lysine, which damages artificial membranes by a Ca(2+)-independent mechanism. Wild-type BthTx-I and the mutants Lys(49)-->Asp, His(48)-->Gln and Lys(122)-->Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom. The Ca(2+)-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His(48)Gln and Lys(49)Asp mutants were similar; however, the Lys(122)Ala mutant demonstrated reduced levels of both activities. Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants. In the case of the Lys(49)Asp mutant, this demonstrates that the absence of catalytic activity in Lys(49)-PLA(2) is not a consequence of the single Asp(49)-->Lys replacement. Furthermore, these results provide unambiguous evidence that the Ca(2+)-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis. The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.

Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.

Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region. Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E. coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes.

Mechanisms of amyloid beta protein-induced modification in ion transport systems: implications for neurodegenerative diseases.

1. Alzheimer's disease (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. Abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss. 2. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AbetaP[1-42] and AbetaP[1-43] and soluble nonpathologic AbetaP[1-40]. Both APP and AbetaP interact with ion transport systems. AbetaP induces a wide range of effects as the result of activating a cascade of mechanisms. 3. The major mechanisms proposed for AbetaP-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of (1) changes in endogenous ion transport systems, e.g. Ca2+ and K+ channels and Na+/K+-ATPase, and membrane receptor proteins, such as ligand-driven ion channels and G-protein-driven releases of second messengers, and (2) formation of heterogeneous ion channels. 4. The consequences of changes in Ca2+-homeostasis-induced generation of ROS are (a) direct modification of intrinsic ion transport systems and their regulatory mechanisms, and (b) indirect effects on ion transport systems via peroxidation of phospholipids in the membrane, inhibition of phosphorylation, and reduction of ATP levels and cytoplasmic pH. 5. We propose that in AD, AbetaP with its different conformations alters cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding plaque formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is not only central for AD but also may explain other malfunctioned protein-processing-related pathologies.

Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism

Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109–200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic β-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca2+-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.

A tale of two toxins—similarities and contrasts in the mechanisms of membrane damage of toxins from Staphylococcus aureus and Bothrops jararacussu

A tale of two toxins—similarities and contrasts in the mechanisms of membrane damage of toxins from Staphylococcus aureus and Bothrops jararacussu

Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl- tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na +,K +-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8±9.3 units/g tissue vs. hypoxic: 57.9±9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639±0.054 μg quinine sulfate/g brain vs. 0.810±0.102 μg quinine sulfate/g brain, p<0.01) and a decrease in Na +,K +-ATPase activity (normoxic: 43.04±2.50 μmol Pi/mg protein/h vs. hypoxic: 33.80±3.51 μmol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na +,K +-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na +,K +-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.

A SequenceSpace analysis of Lys49 phopholipases A2: clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity.

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a sub-family of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Mechanism of membrane damage by (-) epigallocatechin gallate

Mechanism of membrane damage by (-) epigallocatechin gallate

Characterization of the damage to membranes caused by bacterial cytolysins

The damage to cell membranes caused by bacterial cytolysins has been studied for many years. In this review, we attempt to summarize the historical contribution of electron microscopy to our understanding of the modes of action of pore-forming and channel-forming toxins. We describe the ways in which these toxins form holes in membranes by binding to and forming oligomers on biological membranes. We also introduce the new technique of electron spectroscopic imaging (ESI) with an imaging plate (IP), which has been used to analyse the mechanisms of membrane damage by cytolysins.

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

Membrane effects in postharvest physiology

Abstract Plant tissue deterioration is of prime importance to food and horticultural scientists. Experimental evidence continues to be gathered supporting membrane damage as the key event leading to a cascade of biochemical reactions culminating in tissue deterioration and economic loss. In this paper, the sequence of occurrences in plant membranes during natural senescence and resulting from postharvest stresses are reviewed and compared. The picture emerging from numerous studies devoted to this topic is of membrane lipids undergoing extensive chemical degradation. Biophysical changes in membrane lipids and enzymatic and non-enzymatic lipid peroxidation leads to altered membrane properties and result in defects such as ion leakage and cellular decompartmentation. Postharvest stresses including chilling injury and freezing injury often lead to similar patterns of deterioration. However, it is too early to conclude positively that a truly universal mechanism for both senescence and postharvest stress-induced membrane damage exists.

Partially Unfolded Lysozyme at Neutral pH Agglutinates and Kills Gram-Negative and Gram-Positive Bacteria through Membrane Damage Mechanism

The antimicrobial mechanism and structural changes of hen egg white lysozyme irreversibly inactivated at 80 °C and at different pHs were investigated. We found that heat denaturation of lysozyme at increasing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gram-negative bacteria. Interestingly, lysozyme devoid of enzyme activity (heated at 80 °C and pH 7.0 or at pH 6.0 over 90 °C) exhibited strong bactericidal activity against Gram-negative and -positive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positive bacteria was that heated at 80 °C and pH 6.0 (HLz80/6), retaining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz80/6-induced agglutination coincided with severe reduction in colony-forming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptidoglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipid vesicles resulted in a blue shift in the intrinsic tryptophan fluorescence accompanied by an increase in the size of the vesicles, indicating enhanced protein-membrane binding and subsequent fusion of liposomes. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an interesting finding that partial unfolding of lysozyme with the proper acquisition of the hydrophobic pocket to the surface can switch its antimicrobial activity to include Gram-negative bacteria without a detrimental effect on the inherent bactericidal effect against Gram-positive ones. The data suggest that the unique antimicrobial action of unfolded lysozyme attributes to membrane binding and subsequent perturbation of its functions.

Biophysical mechanisms of cell membrane damage in electrical shock.

Biophysical mechanisms of cell membrane damage in electrical shock.

The 1995 Lindberg Award

Joule heating has long been considered the principal component of tissue damage in electrical injury. Recent studies suggest electroporation, a nonthermally mediated mechanism of cell membrane damage, is also a factor. We investigated whether electroporation-mediated muscle necrosis can occur in vivo without significant Joule heating. Pulsed electric fields approximately 150 V/cm were produced in the hind limbs of anesthetized rats. In shocked limbs core muscle temperature rose less than 1.8 degrees C, yet significant damage occurred as determined by technetium-99m pyrophosphate uptake, elevated serum creatine phosphokinase, and prominent hypercontraction band degeneration of myofibers on histopathologic examination. This study is significant because it directly addresses whether nonthermal mechanisms of cell damage can cause tissue necrosis. These results indicate that electroporation effects can mediate skeletal muscle necrosis without visible thermal changes. Thus the phenomenon of "progressive recognition" may be characteristically largely explained by the occurrence of nonthermally mediated tissue damage.

Modeling the bacterial protein toxin, pneumolysin, in its monomeric and oligomeric form.

Pneumolysin is a member of the family of related bacterial thiol-activated toxins, which share structural similarities and a proposed common cytolytic mechanism. Currently the molecular mechanism of membrane damage caused by these toxins remains a matter of controversy. A prerequisite for defining this mechanism is a detailed knowledge of the monomeric and oligomeric pneumolysin structures. We present for the first time details of the monomeric structure of a thiol-activated toxin, pneumolysin. Electron microscope images of metal-shadowed pneumolysin monomers show an asymmetric molecule composed of four domains. We have studied the conformation of pneumolysin monomer by low resolution hydrodynamic bead modeling procedures. The bead model dimensions and shape are derived solely from the electron micrographs. The bead model has been evaluated in terms of the predicted solution properties, which in turn have been compared to the experimental values of the sedimentation coefficient, s(20,w)0, obtained by analytical ultracentrifugation and the intrinsic viscosity, [eta]. Pneumolysin oligomers, observed as ring- and arc-shaped structures, were also examined by electron microscopy. Metal shadowing and negative staining methods were used to establish the overall dimensions of the oligomer and were used to produce a morphological model for the oligomer, incorporating monomer subunits based on the hydrodynamic bead model.

Phenylhydrazine-induced iron release as a mechanism of membrane and DNA damage

Phenylhydrazine-induced iron release as a mechanism of membrane and DNA damage

The role of intracellular free calcium mobilization in the mechanism of action of antitumour ether lipids SRI 62-834 and ET18-OMe

Membrane-active antitumour ether lipids such as ET18-OMe (1- O-octadecyl-2- O-methyl- rac-glycero-3-phosphocholine) and SRI 62–834 ((±)-2-{Hydroxy[tetrahydro-2-(octadecyloxy) methylfuran-2-yl] methoxyl phosphinyloxy}- N, N, N-trimethylethaniminium hydroxide) are selectively cytotoxic to tumour cells in vitro. Their precise mechanisms of action are unclear, but they are known to have effects on cell membranes and cell signalling. A previous report suggested that ether lipids cause a biphasic sustained rise in intracellular free calcium [Lazenby et al., Cancer Res 50: 3327–3330, 1990]. We show here that the second phase is an experimental artefact due to cell membrane permeabilization by ether lipids in serum-free buffers. In serum-free medium, the membrane toxicity of antitumour ether lipids was increased 50–60 fold, when compared to medium containing 10% serum. Membrane disruption was neither dependent on extracellular calcium, nor modulated by preloading cells with the calcium chelators bis( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid or 2-[[2-[bis (carboxymethyl) amino ] — 5 -methylphenoxy] methyl] — 6 -methoxy — 8 -[bis(carboxymethyl) amino] quinoline. This indicates that the mechanism of membrane damage by ether lipids does not involve changes in calcium homeostasis. Using indo-1 and fura-2 as calcium probes, we established that lower concentrations of antitumour ether lipids do elicit a genuine monophasic and transient rise in intracellular free calcium, predominantly mobilized from internal stores. This acute calcium agonist activity of ether lipids is distinct from the inhibitory effects on cell signalling reported previously after more prolonged exposure. It appears that the calcium elevation induced by antitumour ether lipids is unlikely to be instrumental in their selective and potent antitumour activity.

Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57–63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.

Role of free-radical oxidation in the pathology of newborns and infants

The processes of free-radical oxidation of lipids are considered to be the fundamental molecular mechanism of biological membrane damage. At the present stage, significant data have been accumulated on the activity of lipid peroxidation processes in acute pathology in children.

Mechanisms of membrane damage for CHO cells heated in suspension.

Chinese hamster ovary cells were heated for 20 min at 45.5 degrees C in different conditions, and quantitative determinations of cellular membrane blebbing were performed for cells maintained at 25 degrees C and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating was dependent upon the composition of the medium during heating and the posthyperthermia temperature after heating. The total extent of bleb formation after heating was independent of the calcium-ion concentration in the medium during heating; however, differences in the kinetics of bleb disappearance after heating point to the importance of Ca2+ concentration in the expression of heat damage. Without hyperthermia, blebs were formed on the cell-surface membrane with agents which block sulfhydryl groups or release calcium from cellular stores. The cells were protected from bleb formation when cells were incubated with glutathione before addition of sulfhydryl-blocking agents or heat treatment. Oligomycin did not prevent the formation of blebs, suggesting that this phenomenon is not energy-dependent. Only a small percentage of cells were covered with blebs when they were heated in saline solution. When cells were incubated with dbcAMP before heat, blebs did not appear at 25 degrees C. A possible interpretation for these observations is presented.