Complement Activation by Bacterial Surface Glycolipids: A Study with Planar Bilayer Membranes

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd₂ LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd₂ LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway. Received: 16 July 1998/Revised: 28 October 1998     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli

Gram‐negative bacteria can survive in harsh environments in part because the asymmetric outer membrane (OM) hinders the entry of toxic compounds. Lipid asymmetry is established by having phospholipids (PLs) confined to the inner leaflet of the membrane and lipopolysaccharides (LPS) to the outer leaflet. Perturbation of OM lipid asymmetry, characterized by PL accumulation in the outer leaflet, disrupts proper LPS packing and increases membrane permeability. The multi‐component Mla system prevents PL accumulation in the outer leaflet of the OM via an unknown mechanism. Here, we demonstrate that in Escherichia coli, the Mla system maintains OM lipid asymmetry with the help of osmoporin OmpC. We show that the OM lipoprotein MlaA interacts specifically with OmpC and OmpF. This interaction is sufficient to localize MlaA lacking its lipid anchor to the OM. Removing OmpC, but not OmpF, causes accumulation of PLs in the outer leaflet of the OM in stationary phase, as was previously observed for MlaA. We establish that OmpC is an additional component of the Mla system; the OmpC‐MlaA complex may function to remove PLs directly from the outer leaflet to maintain OM lipid asymmetry. Our work reveals a novel function for the general diffusion channel OmpC in lipid transport.     

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Mechanisms of Action of Rabbit CAP18 on Monolayers and Liposomes Made from Endotoxins or Phospholipids

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction. Received: 14 January 2000/Revised: 20 April 2000     

Melatonin Enhances Tamoxifen's Ability to Prevent the Reduction in Microsomal Membrane Fluidity Induced by Lipid Peroxidation

The indoleamine melatonin and the synthetic antiestrogenic drug tamoxifen seem to have similar mechanisms in inhibiting the growth of estrogen receptor positive breast cancer cells. In this study, we compared the ability of these molecules, alone and in combination, in stabilizing microsomal membranes against free radical attack. Hepatic microsomes were obtained from male rats and incubated with or without tamoxifen (50–200 μm), melatonin (1 mm) or both; lipid peroxidation was induced by addition of FeCl₃, NADPH and ADP. After oxidative damage, membrane fluidity, measured by fluorescence polarization techniques, decreased whereas malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations increased. Incubation of the microsomes with tamoxifen prior to exposure to free radical generating processes inhibited, in a dose-dependent manner, the increase in membrane rigidity and the rise in MDA+4-HDA levels. When melatonin was added, the efficacy of tamoxifen in preventing membrane rigidity was enhanced. Thus, the IC₅₀s for preventing membrane rigidity and for inhibiting lipid peroxidation obtained for tamoxifen in the presence of melatonin were lower than those obtained with tamoxifen alone. Moreover, tamoxifen (50–200 μm) in the presence of melatonin reduced basal membrane fluidity and MDA+4-HDA levels in microsomes. These synergistic effects of tamoxifen and melatonin in stabilizing biological membranes may be important in protecting membranes from free radical damage. Received: 7 July 1997/Revised: 12 November 1997     

The Role of Yeast VDAC Genes on the Permeability of the Mitochondrial Outer Membrane

In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the POR1 gene (Δpor1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1 in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability. The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria. Received: 23 May 1997/Revised: 3 October 1997     

Membrane Asymmetry in Isolated Canine Cardiac Sarcoplasmic Reticulum: Comparison with Skeletal Muscle Sarcoplasmic Reticulum

Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent ``stripping' were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR. Received: 19 December 1997/Revised: 3 April 1998     

Influence of Cys-130 S. aureus Alpha-toxin on Planar Lipid Bilayer and Erythrocyte Membranes

Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which the channel becomes unable to discriminate between Cl⁻ and K⁺ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid bilayers was unchanged (maximum efficiency at pH 5.5–6.0). The asymmetry and nonlinearity of the current-voltage characteristics of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and equal to 2.4–2.6 nm. Alterations at the ``trans mouth' were found to be responsible for all observed changes of the channel properties. This mouth is situated close to the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel. Received: 8 September 1995/Revised: 20 November 1996     

Sphingomyelins of Rat Liver: Biliary Enrichment with Molecular Species Containing 16:0 Fatty Acids as Compared to Canalicular-Enriched Plasma Membranes

We harvested canalicular-enriched plasma membranes of hepatocytes and collected fistula bile from male rats and isolated the sphingomyelins. Following sphingomyelinase hydrolysis, we identified the sphingomyelin molecular species on the basis of their benzoylated ceramide derivatives employing high performance liquid chromatography. Sphingomyelin constitutes ≤3% of total biliary phospholipids (which are mostly sn-1 16:0 long-chain phosphatidylcholines) and approximately 30% of canalicular-enriched membranes. In both cases, the principal molecular species were composed of 16:0, 18:0, 20:0, 22:0, 23:0, 24:0, 24:1 and 24:2 fatty acid classes. However, the 16:0 fatty acid species was enriched in biliary sphingomyelin to a significantly greater degree than in sphingomyelins of canalicular-enriched plasma membranes (46% vs. 25% of total). We argue a physical-chemical case for laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane. We bolster our hypothesis by the likelihood that the least hydrophobic, e.g., 16:0 sphingomyelin molecular species, are miscible with biliary phosphatidylcholines, and are secreted into bile. Laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane could provide a means of sequestering cholesterol molecules prior to secretion into bile. Received: 19 March 1998/Revised: 8 October 1998     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

Monitoring of the Membrane Potential in Proteoliposomes with Incorporated Cytochrome-c Oxidase Using the Fluorescent Dye Indocyanine

A method has been developed to monitor changes of the membrane potential across vesicle membranes in real time. Using the potential-sensitive fluorescent dye indocyanine and on the basis of a water/lipid redistribution model, a calculation procedure has been introduced to estimate the membrane potential in vesicles with incorporated cytochrome-c oxidase. Physical parameters, such as vesicle size distribution and density of the lipid bilayer were estimated and used as calculation parameters. By extrapolation of the transient potential change to zero time, the initial rate of the potential change (dU/dt) could be calculated. It is also shown, that the initial potential change (dU/dt) may be used to study the proton/electron stoichiometry of cytochrome-c oxidase incorporated in the vesicles. Received: 28 September 1995/Revised: 6 February 1996     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

1/f-Noise of Open Bacterial Porin Channels

General diffusion pores and specific porin channels from outer membranes of gram-negative bacteria were reconstituted into lipid bilayer membranes. The current noise of the channels was investigated for the different porins in the open state and in the ligand-induced closed state using fast Fourier transformation. The open channel noise exhibited 1/f-noise for frequencies up to 200 Hz. The 1/f-noise was investigated using the Hooge formula (Hooge, Phys. Lett. 29A: 139–140 (1969)), and the Hooge parameter α was calculated for all bacterial porins used in this study. The 1/f-noise was in part caused by slow inactivation and activation of porin channels. However, when care was taken that during the noise measurement no opening or closing of porin channels occurred, the Hooge Parameter α was a meaningful number for a given channel. A linear relationship was observed between α and the single-channel conductance, g, of the different porins. This linear relation between single-channel conductance and the Hooge parameter α could be qualitatively explained by assuming that the passing of an ion through a bacterial porin channel is—to a certain extent—influenced by nonlinear effects between channel wall and passing ion. Received: 8 May 1996/Revised: 27 January 1997     

Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers

In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C₈₀-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C₈₀-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C₈₀-P/DOPC mole ratio, respectively. The TEM micrographs of C₈₀-P, DOPC and C₈₀-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells. Received: 10 January 2000/Revised: 7 June 2000     

Formation of Flat Lamellar Intramembrane Lipid Structures in Microorganisms

Analysis of freeze-fracture replicas and thin sections of cells of the bacteria Sulfobacillus thermosulfidooxidans and Anaerobacter polyendosporus showed that their cytoplasmic membranes contain some regions in the form of flat lamellar inverted lipid membranes a few tenths of nanometers to a few microns in size. The specific features of these membrane structures are as follows: (i) they contain no familiar intramembrane particles commonly present on freeze-fracture replicas; (ii) in cross thin sections, intramembrane structures are bifurcate on the periphery and look like thylakoids; and (iii) the leaflets of intramembrane structures in S. thermosulfidooxidans cells are corrugated. These structures were revealed in bacterial cells cultivated under normal growth conditions. The data obtained suggest the occurrence of a complex type of compartmentalization in biological membranes. Received: 17 July 2000/Revised: 22 November 2000     

A Quantitative Model for the Dependence of Solute Permeability on Peptide and Cholesterol Content in Biomembranes

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and α-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (P _m ) for CMHA by a factor of 6.0 ± 1.8 but did not alter P _m for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the P _m values for both CMHA and MHA by similar factors of 4.2 ± 1.1 and 3.5 ± 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of P _m on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect P _m for both permeants similarly since they are comparable in molecular size, while changes in P _m ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs P _m ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the P _m ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)^1/2, respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent. Received: 20 December 1999/Revised: 21 June 2000     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999     

Cobra Venom Cytotoxin Free of Phospholipase A2 and Its Effect on Model Membranes and T Leukemia Cells

Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A₂ (PLA₂). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA₂ in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA₂ (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studied by EPR of spin probes in oriented lipid multilayers and ¹H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine (PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat cells and normal lymphocytes were killed equivalently when treated with 10⁻⁹ m PLA₂ and 10⁻⁵ m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane. Received: 20 March 1996/Revised: 25 September 1996     

Characterization of the Ryanodine Receptor-Ca2+ Release Channel from the Thoracic Tissues of the Lepidopteran Insect Heliothis virescens

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [³H]-ryanodine binding activity of 1.19 ± 0.21 pmol/mg protein (mean ±se; n= 4). [³H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a K _D of 3.82 nm (n= 4). [³H]-ryanodine binding was dependent on [Ca²⁺] in a biphasic manner and was stimulated by 1 mm ATP. Millimolar caffeine did not stimulate [³H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca²⁺. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca²⁺-sensitive, Ca²⁺-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca²⁺ conductance of ∼110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nm ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [³H]-ryanodine binding properties of a native insect RyR. Received: 3 July 2000/Revised: 17 October 2000