The action of phosphatidylinositol-specific phospholipases C on membranes.

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte "ghosts" and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte "ghosts" together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.     

An EPR investigation of surfactant action on bacterial membranes

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.     

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases.

1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50% of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80% of the sphingomyelin. 2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect. 3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved. 4. Due to its absolute requirement for Ca2+, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca2+ stimulates phospholipase A2 activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25% of the lecithin, half of the phosphatidylethanolamine and some 65% of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.     

Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes.

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.     

Permeabilizing action of an antimicrobial lactoferricin-derived peptide on bacterial and artificial membranes

We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity.     

Persistent organic pollutants in model fungal membranes. Effects on the activity of phospholipases

Soils are the final sink for multiple organic pollutants emitted to the environment. Some of these chemicals which are toxic, recalcitrant and can bioaccumulate in living organism and biomagnify in trophic chains are classified persistent organic pollutants (POP). Vast areas of arable land have been polluted by POPs and the only economically possible means of decontamination is bioremediation, that is the utilization of POP-degrading microbes. Especially useful can be non-ligninolytic fungi, as their fast-growing mycelia can reach POP molecules strongly bond to soil minerals or humus fraction inaccessible to bacteria. The mobilized POP molecules are incorporated into the fungal plasma membrane where their degradation begins. The presence of POP molecules in the membranes can change their physical properties and trigger toxic effects to the cell. To avoid these phenomena fungi can quickly remodel the phospholipid composition of their membrane with employing different phospholipases and acyltransferases. However, if the presence of POP downregulates the phospholipases, toxic effects and the final death of microbial cells are highly probable. In our studies we applied multicomponent Langmuir monolayers with their composition mimicking fungal plasma membranes and studied their interactions with two different microbial phospholipases: phospholipase C (α-toxin) and phospholipase A1 (Lecitase ultra). The model membranes were doped with selected POPs that are frequently found in contaminated soils. It turned out that most of the employed POPs do not downregulate considerably the activity of phospholipases, which is a good prognostics for the application of non-ligninolytic fungi in bioremediation.     

Studies on the hemolytic and hydrolytic actions of phospholipases against mammalian erythrocyte membranes.

The hemolytic actions of three kinds of phospholipase C on horse and sheep erythrocytes were studied in relation to their hydrolytic activities on the phospholipid components of these red cells. Clostridium novyi (oedematiens) type A phospholipase C hemolyzed horse red cells by hydrolyzing phosphatidylcholine. However, the enzyme did not lyse sheep cells nor did it hydrolyze any phospholipid under the same conditions, although this enzyme hydrolyzed both sphingomyelin and phosphatidylethanolamine in the phospholipid mixture extracted from sheep red cells. Clostridium perfringens phospholipase C hemolyzed not only horse red cells by hydrolyzing phosphatidylcholine but also sheep red cells by hydrolyzing sphingomyelin. Sphingomyelin on sheep red cell membrane was hydrolyzed 10 times faster by this enzyme than that on horse red cell membrane. Pseudomonas aureofaciens phospholipase C hemolyzed horse red cells by attacking phosphatidylcholine and phosphatidylethanolamine. The enzyme did not attack sheep red cells but it did hydrolyze phosphatidylethanolamine in the extracted phospholipid mixture from sheep cells. The hemolytic activity of phospholipase C depends not only on the enzyme and the asymmetric distribution of phospholipids in the erythrocyte membrane but also on the accessibility of the enzymes to the phospholipids in the surface of the membranes. Hemolysis by phospholipase C belongs to a hot-cold type of lysis.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.     

Stabilization of synaptic membranes with alpha-tocopherol against the damaging effect of phospholipases. Possible mechanism of the biological action of vitamin E

Using fluorescent and EPR spin probing techniques, the effects of phospholipases A2, C and D on rat brain synaptosomal membranes were investigated. It was shown that treatment of synaptosomal membranes with phospholipases A2, C and D results in their depolarization and increase of their surface negative charge. In case of phospholipases A2 and C, these changes are also accompanied by a decrease of the microviscosity of the synaptosomal membrane lipid bilayer. alpha-Tocopherol protects synaptosomal membranes against the damaging action of phospholipases. The stabilization of synaptosomes by vitamin E consists in the reconstitution of the transmembrane potential and in an increased microviscosity of phospholipase-treated membranes. The stabilizing effect of alpha-tocopherol is due to the binding of phospholipid hydrolysis products rather than to the inhibition of phospholipases. The observed stabilization of synaptosomal membranes by alpha-tocopherol is interpreted as a feasible mechanism of biological effects of vitamin E on biological membranes.     

Analysis of ectatomin action on cell membranes.

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four alpha-helix bundle. At concentrations of 0.05-0.1 microm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 x 10-7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied. Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01-10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of beta-adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol- and forskolin-sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 microm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.     

The role of membrane structure in activation of mitochondrial phospholipases. 2. Mechanism of crambin action on the activities of mitochondrial membrane phospholipases and microviscosity

Effects of crambin on the phospholipase activity, lipid peroxidation and structure of mitochondrial membranes have been investigated. Crambin has been shown to inhibit lipid peroxidation and phospholipase activity induced by Ca2+ and freezing-thawing of mitochondria. As shown by ESR studies, these effects are based on the ability of crambin to cause changes in mitochondrial membrane structure.     

Cholesterol incorporation into bacterial membranes.

The wall-covered bacteria Micrococcus lysodeikticus, Bacillus megaterium, and Proteus mirabilis incorporated exogenous cholesterol into their cytoplasmic membrane in quantities resembling those incorporated by sterol-nonrequiring mycoplasmas. Cholesterol incorporation into the outer membrane of P. mirabilis was much more restricted than into the cytoplasmic membrane.     

Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes.

Tetralin is toxic to bacterial cells at concentrations below 100 mumol/liter. To assess the inhibitory action of tetralin on bacterial membranes, a membrane model system, consisting of proteoliposomes in which beef heart cytochrome c oxidase was reconstituted as the proton motive force-generating mechanism, and several gram-positive and gram-negative bacteria were studied. Because of its hydrophobicity, tetralin partitioned into lipid membranes preferentially (lipid/buffer partition coefficient of tetralin is approximately 1,100). The excessive accumulation of tetralin caused expansion of the membrane and impairment of different membrane functions. Studies with proteoliposomes and intact cells indicated that tetralin makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes, which leads to a partial dissipation of the pH gradient and electrical potential. The effect of tetralin on the components of the proton motive force as well as disruption of protein-lipid interaction(s) could lead to impairment of various metabolic functions and to low growth rates. The data offer an explanation for the difficulty in isolating and cultivating microorganisms in media containing tetralin or other lipophilic compounds.     

Molecular mechanism of cardiotoxin action on axonal membranes.

Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.