Role Of Membrane Phospholipids Research Articles

The role of membrane phospholipids in the implementation of protective strategies of bacteria

To maintain viability under stressful conditions of existence and the implementation of protective strategies, bacteria must receive signals and respond quickly to extreme changes in environmental parameters. The results of recent experimental studies complement the paradigm that has dominated since the 1970s on the predominant role of phospholipids (PL) as molecular building blocks in the formation of the cell wall of bacteria. Specific transformations of these lipid domains have shown to have a significant effect on the shape and function of cells, membrane remodeling, and the ability of bacteria to adapt to environmental stresses. The physiological role of bacterial PLs is pleiotropic and determines both cell integrity and cell function. In addition to the key structural role of membrane PL in the cell, their intermediate metabolites are able to act as secondary messengers and perform important signaling and regulatory functions. Modern studies of the mechanisms of detection and integration of signals from the environment that cause stationary-dynamic changes in phospholipid homeostasis and form pleiotropic resistant cellular bacterial phenotypes are of fundamental and practical interest. PL homeostasis was proved to be crucial for the pathogenesis of bacterial infections and is necessary not only to maintain the viability of bacteria, but also to ensure their growth during infection. The suppression of the biosynthesis of these macromolecules reduces the viability of bacteria. In recent decades, one of the main advances in the concept of "liquid mosaic" model of biological membranes has been the understanding of their domain structure. This discovery is of fundamental and practical interest, since phospholipid domains are a promising target for modern antimicrobial strategies. The aim of this review is to summarize modern ideas about the structural, metabolic and signaling role of membrane PL in the implementation of the protective mechanisms of bacteria and maintaining their viability in adverse environmental conditions.

Tissue Factor encryption and decryption: Facts and controversies

Tissue factor (TF)-initiated coagulation plays a critical role in both hemostatsis and thrombosis. It is generally believed that most of the tissue factor expressed on cell surfaces is maintained in a cryptic, i.e., coagulantly inactive state and an activation step (decryption) is required for the expression of maximum TF procoagulant activity. However, what exactly constitutes cryptic or procoagulant TF, molecular differences between these two forms and mechanisms that are responsible for transformation from one to the other form are not entirely clear and remain highly controversial, thus are a matter of ongoing debate. This brief review discusses pertinent literature on TF encryption/decryption with specific emphasis on the role of membrane phospholipids and reduction/oxidation of the TF Cys186-Cys209 disulfide bond in regulating TF activity at cell surfaces.

Phosphatidylinositol 4,5-Bisphosphate Regulates Activation-Induced Platelet Microparticle Formation

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.

The amphipathic peptide mellitin as a tool to study the membrane-dependent activation of tissue transglutaminase

The role of membrane phospholipids on the cross-linking activity of guinea pig liver (tissue) transglutaminase has been investigated using the amphipathic model peptide melittin as glutaminyl substrate and the primary amine monodansylcadaverine as extrinsic amine donor. A marked increase of transglutaminase catalytic activity was observedin vitro assays in the presence of neutral membrane phospholipids. In contrast, activation was abolished in the presence of membranes containing pure anionic lipids. Enzyme activation could be ascribed to a direct binding of the lipid to the protein as demonstrated in enzymatic assays using a non membrane-interacting peptide (Cbz-Gln-Gly). The data obtained with model peptides suggest that the cross-linking activity of tissue transglutaminase could be modulated by the local microenvironment composition of the lipid bilayer and indicate that membrane phospholipids should be taken into account for further experiments directed to asess, the still poor understood, physiological role of tissue transglutaminase.

Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3β-ol (andien-β) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled < 10% of the pregnenolone metabolites. No estrogens or 5α-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3β-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-β to dienone but did not affect 17-hydroxylase or ‘andien-β-synthetase’. However, when the C-17,20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.

Role of membrane phospholipids and glycolipids in the Vero cell surface receptor for rubella virus

Membrane receptors for rubella virus (RV) in Vero cells were studied by means of two different approaches: (i) by enzyme treatment of the whole cell membrane and (ii) by testing the ability of isolated plasma membrane molecules to compete with cells for virus binding. The replication of RV was studied with both indirect immunofluorescence assay and molecular hybridization techniques. Phospholipases A2 and C digestion of cells greatly reduced the infectivity by the virus, pointing towards the involvement of lipid structures as receptor sites for RV. Furthermore, susceptibility of Vero cells to virus infection was also reduced after beta-N-acetyl-D-glucosaminidase, alpha-glucosidase and beta-galactosidase treatment, suggesting that carbohydrate residues may participate in a complex cellular receptor structure for RV. When the major membrane lipids were examined separately for their ability to inhibit viral infectivity, several phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin) and glycolipids (gangliosides, lactosylceramide, cerebroside sulphate) showed a strong neutralizing activity, confirming the role of membrane lipid moiety in the cell surface receptor for RV.

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro.

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.

Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion.

Depletion of membrane phospholipids is known to be associated with myocardial ischemia, but its relationship to the injury involved with the reperfusion of ischemic myocardium is not known. The present study was designed to relate phospholipid degradation with reperfusion injury. The isolated in situ pig heart was subjected to 60 min of regional ischemia induced by occluding the left anterior descending (LAD) coronary artery and 60 min of global ischemia by hypothermic cardioplegic arrest followed by 60 min of reperfusion. The pigs were divided into two groups. In the treatment group, the heart was preperfused with mepacrine (0.05 mM), a known phospholipase inhibitor, for 15 min prior to LAD occlusion. In the control group, the total phospholipid content was not significantly decreased during LAD occlusion and arrest, but was reduced appreciably after reperfusion. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol followed a similar pattern. The lowering of these phospholipids during reperfusion was accompanied by enhancement of lysophosphatidylcholine. Mepacrine restored the normal levels of these phospholipids. During reperfusion, fatty acyl CoA synthetase, lysophospholipase, and lysophosphatidylcholine acyltransferase were depressed, whereas phospholipase A2 was enhanced. Mepacrine inhibited phospholipase A2, but had no effects on the other enzymes. Mepacrine also provided significant protection against reperfusion injury, as documented by the preservation of high-energy phosphate compounds and inhibition of the appearance of creatine kinase activity in the perfusate. These results suggest that membrane phospholipids play an important role in myocardial injury associated with ischemia and reperfusion, primarily because the deacylation-reacylation cycle of phospholipid biosynthesis becomes defective.

The role of membrane phospholipids in the induction of creatine kinase activity and DNA synthesis in cultured rat bone cells by PTH

The role of membrane phospholipids in the induction of creatine kinase activity and DNA synthesis in cultured rat bone cells by PTH

Attenuation of enkephalin activity in neuroblastoma X glioma NG108-15 hybrid cells by phospholipases.

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.

Catalytic properties of carp brain monoamine oxidase in delipidated and reconstituted mitochondrial membranes

A possible role of membrane phospholipids in catalytic properties of carp brain mitochondrial monoamine oxidase (MAO) in both intact and delipidated membranes were studied before and after addition of some phospholipids. Deaminations of 5-HT and tyramine were greatly decreased by treatment with either phospholipase A2, C or D, but that of PEA was to a lesser extent. The sensitivity of MAO in the delipidated preparation to clorgyline and deprenyl was similar to that in the intact one; however, the amount of 3H-pargyline bound to MAO was found to be about half, and the Km values for the three substrates were extremely increased after delipidation. Among phospholipids tested, phosphatidylinositol(PI) most effectively and dose-dependently activated activity towards 5-HT in both intact and delipidated mitochondria, but the inhibitor sensitivity of MAO and the Km values for these substrates were not changed as compared before and after the PI addition. As a result, delipidation accounts for loss of activity through decreases in both the Km values and amount of active enzyme, but some phospholipids activate MAO activity without altering affinities towards substrates and selective MAO inhibitors.

Topological location and biological significance of phospholipids in the membrane of Newcastle disease virus. Hydrolysis of phospholipids in intact virion with pure phospholipases A2, C, and D.

The composition, topological distribution and biological significance of phospholipids in the membrane of Newcastle disease virus (NDV) grown in embryonated chicken eggs were investigated. Phosphatidylethanolamine and sphingomyelin were the predominant phospholipids in NDV membrane. The location of phospholipids in the lipid bilayer of the membrane was studied by assessing their reactivities with highly purified phospholipase A2 (Agkistrodon halys blomhoffi) and phospholipase D (Streptomyces chromofuscus), and the biological role of membrane phospholipids was also investigated by using pure phospholipases A2, C (Bacillus cereus) and D. Choline-containing phospholipids were found predominantly in the outer layer of the membrane. The inner layer was composed mainly of aminoglycerophospholipids, though a fair amount of them also appeared to be located in the outer half of the bilayer. When intact virion was treated with phospholipase C, marked decreases in hemolytic activity and infectivity mediated by viral fusion (F) glycoprotein were observed, but hemagglutinating and neuraminidase activities did not change significantly. Apparently complete hydrolysis of phospholipids in the outer half of the lipid bilayer with phospholipase D caused about 22% decrease in the original hemolytic activity. On the other hand, when all phosphatidylcholine and aminoglycerophospholipids in the outer half of the viral membrane were hydrolyzed with purified phospholipase A2, no significant change in viral hemolytic activity or morphology was detected. No marked change of hemagglutinating and neuraminidase activities was detected on treatment of NDV with phospholipases A2 and D. The above results suggest that the integrity of fatty acid ester of glycerophospholipids in NDV membrane is not essential for the manifestation of viral activities, though polar groups of the phospholipids in the outer half of the membrane may be involved in the function of fusion (F) glycoprotein, but not in that of hemagglutinating and neuraminidase (HN) glycoprotein of NDV.

Effect of various hepatic membrane fractions on microtubule assembly-with special emphasis on the role of membrane phospholipids.

This report describes an interaction between rat brain microtubule protein and various hepatic fractions in vitro. Purified preparations of Golgi membranes, plasma membrane, rough and smooth endoplasmic reticulum, nuclear membranes, and mitochondria were obtained from the livers of 200-g rats. Several concentrations of fresh or sonicated frozen membranes were incubated with twice-cycled rat brain microtubule protein in a microtubule assembly buffer for 60 min at 30 degrees C. Changes in microtubule assembly were assessed either by quantitative electron microscopy on negatively stained samples or by spectrophotometric methods. The results show that all the tested membranes "bound" microtubule protein, preventing assembly: Golgi and plasma membranes, as well as mitochondria, were especially potent in this regard. To identify the membrane-associated components responsible for microtubule protein binding, the membranes were extracted with methanol-chloroform, and liposomes were prepared from the resulting lipids. Microtubule protein incubated with these liposomes showed a differential ability to assemble that was similar to the effect obtained with intact membranes. Membrane-extracted phospholipids were identified as the lipid component responsible for these changes, with the negatively charged phospholipids (cardiolipin and phosphatidylserine) being uniquely active. These findings indicate that hepatic membranes differentially interact with brain microtubule protein; this interaction may be dependent on membrane phospholipids.

Effects of phosphatidylcholine liposomes on the fatty acid synthetase complex from Mycobacterium smegmatis

Phosphatidylcholine liposomes stimulate fatty acid synthesis catalyzed by the multienzyme complex from Mycobacterium smegmatis up to 3-fold and raise the proportion of shorter chain fatty acids (C 14, C 16, C 18) from 15 to 72%. Palmitoyl-CoA in a concentration of 10 μM inhibits the synthetase completely. This inhibition is fully relieved by liposomes. Increasing the incubation temperature from 19 to 45° C, raises the rate of synthesis (2-fold) and the proportion of long chain fatty acids (C 24 and C 26). Liposomes (250 μM) potentiate the effect of temperature on rate by another factor of 2 and have a chain-shortening effect in parallel with increasing temperature. In short term experiments (30 s) an increase in the concentration of acetyl-CoA (from 50 to 1000 μM) does not significantly affect the overall rate of synthesis but it doubles the percentage of short chain fatty acids. Under these conditions there is no superimposed liposome effect either on rate or product distribution. During longer periods (15 min) the overall rate increases as a function of acetyl-CoA concentration. This rate increase is enhanced by liposomes. At the same time, liposomes substantially raise the percentage of short chain acids. It is concluded that liposomes affect the de novo synthesis rate by alleviating feedback inhibition of the synthetase caused by C 16- and C 18-CoA. By sequestering these products, liposomes minimize the further elongation of C 16 chains. The possible role of membrane phospholipids in controlling the rate of synthesis and the length of fatty acid chains is discussed.

The role of membrane phospholipids in the interaction of ribosomes with endoplasmic-reticulum membrane.

It is shown that the ionic head groups of the membrane phospholipids cannot be solely responsible for the attachment of the ribosome and that other membrane components must also be involved in the binding process.

The action of phospholipases on the inner and outer surface of the squid giant axon membrane.

1. The effects of phospholipases on resting potential, action potential and membrane ionic conductances of squid giant axons have been studied by means of internal perfusion and voltage clamp techniques. The sample of phospholipases used exhibited both phospholipase A and B activities.2. When applied externally, phospholipases had no effect on the resting and action potentials at a concentration of 100 mug/ml.3. When applied internally, the enzymes effectively suppressed the amplitude and maximum rate of rise of the action potential at a concentration of 100 mug/ml. The resting potential started decreasing appreciably only after the action potential had been suppressed to a considerable extent.4. Under voltage clamp conditions, the peak transient current was suppressed following internal perfusion of phospholipases, 150 mug/ml. The steady-state current began to decrease, and the leakage current to increase only after the transient current had been suppressed to a considerable extent.5. The time course of sodium inactivation was not significantly slowed by internal perfusion of the enzymes.6. The curve relating the peak transient conductance to the membrane potential was shifted in the direction of depolarization after internal perfusion of phospholipases. No appreciable shift of the steady-state conductance curve was observed.7. These experimental results are discussed in connexion with the possible role of membrane phospholipids in the conductance changes associated with excitation.