Sodium channel activity of lobster nerve membrane treated with purified phospholipase A2

Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in reconstituted vesicles prepared from lobster nerve membrane and soybean lipids. The sodium channel activity was abolished by treatment of membranes, prior to reconstitution, with purified phospholipase A2. When the treatment with phospholipase A2 was carried out in the presence of bovine serum albumin the channel activity was fully preserved. Electron microscopy revealed that the normal vesicular appearance of the membranes was changed to an amorphous mass by treatment of the membranes with enzyme alone. A population of preserved vesicular structures was observed when bovine serum albumin was present during the enzyme treatment. Analysis of the membrane components indicate that there is no relationship between phospholipid composition and sodium channel activity.     

Cytoplasmic phospholipase A2 antagonists inhibit multiple endocytic membrane trafficking pathways

Previous studies have suggested a role for cytosolic Ca²⁺-independent phospholipase A₂ (PLA₂) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA₂ requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA₂ antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA₂ plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules.     

Effect of liver plasma membrane fluidity on endogenous phospholipase A2 activity

Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzyme's microenvironment.     

Isolation of rhodopsin by the combined action of cardiotoxin and phospholipase A2 on rod outer segment membranes

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom V₄^II cardiotoxin in rod outer segment membrane preparations. The extent of the morphological changes depended on the purity of the cardiotoxin. Pure cardiotoxin had no detectable effect upon the preparation, but, when contaminated with venom phospholipase A₂, let to a rapid disintegration of the membrane vesicles. With trace amounts (up to about 0.5% of the cardiotoxin) of phospholipase A₂, the membrane vesicles disintegrated into smooth lamellae and particles in solution. These two components were separated by centrifugation. The pellet, which showed the presence of smooth lamellae and aggregated particles, was composed of unbleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. The supernatant, which showed only the presence of dispersed particles, was composed of unbleached rhodopsin, lysolipids and cardiotoxin. With cardiotoxin containing larger amounts of phospholipase A₂ (more than 0.5% of the cardiotoxin), membrane vesicles were disintegrated into large aggregates of amorphous material, composed of bleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. These results confirm our previous observation on the release of integral membrane proteins from membrane vesicles by the action of cardiotoxin containing traces of phospholipase A₂ (Gulik-Krzywicki, T., Balerna, M., Vincent, J.P. and Lazdunski, M. (1981) Biochim. Biophys. Acta 643, 101–114) and suggest its possible use for isolation and purification of integral membrane proteins.     

Polarographic assay for phospholipase A2

A rapid, specific, and quantitative assay for phospholipase A₂ from Naja naja venom has been devised, in which phospholipid hydrolysis is measured as soybean lipoxidase-catalyzed oxygen incorporation into the ensuing unsaturated fatty acids. Under conditions where phospholipid was limiting, a linear relationship developed between the extent of oxygen uptake and the amount of egg lecithin metabolized. When phospholipase was rate limiting, the initial rate of oxygen consumption was a linear function of phospholipase concentration over a 14-fold range from 30 to 420 ng/ml. This linear relationship did not exist at higher phospholipase levels, probably due to the micellar nature of the phospholipid. Since this assay can readily detect as little as 17 ng/ml of phospholipase A₂ (Naja naja venom) and is insensitive to most potential interfering materials, it should be useful in a variety of applications.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

A rapid simple radiometric assay for phospholipase A2 activity

A rapid, simple radiochemical assay for phospholipase A₂ (PLA₂) activity is described in which incubations of homogenized tissue were directly applied to columns of silica gel absorbent. The metabolites and substrate were eluted from the columns with two different solvent systems so that each could be separated and quantified.     

Involvement of phospholipase A2 in neurodegeneration

Phospholipases A₂ are a heterogeneous class of enzymes that hydrolyse fatty acids from the sn-2 position of membrane phospholipids. Prolonged stimulation of phospholipase A₂ may damage membrane integrity, not only because of the loss of essential phospholipid from the lipid bilayer but also as a result of an uncontrollable Ca²⁺ influx. The increased levels of intracellular Ca²⁺ may be responsible for enhanced lipolysis, proteolysis and DNA fragmentation. This process along with the accumulation of lipid peroxidation products may be associated with neurodegeneration in acute neural trauma (ischemia, head and spinal cord injuries) and neurodegenerative diseases (Alzheimer's disease).     

Liquid-liquid immiscibility in model membranes activates secretory phospholipase A2

Secretory phospholipase A₂ (sPLA₂) hydrolyzes phosphatidylcholines (PC) within lipid bilayers to produce lyso-PC and a fatty acid, which can act as signaling molecule in biological membranes. The activity of sPLA₂ depends on the membrane structure. Bilayer defects, curvature, and gel-fluid micro-heterogeneity are known to activate sPLA₂. Here, we investigate if liquid-liquid immiscibility within model membranes is sufficient for sPLA₂ activation. The onset of the hydrolytic activity of cobra-venom sPLA₂ towards mixed monolayers of dimyristoyl-PC (DMPC)/cholesterol 2:1 (mol/mol) has been determined using infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated (PM-) IRRAS. The lag phase of sPLA₂ activity increases exponentially with rising surface pressures starting at 12 mN/m. This indicates that enzyme activation is hampered at higher surface pressures. Below 12 mN/m, no lag phase is observed, and sPLA₂ is efficiently activated. The surface pressure that is critical for sPLA₂ activation correlates with the critical miscibility pressure according to the phase diagram of DMPC and cholesterol. Thus, coexisting, liquid-phase domains provide sufficient boundaries to activate sPLA₂. Moreover, liquid-liquid immiscibility is an activating mechanism for sPLA₂ that also applies to biological membranes under physiological conditions because the corresponding bilayer structure is associated with that of membrane rafts.     

Electron microscopy of Bacillus subtilis protoplast membrane after treatment with phospholipase A2 and phospholipase C

The effects of phospholipase A₂ and phospholipase C on Bacillus subtilis protoplast membrane have been studied by electron microscopy and by chemical methods. Phospholipase A₂ (from porcine pancreas) almost quantitatively converted cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol to fatty acids and lysoderivatives. The fatty acids like the lysophospholipids remained in the membrane. Phospholipase C (from Bacillus cereus) hydrolyzed about 80% of the phosphatidylethanolamine and about 40% of the cardiolipin. Electron microscopy has been carried out with respect to general morphology of the affected protoplasts, the occurrence of a triple-layered membrane structure in thin sections, and the ultrastructure of membrane fracture faces upon freeze fracturing. Phospholipase A₂ treatment resulted in fragmentation of the protoplasts. In all cases the triple-layered membrane profile was preserved in thin sections. The membrane fracture faces appeared normal, i.e. they showed a convex face with many particles and a concave face with few particles. This indicated that the hydrophobic interior of the membrane was not too much damaged after incubation with phospholipases, presumably because of the stabilizing action of membrane proteins.     

Inhibition of phospholipase A2 and phospholipase C by polyamines

The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A₂ (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca²⁺, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Interaction of phospholipase A2 and phospholipid bilayers

Binding of phospholipase A₂ from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC₁₄) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC₁₄ alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC₁₄ could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.     

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A₂ (PLA₂) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA₂. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Na+-Ca2+ exchange in the axolemma-rich membrane vesicle preparations from the walking-leg nerves of the american lobster

An axolemma-rich membrane vesicle fraction was prepared from the leg nerve of the lobster, Homerus americanus. In this preparation Ca²⁺ transport across the membrane was shown to require a Na⁺ gradient (Na⁺-Ca²⁺ exchange), and external K⁺ was found to facilitate this Na⁺-Ca²⁺ exchange activity. In addition, at high Ca²⁺ concentrations (20 mM) a Ca²⁺-Ca²⁺ exchange system was shown to operate, which is stimulated by Li⁺. The Na⁺-Ca²⁺ exchange system is capable of operating in the reverse direction, with Ca²⁺ uptake coupled with Na⁺ efflux. Such a vesicular preparation has the potential for providing useful experimental approaches to study the mechanism of this important Ca²⁺ extrusion system in the nervous system.     

An affinity ligand for phospholipase A2 purification

An alkyl ether analog of phosphatidylcholine was synthesized and used as a ligand to purify acid-extracted phospholipase A₂ from bovine ileum smooth muscle by affinity chromatography in the presence of cholate. This ligand contains a primary amino group at the ω-position of the acyl chain in position 1 and so permits direct covalent coupling with the ester group of Affi-Gel-10. An endogenous membrane bound phospholipase A₂ has been purified 32-fold in a good yield (70%) employing this ligand in an affinity chromatography step.     

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

1. 1. Cobra venom phospholipase A₂ from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography.

2. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine hydrolysis in those cells.

3. 3. It is argued that the plateaus observed in dose-response curves for such treatments may be caused by an increase in lateral surface pressure within the outer half of the membrane due to the production of free fatty acids and lyso-compounds.