Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.     

The N-terminal alpha-helix of pancreatic phospholipase A2 determines productive-mode orientation of the enzyme at the membrane surface

Phospholipase A(2) (PLA(2)) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA(2) activity strongly increases upon binding to the surface of aggregated phospholipid. The N-terminal approximately ten residue alpha-helix of certain PLA(2) isoforms plays important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA(2) and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal alpha-helix and the rest of the protein in membrane binding of PLA(2) and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA(2). While the full-length PLA(2) binds to membranes with a defined orientation, an engineered PLA(2) fragment DeltaN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits approximately 100-fold lower enzymatic activity compared to the full-length PLA(2), indicating the key role of the N terminus in PLA(2) function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA(2) and identification of its interfacial binding surface. Moreover, the full-length PLA(2) demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the DeltaN10 fragment is more rigid than the full-length PLA(2) both in free and membrane-bound states. Our results suggest that the N-terminal alpha-helix supports the activation of PLA(2) by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA(2) at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme.     

Phospholipase A2 products retain a neuron specific gamma isoform of PKC on the plasma membrane through the C1 domain--a molecular mechanism for sustained enzyme activity

To clarify molecular mechanism for sustained activation of gamma protein kinase C (gammaPKC), a neuron-specific subtype, we investigated the involvement of phospholipase A2 (PLA2) products in the membrane association of gammaPKC upon activation of G protein coupled purinoceptors in CHO-K1 and NG 108-15 cells. In addition, the functional domain responsible for PLA2-product mediated retention of gammaPKC on the plasma membrane was determined by simultaneously monitoring two different fluorescence-tagged gammaPKCs and mutants in the same living CHO-K1 cells. Purinoceptor activation by UTP induced a transient translocation of gammaPKC from the cytoplasm to the plasma membrane. Interestingly, PLA2 inhibitors, bromoenol lactone (BEL) and arachidonyl-trifluoromethyl ketone (AACOF3), shortened the retention time of gammaPKC on the plasma membrane in cells treated with UTP, while a DAG kinase inhibitor did not affect it. The C1 domain deficient mutant (DeltaC1-gammaPKC) also showed short membrane association compared with wild type gammaPKC, when cells are treated with UTP or arachidonic acid (AA) plus a Ca(2+) ionophore. However, deletion of C1A or C1B subdomains (DeltaC1A-gammaPKC or DeltaC1B-gammaPKC) did not alter the retention time on the plasma membrane, whereas PLA2 inhibitor shortened the retention times of both mutants. These results indicate that PLA2 products prolong the retention of gammaPKC on the plasma membrane through the C1A and/or C1B subdomain in purinoceptor-stimulated CHO-K1 cells. The importance of PLA2 product and C1 domain for the retention of gammaPKC on the membrane was also confirmed using neuronal cell line, suggesting that these are part of molecular machinery for sustaining enzyme activity in neurons.     

Toward understanding interfacial activation of secretory phospholipase A2 (PLA2): membrane surface properties and membrane-induced structural changes in the enzyme contribute synergistically to PLA2 activation

Phospholipase A2 (PLA2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA2 to the interfacial activation of PLA2, that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA2, membrane-induced structural changes in PLA2, and the enzyme activation.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Effects of lipid phase transition and membrane surface charge on the interfacial activation of phospholipase A2

Phospholipase A2 (PLA2) enzymes act at the membrane-water interface to access their phospholipid substrate from the membrane. They are regulated by diverse factors, including the membrane charge, fluidity, mode of membrane binding (insertion, orientation), and allosteric conformational effects. Relative contributions of these factors to the complex kinetics of PLA2 activation are not well understood. Here we examine the effects of thermal phase transitions and the surface charge of phospholipid membranes on the activation of human pancreatic PLA2. The temperature dependence of the initial catalytic rate of PLA2 peaks around the lipid phase transition temperature (Tm) when Tm is not too far from physiological temperatures (30-40 degrees C), and the peak is higher in the presence of anionic membranes. High PLA2 activity can be induced by thermal perturbations of the membrane. Temperature-dependent fluorescence quenching experiments show that despite dramatic effects of the lipid phase transition on PLA2 activity, the membrane insertion depth of PLA2 increases only modestly above Tm. The data show that membrane structural disorder, and not the depth of membrane insertion, plays a major role in PLA2 activity.     

Human fetal skin fibroblast migration stimulated by the autocrine growth factor bFGF is mediated by phospholipase A(2) via arachidonic acid without the involvement of pertussis toxin-sensitive G-protein

We reported previously that human fetal skin fibroblast migration into a denuded area was stimulated by an autocrine factor, basic fibroblast growth factor (bFGF). Since the signal transduction pathway of this migration is unknown, we attempted to clarify it by comparing this fibroblast migration with a previously reported bovine endothelial cell migration into a wounded area stimulated by an addition of bFGF, in which the bFGF signal was mediated by phospholipase A(2)-coupled G-protein and phospholipase A(2) (PLA(2)) via arachidonic acid. Our study demonstrated that pertussis toxin, a specific inhibitor of PLA(2)-coupled G-protein, did not suppress human fetal skin fibroblast migration, but 2-(p-amylcinnamyl)amino-4-chlorobensoic acid (ONO-RS-082), a PLA(2) inhibitor, did. Since ONO-RS-082 is a non-specific PLA(2) inhibitor, a cytoplasmic, Ca-dependent PLA(2) (cPLA(2)) inhibitor, AACOCF3, was examined. AACOCF3 suppressed cell migration in certain concentrations. The PLA(2) inhibitor-suppressed cell migration was restored by adding arachidonic acid, and cell migration suppressed by anti-bFGF antibodies was restored by adding arachidonic acid. In addition, pertussis toxin did not suppress arachidonic acid release, which shows an action of PLA(2), but AACOCF3 did. These results indicate that human fetal skin fibroblast migration stimulated by an autocrine factor, bFGF, was mediated by PLA(2) via arachidonic acid without the involvement of PLA(2)-coupled G-protein.     

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.     

Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells

In murine P388D1 macrophages, the generation of prostaglandin E2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A2 (PLA2), Group IV cytosolic PLA2 (cPLA2), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA2 that induces expression of Group V PLA2, which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E2 generation. Because Group V PLA2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA2, blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA2 previously associated with the outer cellular surface. Localization of Group V PLA2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA2. Collectively, these results suggest that encapsulation of Group V PLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA2 and COX-2 for efficient prostaglandin synthesis.     

Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.     

Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus

Mammalian Group IIA phospholipases A(2) (PLA(2)) potently kill Staphylococcus aureus. Highly cationic properties of these PLA(2) are important for Ca(2+)-independent binding and cell wall penetration, prerequisites for Ca(2+)-dependent degradation of membrane phospholipids and bacterial killing. To further delineate charge properties of the bacterial envelope important in Group IIA PLA(2) action against S. aureus, we examined the effects of mutations that prevent specific modifications of cell wall (dltA) and cell membrane (mprF) polyanions. In comparison to the parent strain, isogenic dltA(-) bacteria are approximately 30-100x more sensitive to PLA(2), whereas mprF(-) bacteria are <3-fold more sensitive. Differences in PLA(2) sensitivity of intact bacteria reflect differences in cell wall, not cell membrane, properties since protoplasts from all three strains are equally sensitive to PLA(2). A diminished positive charge in PLA(2) reduces PLA(2) binding and antibacterial activity. In contrast, diminished cell wall negative charge by substitution of (lipo)teichoic acids with d-alanine reduces antibacterial activity of bound PLA(2), but not initial PLA(2) binding. Therefore, the potent antistaphylococcal activity of Group IIA PLA(2) depends on cationic properties of the enzyme that promote binding to the cell wall, and polyanionic properties of cell wall (lipo)teichoic acids that promote attack of membrane phospholipids by bound PLA(2).     

Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187.     

Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites

Phospholipase A(2) (PLA(2)) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA(2) was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His(48) modification affected PLA(2) structure, its membrane-binding affinity, and the effects of PLA(2) on the membrane structure. The native PLA(2) increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA(2) and the formation of PLA(2)-membrane hydrogen bonding. The inhibited PLA(2) had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible alpha-helices in the native PLA(2), which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA(2) induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA(2) acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA(2), including the alpha-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA(2), which contributes to the interfacial activation of the enzyme.     

Purification and characterization of a membrane-associated 48-kilodalton phospholipase A(2) in leaves of broad bean

Several lines of evidence indicate that phospholipase A(2) (PLA(2)) plays a crucial role in plant cellular responses through production of linolenic acid, the precursor of jasmonic acid, from membrane phospholipids. Here we report the purification and characterization of a 48-kD PLA(2) from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA(2) was purified to near homogeneity by sequential column chromatographies from the membrane extracts. The purified 48-kD protein migrated as a single band on a SDS-PAGE gel and its density correlated with the PLA(2) activity. It was further confirmed that this 48-kD protein is the PLA(2) enzyme based on immunoprecipitating the activity with a monoclonal antibody against it and purifying the enzyme to homogeneity with the antibody affinity column. The purified plant PLA(2) preferred 2-linolenoyl-sn-glycerol-3-phosphocholine (GPC) to 2-linoleoyl-GPC, 2-palmitoyl-GPC and 2-arachidonyl-GPC as substrates with a pH optimum at pH 7.0 to 8.0. The plant PLA(2) was activated by calmodulin and inhibited by pretreatment of 5,8,11, 14-eicosatetraynoic acid known as an inhibitor of mammalian PLA(2)s. The enzyme was characterized as a Ca(2+)-independent PLA(2) different from mammalian PLA(2)s. This membrane-associated and Ca(2+)-independent PLA(2) is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.     

Differential roles of ionic, aliphatic, and aromatic residues in membrane-protein interactions: a surface plasmon resonance study on phospholipases A2

The roles of cationic, aliphatic, and aromatic residues in the membrane association and dissociation of five phospholipases A(2) (PLA(2)), including Asp-49 PLA(2) from the venom of Agkistrodon piscivorus piscivorus, acidic PLA(2) from the venom of Naja naja atra, human group IIa and V PLA(2)s, and the C2 domain of cytosolic PLA(2), were determined by surface plasmon resonance analysis. Cationic interfacial binding residues of A. p. piscivorus PLA(2) (Lys-10) and human group IIa PLA(2) (Arg-7, Lys-10, and Lys-16), which mediate electrostatic interactions with anionic membranes, primarily accelerate the membrane association. In contrast, an aliphatic side chain of the C2 domain of cytosolic PLA(2) (Val-97), which penetrates into the hydrophobic core of the membrane and forms hydrophobic interactions, mainly slows the dissociation of membrane-bound protein. Aromatic residues of human group V PLA(2) (Trp-31) and N. n. atra PLA(2) (Trp-61, Phe-64, and Tyr-110) contribute to both membrane association and dissociation steps, and the relative contribution to these processes depends on the chemical nature and the orientation of the side chains as well as their location on the interfacial binding surface. On the basis of these results, a general model is proposed for the interfacial binding of peripheral proteins, in which electrostatic interactions by ionic and aromatic residues initially bring the protein to the membrane surface and the subsequent membrane penetration and hydrophobic interactions by aliphatic and aromatic residues stabilize the membrane-protein complexes, thereby elongating the membrane residence time of protein.     

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.     

Localization of phospholipase A2 in normal and ras-transformed cells

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.