Selectivity and Regulation in the Phospholipase A2-Mediated Attack on Cholinergic Synaptic Vesicles by β-Bungarotoxin

Abstract The total fatty acid composition of purified Torpedo californica electric organ synaptic vesicles was determined by GLC analysis of methyl esters. Limit amounts of fatty acids released by high concentrations of either β-bungarotoxin (β-BuTx) or Naja naja venom phospholipase A₂ (PLA₂) acting in deoxycholate are reported. The time and enzyme concentration dependence for β-BuTx- and PLA₂-induced release of fatty acids from intact synaptic vesicles indicate that PLA₂ is 100- to 1,000-fold more active. The Ca²⁺ dependence for β-BuTx-induced release of fatty acids also was determined. ATP inhibits β-BuTx- but not PLA₂-induced release of fatty acids from vesicles in a manner that can not be ascribed only to chelation of the required Ca²⁺. ATP, other nucleotides, and adenosine have complex effects on β-BuTx-induced release of fatty acids from egg yolk phosphatidylcholine dispersed in deoxycholate. The results suggest that β-BuTx-mediated hydrolysis of the cholinergic synaptic vesicle membrane is ～10- to 100-fold more effective at causing uncoupling of vesicles than is PLA₂ and that the enzymatic activity of β-BuTx is subject to regulation by nucleotide-like factors.     

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.     

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms

We describe the interaction of various phospholipases A₂ (PLA₂) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA₂s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA₂ with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA₂, S49 PLA₂ and K49 PLA₂) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA₂ compared with D49 PLA₂, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A₂ (PLA₂). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA₂ toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA₂. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA₂. Moreover, it was found that PLA₂ and N-terminally mutated PLA₂ adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA₂ and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA₂, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA₂ at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA₂.     

Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids

Phospholipids are key components of biological membranes and their lipolysis with phospholipase A₂ (PLA₂) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA₂-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA₂-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S_CD order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them.     

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A₂ (PLA₂) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA₂ molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA₂. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm²/s on the L-DPPC and 4.9 μm²/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm²/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.     

In vitro secretion of digestive phospholipase A2 by midguts isolated from tobacco hornworms,Manduca sexta

We report on secretion of phospholipase A₂ (PLA₂) by in vitro preparations of midguts isolated from tobacco hornworms, Manduca sexta. This enzyme is responsible for hydrolysis of fatty acids from the sn‐2 position of phospholipids, a necessary step in fatty acid absorption. The in vitro midgut preparations are competent to secrete PLA₂ into incubation buffer. Secretion began within the first 30 min of incubation and increased to a maximum at 8 h. We selected 2 h incubations because substantial loss of tissue integrity was observed after 8 h incubations. Using 2 h incubations, we recorded increased secretion of digestive PLA₂ from midguts incubated in buffer amended with diet or with yeast as a component of the diet. We also recorded small increases in secretion of PLA₂ from midguts incubated in buffer amended with a specific phospholipid, phosphatidylcholine. Midguts incubated in buffer amended with increased concentrations of phospholipid did not yield higher levels of PLA₂ activity. Lepidopteran midguts can be divided into three regions, and we recorded the highest secretion of PLA₂ from the middle region and lowest secretion from the anterior region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA₂, we suggest that secretion of digestive enzymes in tobacco hornworms is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Arch. Insect Biochem. Physiol. 42:179–187, 1999 .© 1999 Wiley‐Liss, Inc.     

Regulation of free arachidonic acid levels in isolated salivary glands from the lone star tick: A role for dopamine

An important regulatory step for prostaglandin synthesis is the availability of the precursor, free arachidonic acid (AA). In isolated salivary glands of the lone star tick, Amblyomma americanum (L.), the level of free AA appears to depend on higher phospholipase A₂ (PLA₂) activity rather than decreased rates of re-esterification by lysophosphatide acyl transferase (LAT). This conclusion is supported by experiments where inhibition of LAT with merthiolate was without effect, while the calcium ionophore A23187, a PLA₂ stimulant, increased levels of free AA. The PLA₂ activity in A. americanum was reduced by the substrate analog, PLA₂ inhibitor, oleyloxyethyl phosphorylcholine in a dose-dependent manner, but was insensitive to the other mammalian PLA₂ inhibitors mepacrine (20μM), aristolochic acid (45μM), and dexamethasone (50μM). No substrate preference was observed for the functional group of the phospholipid, with phosphatidylcholine and phosphatidylethanolamine being equal sources of AA in A23187-stimulated glands. Compared to phospholipids containing other fatty acids, only arachidonyl-phospholipid (arachidonyl-PL) was significantly hydrolyzed by PLA₂ activity in A23187-stimulated glands. Dopamine was as effective as A23187 as a stimulant of PLA₂ activity in isolated glands, but this effect was abolished in the presence of the calcium channel blocking agent verapamil. It is concluded that free AA levels in tick salivary glands are increased through activation of a Type IV-like PLA₂ following an increase of intracellular calcium caused by the opening of voltage-dependent calcium channels due to dopamine stimulation. © 1995 Wiley-Liss, Inc.     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Cytidine 5′-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor- (TNF-) and interleukin-1 (IL-1/) activate phospholipase A₂ (PLA₂) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA₂ releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA₂ stimulation and loss of CCT activity. TNF- also stimulates proteolysis of CCT. TNF- and IL-1 are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA₂ and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF- and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.     

Phospholipase A2 induced effects on the structural organization and physical properties of pea chloroplast membranes

Phospholipase A₂ (PLA₂)-induced effects on the membrane organization, fluidity properties and surface charge density of pea chloroplasts were investigated. It was observed that lipolytic treatment with PLA₂ altered the chloroplast structure having as a result a swelling of thylakoids and a total destruction of normal granal structure. In spite of this, the thylakoid membranes remained in close contact. At the same time, a slight decrease of surface charge density was registered, thus explaining the adhesion of swelled membranes. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured during PLA₂ treatment. A pronounced decrease of DPH fluorescence polarization was found, indicating that phospholipase treatment resulted in considerable disordering and/or fluidization of the thylakoid membranes. The increased fluidity could be attributed to the destabilizing effect of the products of enzymatic hydrolysis of the phospholipids (free fatty acids, lysophospholipids) on the bilayer structure of thylakoids membranes.Abbreviations 9-AA 9-aminoacridine - BSA bovine serium albumin - DCMU 3-/3,4-dichlorophenyl-1,1-dimethyl/urea - DPH 1,6-diphenyl-1,3,5-hexatriene - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b-protein complex of PS II - MES 2/N-morpholine/ethanesulfonic acid - PLA₂ phospholipase A₂ - PS I, PS II photosystem I and photosystem II, respectively - S lipid structural order parameter - THF tetrahydrofuran - TRICINE N-/tris/hydroxymethyl/methyl/glicine     

Changes in PLA(2) activity after interacting with anti-inflammatory drugs and model membranes: evidence for the involvement of tryptophan residues

Phospholipase A₂ (PLA₂) lipolytic activity can be regarded as a limiting factor for the development of inflammatory processes by restricting the production of pro-inflammatory mediators, hence representing a valuable therapeutic target for drugs that are able to modulate the activity of this enzyme. In the current work, the hydrolysis of phospholipids by PLA₂ was monitored with acrylodan-labelled intestinal fatty acid binding protein (ADIFAB) and this fluorescence based technique was also used to access the enzymatic inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs). The intrinsic fluorescence of PLA₂ tryptophan residues was further used to gain complementary information regarding the accessibility of these residues on the PLA₂ structure upon interaction with the NSAIDs tested; and to calculate the NSAIDs-PLA₂ binding constants. Finally, circular dichroism (CD) measurements were performed to evaluate changes in PLA₂ conformation resultant from the inhibitory effect of the drugs tested. Overall, results gathered in this study point to the conclusion that the studied NSAIDs inhibit PLA₂ activity due to a disturbance of the enzyme binding efficiency to membrane interface possibly by a shielding effect of the Trp residues required for the membrane interfacial binding step that precedes lipolysis process.     

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin

The myotoxic mechanism for PLA₂-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA₂-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA₂-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA₂-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA₂-like toxins in general.     

Phospholipase A2 and Its Role in Brain Tissue

Abstract: Phospholipase A₂ (PLA₂) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA₂-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA₂ in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA₂ has during both physiologic and pathologic states. Several different PLA₂ enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA²⁺-dependent and Ca²⁺-independent, based on their catalytic dependence on Ca²⁺. Under physiologic conditions, PLA₂ may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA₂ activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA₂ found in the central nervous system and attempts to define the role of PLA₂ during both physiologic and pathologic conditions.     

Chronic Lithium Treatment Decreases Brain Phospholipase A2 Activity

Chronic lithium administration decreases the turnover of arachidonic acid (AA) in several brain phospholipids. This suggests that lithium may attenuate phospholipase A₂ (PLA₂) activity in brain. We now report effects of chronic lithium treatment on PLA₂ activity in postnuclear supernatant from rat brain: Enzyme activity was determined by two assay methods, radiometric and fluorometric, and measured the release of the fatty acid on the second acyl position (sn2) from choline and ethanolamine phospholipids. PLA₂ activity in brain postnuclear supernatant from rats chronically treated with lithium in the diet was significantly decreased (20–50%) when compared with controls. In vehicle or lithium-treated rats, PLA₂ activity was not significantly augmented or attenuated by the addition of calcium chelators, divalent cations or LiCl supplementation (1.0 mM) to postnuclear supernatant. These results suggest that a major therapeutic effect of lithium is to attenuate brain PLA₂ activity involved in signal transduction.     

Thapsigargin-induced calcium entry in FRTL-5 cells: Possible dependence on phospholipase A2 activation

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A₂ (PLA₂) is activated. Since PLA₂ is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA₂ activity and the entry of calcium. Stimulating ³H-arachidonic acid (³H-AA)-labelled cells with thapsigargin resulted in a substantial release of ³H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA₂ activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA₂ in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA₂ activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca²⁺ in FRTL-5 cells. © 1994 Wiley-Liss, Inc.     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

Modeling and optimization of phospholipase A1‐catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production

Modeling the phospholipase A₁ (PLA₁)‐catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA₁ (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:35–41, 2015