The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction.

Phospholipase A2 present in a highly purified, potently bactericidal, fraction from rabbit graulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A2 but also by phospholipase A1, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30-60 min and at higher concentrations of the fraction. Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains. The addition of 0.04 M Mg2+, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg2+ in either strain. Less than 5 micrograms of granulocyte fraction causes loss of viability of from 90 to 99% of 1 X 10(8) microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.     

Reversible envelope effects during and after killing of Escherichia coli W by a highly-purified rabbit polymorphonuclear leukocyte fraction

The effects of a highly-purified, potently bactericidal fraction from rabbit polymorphonuclear leukocytes on the envelope of Escherichia coli (W) have been examined. This leukocyte fraction has equally enriched bactericidal, permeability-increasing and phospholipase A₂ activities, and is essentially devoid of lysozyme, myeloperoxidase and protease activities (Weiss, J., Franson, R.C., Beckerdite, S., Schmeidler, K. and Elsbach, P. (1975) J. Clin. Invest. 55, 33–42). Rapid killing of E. coli by this fraction is accompanied by two almost immediate alterations in the bacterial envelope: (1) a discrete increase in envelope permeability (measured by inhibition of bacterial leucine incorporation by normally impermeant actinomycin D), and, (2) hydrolysis of ¹⁴C-labeled fatty acid-prelabeled E. coli phospholipids. Both envelope effects are promptly reversed during further incubation at 37 °C, but not at 0 °C, with 40 mM Mg²⁺. Reversal is also produced by Ca²⁺ (40 mM) and trypsin (200 μg/ml), but 200 mM K⁺ causes only partial recovery and Na⁺ and hyperosmolar sucrose are ineffective. Upon addition of Mg²⁺, phospholipid degradation ceases abruptly and the labeled products of hydrolysis (free fatty acids and lysocompounds) disappear with a corresponding reaccumulation of radioactive diacylphosphatides. The time course of resynthesis of phospholipids coincides with that of restoration of the permeability barrier. Higher concentrations of the leukocyte fraction and prolonged incubation increase both the extent of phospholipid degradation and the time required for reversal of both envelope effects. These findings suggest that both the initiation of the increased permeability and its reversal are linked to respectively the breakdown and resynthesis of major E. coli membrane phospholipids, and thus depend on the fact that the biochemical apparatus of E. coli remains capable of biosynthesis despite loss of viability.Treatment of E. coli, exposed to the leukocyte fraction, with albumin results in extracellular sequestration of the products of hydrolysis and also restores the permeability barrier to actinomycin D, suggesting that the accumulation of lytic products of lipid hydrolysis within the bacterial envelope, rather than the loss of phospholipids per se, causes increased permeability.     

Acylation of monoacylglycerophosphoethanolamine in the inner and outer membranes of the envelope of an Escherichia coli K12 strain and its phospholipase A-deficient mutant

The site of the Escherichia coli envelope of the conversion of 1-acylglycero-3-phosphoethanolamine to diacylglycerophosphoethanolamine was explored, using two K12 strains with a wild-type phospholipid-degradative apparatus and a K12 mutant lacking detectable phospholipase A₁ and A₂ activity.Experiments with various radioactively labeled substrates show that acylation by crude envelope preparations as well as isolated inner and outer membranes of parent and mutant strains involves neither exogenous fatty acids nor a transacylation reaction with added monoacylglycerophosphoethanolamine. Furthermore, acylation exhibits no absolute requirement for added ATP and coenzyme A.Specific activity of acylating activity is the same in inner membrane preparations of parent and mutant strain and in outer membrane preparations of the mutant deficient in phospholipase A. Although clearly evident, net diacylglycerophosphoethanolamine formation by outer membranes of the parent strain, however, was about 6-fold less. This lower conversion may be attributed to activation during incubation of phospholipases A within the outer membrane, resulting in breakdown of the diacylcompound formed.Reacylation of lysophospholipids formed in the E. coli envelope by the action of endogenous or exogenous phospholipases A provides the organism with the potential of biochemically inexpensive repair and modification of the envelope phospholipids. Moreover, major phospholipids hydrolyzed in the outer membrane of E. coli can be resynthesized in the same location, without need for the transport of the products of hydrolysis to the lipid biosynthetic apparatus associated with the cytoplasmic membrane.     

A lipid requirement for the (Ca2+ + Mg2+)-activated ATPase of erythrocyte membranes.

The lipid requirement of the (Ca²⁺ + Mg²⁺)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A₂ from Naja naja and serum albumin. Optimal restoration of the (Ca²⁺ + Mg²⁺)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.     

Effect of phospholipase A2 on purified gastric vesicles

The phospholipid and fatty acid composition and role of phospholipids in enzyme and transport function of gastric (H⁺+K⁺)-ATPase vesicles was studied using phospholipase A₂ (bee venom). The composition (%) was phosphatidylcholine (PC) 33%; sphingomyelin (sph) 25%; phosphatidylethanolamine (PE) 22%; phosphatidylserine (PS) 11%; and phosphatidylinositol (PI) 8%. The fatty acid composition showed a high degree of unsaturation. In both fresh and lyophilized preparations, even with prolonged incubation, only 50% of phospholipids were hydrolyzed, but the amount of PE and PS disappearing was increased following lyophilization. There was a marked decrease in K⁺-ATPase activity (75%) but essentially no loss of the associated K₊ p-nitrophenyl phosphatase was found. ATPase activity could be largely restored by various phospholipids (PE > PC > PS). There was also an increase in Mg²⁺-ATPase activity, partially reversed in fresh preparations by the addition of phospholipids (PE > PS > PC). Proton transport activity of the preparation was rapidly inhibited, initially due to a large increase in the HC1 permeability of the preparation. Associated with these enzymatic and functional changes, the ATP-induced conformational changes, as indicated by circular dichroism spectra were inhibited.     

Action of phospholipase A2 and phospholipase C on Escherichia coli

The action of exogenous phospholipases on Escherichia coli has been examined. Cells harvested in late log phase were found to be completely resistant to the action of phospholipases A₂ and C. Treatment of cells with Tris and EDTA was required to make the phospholipids in the cell accessible to these phospholipases. Phospholipase A₂ hydrolyzed mainly phosphatidylethanolamine and phosphatidylglycerol, whereas phospholipase C preferentially degraded phosphatidylethanolamine.During the EDTA treatment, an endogenous phospholipase A₁ or a lysophospholipase (or both) was unmasked which caused the formation of free fatty acids in experiments in which no phospholipase was added and which degraded some of the lysophospholipids formed by phospholipase A₂.The cells were rapidly killed by the successive Tris-EDTA-phospholipase treatment, but no cell disintegration was observed.     

Tissue polyunsaturated fatty acids and a digestive phospholipase A2 in the primary screwworm,Cochliomyia hominivorax

We report on the presence of arachidonic acid in larval and adult tissues of the primary screwworm, Cochliomyia hominivorax and of the secondary screwworm, C. macellaria. Arachidonic acid is present in the phospholipids of whole animal extracts of both species. This fatty acid appears to be accumulated during the larval stages, because proportions of arachidonic acid were higher in adults than in larvae. These insects probably obtain the arachidonic acid from dietary phospholipids. We also report on a phospholipase A₂ activity in midgut preparations from third instars of the primary screwworm. Phospholipase A₂ is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids to release essential fatty acids. The screwworm enzyme is similar to mammalian digestive phospholipase A₂s because it depends on calcium for high catalytic activity, it is sensitive to the site-specific inhibitor oleyloxyethylphosphorylcholine, and it interacts with heparin. We further characterized the screwworm midgut phospholipase A₂ by altering the reaction conditions, including reaction time, radioactive substrate concentration, protein concentration, pH and temperature. We speculate that the biological significance of this enzyme relates to acquiring essential fatty acids, including arachidonic acid, from dietary phospholipids.     

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.     

The preservation of Mycoplasma capricolum cell intactness after phospholipase A2 treatment

(1) By treating Mycoplasma capricolum cells with phospholipase A₂ about 80% of membrane phospholipids were rapidly hydrolyzed. The rate and extent of hydrolysis (at 37°C) were the same in intact cells and in isolated unsealed membranes. (2) Due to the low endogenous lysophospholipase activity detected in M. capricolum, phospholipase A₂ treatment resulted in the accumulation of lysophospholipids and free fatty acids. The free fatty acids were efficiently extracted from the cells by 1% bovine serum albumin whereas the lysophospholipids were almost fully retained within the cell membrane. (3) Following phospholipase A₂ treatment in the presence of 1% bovine serum albumin, cell intactness was preserved as indicated by the constant absorbance of the cell suspension and the retention of nucleic acids and NADH dehydrogenase activity within the cells. The treated cells showed, however, a slight decrease in K⁺ content and a decrease in cell viability. Viability was fully preserved after phospholipase A₂ treatment of cells grown with exogenous sphingomyelin. (4) Adapting M. capricolum to a cholesterol-poor medium resulted in a marked decrease in the cholesterol to phospholipid molar ratio (from about 1.1 to 0.3). Phospholipase A₂ treatment of the cholesterol-poor cells resuted in cell lysis. Cell lysis was induced in the cholesterol-rich cells by hydrolysing the lysophospholipids accumulated following phospholipase A₂ treatment. (5) It is suggested that after phospholipase A₂ treatment of M. capricolum cells, a relatively stable cell membrane is maintained and cell intactness is preseved due to the interaction of cholesterol, present in high amount in this membrane, with the lysophospholipids formed.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A₂ from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only.The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A₂ from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.     

Lung surfactant synthesis: a Ca++-dependent microsomal phospholipase A2 in the lung.

We present the first direct evidence for a highly active, Ca⁺⁺-dependent phospholipase A₂ in the microsomal fraction of rat lung homogenate. Several previously reported studies from other laboratories strongly implicate this enzyme as a key metabolic step in the biosynthesis of dipalmitoyl lecithin, the primary component of pulmonary surfactant. In the present study, stoichiometric amounts of [³H]lysophosphatidylethanolamine and [¹⁴C]fatty acid were released during incubation of 1-[9, 10-³H]palmitoyl-2-sn-[1′-¹⁴C]linoleoyl phosphatidylethanolamine with the lung microsomal fraction. Marker enzyme measurements showed that the microsomal activity cannot be due to contamination with mitochondria, which also show phospholipase A₂ in both lung and liver. In contrast, liver microsomes show predominantly a phospholipase A₁ activity.     

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.     

Skeletal muscle mitochondrial phospholipase A2 and the interaction of mitochondria and sarcoplasmic reticulum in porcine malignant hyperthermia

Comparative studies were carried out on the Ca²⁺-transport systems of mitochondria and sarcoplasmic reticulum from longissimus dorsi muscle of genetically selected malignant hyperthermia-prone and normal pigs in order to identify the biochemical lesion responsible for the enhanced release of Ca²⁺ in the sarcoplasm occurring in porcine malignant hyperthermia. Mitochondria isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs contained a significantly (P < 0.001) higher amount of endogenous long-chain fatty acids. Similar amounts of endogenous mitochondrial phospholipase A₂ were observed in both types of pigs, but the total activity in malignant hyperthermia-prone pigs was at least twice that of normal. Spermine, a phospholipase A₂ inhibitor, lowered the activity in both types of mitochondria to a similar final level. Mitochondria of malignant hyperthermia-prone pigs showed a significantly (P < 0.001) higher oligomycin-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity, but the Mg²⁺-ATPase and the (Ca²⁺ + Mg²⁺)-ATPase activities were similar in both types of pigs. Sarcoplasmic reticulum isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs showed a significantly higher (Ca²⁺ + Mg²⁺)-ATPase activity and a lower rate of Ca²⁺ uptake; the maximal amount and the rate of Ca²⁺ uptake by sarcoplasmic reticulum of malignant hyperthermia-prone pigs were half that of normal. Mitochondria from longissimus dorsi muscle of malignant hyperthermia-prone pigs inhibited the Ca²⁺-transport system of the sarcoplasmic reticulum of longissimus dorsi from both normal and malignant hyperthermia-prone pigs, but mitochondria from normal pigs had no influence on the sarcoplasmic reticulum from either type. Experimental evidence favours the concept that long-chain fatty acids released from skeletal muscle mitochondria by endogenous mitochondrial phospholipase A₂ are responsible for the enhanced release of Ca²⁺ from mitochondria (Cheah, K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84), and also additional release of Ca²⁺ from sarcoplasmic reticulum into the sarcoplasm during porcine malignant hyperthermia syndrome.     

Selectivity of phospholipid hydrolysis by phospholipase A2 enzymes in activated cells leading to polyunsaturated fatty acid mobilization

Phospholipase A₂s are enzymes that hydrolyze the fatty acid at the sn-2 position of the glycerol backbone of membrane glycerophospholipids. Given the asymmetric distribution of fatty acids within phospholipids, where saturated fatty acids tend to be present at the sn-1 position, and polyunsaturated fatty acids such as those of the omega-3 and omega-6 series overwhelmingly localize in the sn-2 position, the phospholipase A₂ reaction is of utmost importance as a regulatory checkpoint for the mobilization of these fatty acids and the subsequent synthesis of proinflammatory omega-6-derived eicosanoids on one hand, and omega-3-derived specialized pro-resolving mediators on the other. The great variety of phospholipase A₂s, their differential substrate selectivity under a variety of pathophysiological conditions, as well as the different compartmentalization of each enzyme and accessibility to substrate, render this class of enzymes also key to membrane phospholipid remodeling reactions, and the generation of specific lipid mediators not related with canonical metabolites of omega-6 or omega-3 fatty acids. This review highlights novel findings regarding the selective hydrolysis of phospholipids by phospholipase A₂s and the influence this may have on the ability of these enzymes to generate distinct lipid mediators with essential functions in biological processes. This brings a new understanding of the cellular roles of these enzymes depending upon activation conditions.