Hydrolysis of a fluorescent substance formed from an oxidized phospholipid and an amino compound by phospholipase A2

Phosphatidylcholine hydroperoxide produced a fluorescent substance (FS-III) through reaction with 1-aminopentane after preincubation with heme methyl ester as a model system. The FS-III was retained at the 2-position of the glycerol backbone of phosphtidylcholine without breakdown into low molecular weight compounds. Phosphatidylcholine oxidized by catalysis with ferrous ion and ascorbic acid also produced the same fluorescent substance (FS-III). Phospholipase A₂ specifically hydrolyzed the FS-III attached to the phospholipid, making it possible to elute the same fluorescent substance (FS-II) as that obtained from oxidized methyl linoleate. The release of FS-II by hydrolysis of FS-III attached to phospholipid increased with greater phospholipase A₂ activity. It is suggested that, with aging, the accumulation of fluorescent lipofuscin pigments in biomembranes may be related to changes in the peroxidized phospholipid content and that phospholipase A₂ may play a role in decreasing the formation and accumulation of fluorescent phospholipids in biomembranes.     

Study of the two pathways for arachidonate oxygenation in blood platelets

During collagen-induced blood platelet aggregation, arachidonic acid is set free from membrane phospholipids and subsequently converted into 12-hydroxyeicosatetraenoic acid by arachidonate lipoxygenase and into thromboxane A₂, 12-hydroxyheptadecatrienoic acid (HETE) and malondialdehyde by cyclooxygenase and thromboxane synthase. Lipoxygenase and cyclooxygenase have optimal activity at neutral to basic pH, while the thromboxane synthase is pH-independent between 5 and 9. These enzymes are membrane-bound. The cyclooxygenase is rapidly inactivated upon membrane disruption by nonionic detergents or phospholipid degradation with phospholipase A₂. It was found that platelet phospholipase A₂ preferentially splits off fatty acid with four double bonds. Eicosatetraynoic acid was used to investigate the physiological function of the arachidonate lipoxygenase during collagen-induced aggregation of rat blood platelets. This fatty acid is a more efficient inhibitor of lipoxygenase than of cyclooxygenase. At an inhibitor concentration of 0.6 μg/ml, platelet aggreation, 12-hydroxyeicosatetraenoic acid production as well as 15-hydroxytryptamine release are completely inhibited, while there is an apparent stimulation of the cyclooxygenase. These results indicate that arachidonate lipoxygenase is essential for irreversible blood platelet aggregation.     

Dietary induced alterations in swelling characteristics and endogenous phospholipase A2 activity of rat liver mitochondria

The present study describes the rapid alterations in the fatty acid patterns of phosphatidyl choline and phosphatidyl ethanolamine from rat liver mitochondria induced by corn oil feeding to EFA-deficient rats. Simultaneous changes occurring with comparable rates were observed in the swelling properties and phospholipase A₂ activity of the mitochondria. Mitochondria isolated from the liver of EFA-deficient rats exhibited a high tendency to swell and a high phospholipase A₂ activity in comparison with those prepared from normal rat liver. Feeding of corn oil for 48 hr to the EFA-deficient rats completely reduced this high rate of swelling and phospholipase A₂ activity to the normal level. In the same time eicosatrienoic acid, a characteristic fatty acid constituent of phospholipids from EFA-deficient animals, was replaced by the more common fatty acids, linoleic and arachidonic. The possible relationship between fatty acid constituents of phospholipids, swelling properties and phospholipase A₂ activity in rat liver mitochondria are discussed.     

Vergleich zur Identität von Phospholipiden aus Humanseren und daraus hergestellten HDL-Fraktionen

A Comparison of Phospholipids in Human Sera with Phospholipids in HDL-Fractions The phospholipid composition of human sera and HDL-fractions was analyzed and characterized. The phospholipids were extracted with Extrelut (Merck) according to the manufacturer's recommendation. The recovery of phospholipids (expressed as P_i before and after passage through the elution-column) was rather poor for sera (25%) and distinctly better for HDL-fractions (46%). Phosphatidylcholine is the main phospholipid in human sera and in a similar manner in the HDL-fraction. The fatty acid distribution of phosphatidylcholine in both fractions was assessed and the fatty acids in sn2-position were evaluated after hydrolysis with phospholipase A₂. The fatty acid profile for native lysophosphatidylcholine, extracted from sera, was compared to the fatty acid distribution of lysolecithin formed from lecithin of sera after hydrolysis with phospholipase A₂. Both lysolecithins differed distinctly in their fatty acid distribution. Phosphatidylcholine from sera was identical with phosphatidylcholine from the HDL-fractions concerning the fatty acid distribution and their positional isomerism. The small recovery of phospholipids after passage through an Extrelut-column is due to the strong protein-phospholipid interaction in sera and HDL-fractions.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

The effect of phospholipase D on the function of fragmented sarcoplasmic reticulum

Incubations of fresh preparations of fragmented sarcoplasmic reticulum (FSR) were carried out at pH 5.7. This pH was necessary for hydrolysis of phospholipids by phospholipase D. The pH did not influence calcium uptake or the activity of calcium-stimulated ATPase of FSR. Treatment of FSR with phospholipase D caused hydrolysis of the membrane phospholipids. The phosphatidic acid produced remained bound to the membrane. Increasing phospholipid cleavage was paralleled by loss of calcium uptake, which was complete when about two-thirds of the membrane phospholipids were hydrolyzed.     

Resistance to the effect of phospholipase A2 of the biliary phospholipids during incubation of bile

Mixtures of fresh bile of the rat and of isolated hepatic phospholipids (one or the other of these components having been labeled with³H oleic acid) were incubated either with heated rat pancreatic juice, at 37 C during periods of 1 and 3 hr, or with snake venom, at 25 C during periods of 17 and 36 hr, as sources of phospholipase A₂. After incubation, tritiated free oleic acid was measured since this acid was in the 2 position of both phospholipidic substrates. With heated pancreatic juice, no significant enzymatic hydrolysis of the bile phospholipids occured, but isolated hepatic phospholipids were readily attacked. With snake venom, the whole isolated hepatic phospholipids were very strongly hydrolyzed while biliary phospholipids were hydrolyzed to a much lesser extent.     

Specificity of polyunsaturated fatty acid release from rat brain synaptosomes

Release of specific polyunsaturated fatty acids from cell membranes may have a significant implication in biological function, considering the involvement of various fatty acids in cell signal transduction. In the present study, release of polyunsaturated fatty acids from rat brain synaptosomes by endogeneous synaptosomal lipase activity was examined in comparison to that by cobra venom phospholipase A₂ (Naja naja naja). Cobra venom phospholipase A₂ (Naja naja naja) preferentially hydrolyzed docosahexaenoic acid (22∶6n−3) from both synaptosomes and lipid muxtures containing similar classes of lipids commonly found in the brain. Arachidonic acid (20∶4n−6) and oleic acid (18∶1n−9) were also hydrolyzed; however, monoene species was hydrolyzed slower than were polyenoic species in synaptosomes. Phosphatidylethanolamine was the most preferred phospholipid class for release of 22∶6n−3 fatty acid from both lipid mixtures and synaptosomes. In contrast to hydrolysis by cobra venom phospholipase A₂, endogenous synaptosomal lipase activity preferentially hydrolyzed 20∶4−6 from rat brain synaptosomes, despite the high abundance of 22∶6n−3 in synaptosomal membranes. Preferential release of 20∶4n−6 was observed over a wide range of pH values and calcium concentrations. Synaptosomal 22∶6 species appeared to be resistant to hydrolysis even after stimulation with various agents such as phorbolmyristate, suggesting that physiological importance of 22∶6−3 in neuronal membranes may not be as the release fatty acid.     

Influence of hypo- and hyperthyroidism on rat liver glycerophospholipid metabolism

The effects of hyper- and hypothyroidism on enzyme activities involved in phospholipid metabolism in the rat liver were studied. Hyperthyroidism significantly decreases activities of both microsomal acyl-CoA:glycero-3-phosphate acyltransferase (GPAT) (34%, p<0.01) and microsomal acyl-CoA:1-acylglycero-3-phosphocholine acyltransferase (GPCAT) (28–33%, p<0.01). This may contribute to the decreased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hyperthyroidism. Mitochondrial GPAT, phospholipase A₂ and cytosol lysophospholipase are unaffected by hyperthyroidism. In contrast, hypothyroidism stimulates mitochondrial GPAT (38%, p<0.01) and microsomal GPCAT (14–19%) activities but decreases both mitochondrial phospholipase A₂ (36%, p<0.01) and cytosol lysophospholipase (56%, p<0.01) activities. The increased GPCAT activity may contribute to the increased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hypothyroidism. Triiodothyronine (T₃) treatment of the hypothyroid rat (25 μg/100 g body weight/day for four days) corrected phospholipase A₂ and lysophospholipase activities to the level of the control rat, but failed to correct the increased mitochondrial GPAT activity and not only corrected but lowered GPCAT activity to the level of the hyperthyroid rat.     

Leukotriene B4 promotes phospholipid acylation in human neutrophils

The present study was undertaken to test the hypothesis that leukotriene B₄ (LTB₄) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB₄ (1.5 to 150 nM) for 1 for 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferases catalyzing acylation of the respective lysophospholipids. LTB₄ stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid but not by arachidonic acid. LTB₄ and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB₄ or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB₄-activated neutrophils reflects activation of phospholipase A₂ and acyltransferases as well as ofde novo phospholipid synthesis.     

Phospholipid hydroperoxides are detoxified by phospholipase A<Subscript>2</Subscript> and GSH peroxidase in rat gastric mucosa

The aim of this study was to determine the metabolic fate of phospholipid hydroperoxides (PLOOH) in rat gastric mucosa. Here we report evidence concering the mechanism for PLOOH detoxification in gastric mucosa homogenate. Analysis by the TLC blot technique showed that the gastric mucosa has the highest potential to eliminate 1-palmitoyl-2-linoleoyl-phosphatidylcholine hydroperoxides (PL-PtdChoOOH) compared with the intestinal mucosa and liver. Major products detected after incubation with gastric mucosa were the partially reduced linoleic acid hydroperoxides (LAOOH) and lysophosphatidylcholine, indicating the involvement of phospholipase A₂ (PLA₂) in the elimination pathway. Using unilamellar vesicles, we demonstrated that gastric mucosal PLA₂ does not distinguish between PLOOH and intact phospholipids. Although gastric mucosal PLA₂ activity efficiently eliminated excess amounts of PLOOH, the complete reduction of LAOOH was dependent on the supply of exogenous GSH. In a separate experiment, administration of egg yolk PtdChoOOH to rats for 6 d significantly elevated GSH peroxidase (GPx) activity in the gastric mucosa. We concluded that excess amounts of PLOOH are efficiently eliminated through the hydrolysis by PLA₂, and the subsequent reduction of FA hydroperoxide by GPx is the critical step for complete detoxification of oxidized phospholipids in the stomach.     

Beeinflussung des Lands Zyklus durch Inhibitoren und Cofaktoren

The Influence of Activators and Inhibitors on Enzymes of the Land's Cycle in Microsomal Membranes The fatty acid exchange of phospholipids in biological membranes is described by the Land's cycle. Acyltransferases and phospholipase A₂ are involved in the reacylation and deacylation of phospholipids. The membranes of a microsomal preparation from porcine heart muscle was separated on a Ficoll gradient in plasma membranes (on 10% Ficoll) and membranes of endoplasmic reticulum (on 15% Ficoll). Membranes on 10% Ficoll are designated as fraction F¹ and those on 15% Ficoll as fraction F₂. Both membrane fractions showed quite different activities for enzymes belonging to the Land's cycle. Enzymes of the Land's cycle of fraction F₁ were dependent on magnesium. ATP and CoA. A dependence of ATP but not of CoA could be detected for enzymes in membrane fraction F₂. Bromopalmitic acid as inhibitor showed the same results. In a comparable way the inhibitor effect of EDTA was much stronger on membrane fraction F₁ than on fraction F₂. The stimulation of enzyme activities by calcium effects especially the activities of phospholipase A₂ activity are present in both membrane fractions. In addition there might be an activity of acyl-CoA-synthetase in membrane fraction F₁ but a comparable activity could not be detected for F₂. We suppose that the fatty acid exchange in phospholipids of F₂ is not effected by the addition of coenzyme A. Both membrane systems seem to be quite different concerning the enzyme activities representing the Land's cycle.     

Effects of streptozotocin-induced diabetes on phosphoglyceride metabolism of the rat liver

We have studied the effect of streptozotocin (SZ)-induced diabetes on fatty acyltransferase and phospholipase enzyme activities involved in the synthesis and degradation of rat liver phosphoglycerides. Neither mitochondrial nor microsomal acyl-CoA: glycerol 3-phosphate acyltransferase (GPAT) activity was altered, although insulin treatment stimulated mitochondrial GPAT activity. However, microsomal acyl-CoA: 1-acylglycerol 3-phosphate acyltransferase (1-acyl-GPAT) activity increased (24–33 per cent, p<0.01) in the diabetic animals using 3 different acyl-CoA donors: palmitoyl-CoA, oleoyl-CoA and linoleoyl-CoA. SZ-induced diabetes also increased acyl-CoA:1-acylglycerol 3-phosphorylcholine acyltransferase (GPCAT) activity (38–45 per cent, p<0.01) with 3 different acyl-CoA donors: oleoyl-CoA, linoleoyl-CoA and arachidonoyl-CoA. 1-acyl-GPAT and GPCAT activity returned to normal with insulin treatment. In contrast to the increased activity of the microsomal fatty acyltransferases 1-acyl-GPAT and GPCAT, SZ-induced diabetes decreased mitochondrial phospholipase A₂ activity and lysophospholipase activity (49–70 per cent, p<0.01). Insulin treatment of the diabetic rats corrected the decreased lysophospholipase and stimulated phospholipase A₂ activity 35 per cent higher than controls. Since microsomal 1-acyl-GPAT and GPCAT are known to have higher activity toward unsaturated fatty acyl-CoA donors, the increased GPCAT activity coupled with the decreased lysophospholipase activity and the increased 1-acyl-GPAT activity in diabetes would tend to increase the formation of newly synthesized phospholipids containing unsaturated fatty acids. This mechanism plus the decreased fatty acid desaturase (4) may be the factors which alter the fatty acid composition of phosphoglycerides in diabetic rat liver microsomes.     

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A₂ incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A₂ (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A₂, and linoleic and linolenic acids by phospholipase A₂ alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.     

Phorbol ester stimulates PAF synthesisvia the activation of protein kinase C in rat leukocytes

When rat pleural mononuclear leukocytes were stimulated with 1 μM phorbol myristate acetate (PMA), platelet-activating factor (PAF)-like activity was detected in the supernatant and the cellular fractions of the incubation mixture, as measured by rabbit platelet aggregation. C₁₆PAF activity peaked at 30 min in both fractions. Acetyltransferase activity in the microsomal fraction of the stimulated cells also increased rapidly and showed a peak at 10 min. A protein kinase C inhibitor, staurosporine, and an inhibitor of phospholipase A₂,p-bromophenacylbromide, inhibited stimulated PAF formation in both fractions. Staurosporine also inhibited PMA induced acetyltransferase activity. The data suggest that PMA stimulates PAF synthesis by the remodeling pathway in rat pleural cells through activation of both phospholipase A₂ and acetyltransferase, and that the acetyltransferase, in turn, may be activated through activation of protein kinase C. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Enzyme‐catalyzed synthesis of structured phospholipids with conjugated linoleic acid

Structured phospholipids were synthesized with the functional lipid conjugated linoleic acid (CLA). The lipase‐ and phospholipase A₂‐catalyzed enzymatic acidolysis reaction between phospholipids (PL) and CLA was used for fatty acid modification. Enzymatic processes were an effective way to produce structured PL. Screening of four lipases and immobilized phospholipase A₂ and a combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PL. The maximum incorporation achieved by the latter enzyme was 16% with soy PL in 72 h.     

Changes in fatty acid composition of phospholipids from liver microsomes and nuclei in rats fed a choline-free diet

Male F-344 rats were fed a choline-free (CF) diet, and changes in phospholipid content, phospholipid fatty acids and phospholipase A₂ activity in liver nuclei and microsomes were examined during the first 72 hr. Both nuclei and microsomes showed a decrease in phosphatidylcholine (PC) content. Microsomes showed an increase in PC arachidonate while nuclei showed a decrease. Also, microsomes showed increased activity of phospholipase A₂ (PLA₂) while nuclei did not. These observations are consistent with the hypothesis that the absence of diene conjugates in liver microsomes in the rats on the CF diet may reflect the increased rate of removal of peroxidized fatty acids by phospholipase A₂.     

Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii

Hyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A² in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn-2 fatty acids in the following order: linoleoyl> oleoyl>arachidoyl phosphatidylcholine. Phospholipase A₂ had similar activities toward l-α-1-palmitoyl-2-arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A₂ activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium. The activity was enhanced more than 5–18-fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544 disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A₂ was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using 10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). No activity of phospholipase A₂ activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS-PAGE. Portions of this article were presented at the Biocatalysis Symposium at the 91st Annual Meeting and Expo of the American Oil Chemists’ Society in San Diego, CA, April, 2000.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

On the specificity of a phospholipase A2 purified from the 106,000×g pellet of bovine brain

Assessment has been made of the specificity of a purified phospholipase A₂ from the 106,000×g pellet (microsomal fraction) of bovine grey matter which shows strong activity toward phosphatidylinositol (PI). In the first series of experiments involving the utilization as substrates of PI with different¹⁴C- or³H-labeled fatty acids in the 2-position, the purified phospholipase A₂ most readily removed 16∶0 palmitic acid, followed by 18∶0 stearic acid, 18∶1 oleic acid and 20∶4 arachidonic acid. In the second series of experiments, the purified phospholipase A₂ showed preferential action toward PI (100%) compared to phosphatidylcholine (PC, 62.5%), phosphatidic acid (PA, 32.6%), phosphatidylethanolamine (PE, 25.1%) and phosphatidylserine (PS, 21.5%), where each phosphoglyceride was labeled in the 2-position with [1-¹⁴C] oleic acid. In the third series of experiments, fatty acids were shown to cause inhibition of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C] oleoyl PI in the order 20∶4>18∶1>18∶0>16∶0 which is the reverse order to that just noted. In the final series of experiments, the addition of the phosphoglycerides PC, PE, PS and PA in amounts of 5 or 10 μM caused either no inhibition (PE, 2%), slight inhibition (PC, 15%) or reasonably significant inhibition (PA, 20% and PS, 40%) of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C]-oleoyl PI. The pattern of specificity observed for the purified phospholipase A₂ combined with its microsomal location are the expected properties of a phospholipase A₂ that might function in a deacylation-reacylation cycle for modifying the fatty acid distribution in PI.