Phospholipases A1 and A2 in bovine thyroid.

In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.     

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.     

Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Purification and characterization of lysophospholipase released from rat platelets

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.     

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

The enzymatic system of phospholipid deacylation-reacylation in rat liver lysosomal membranes

It has been shown for the first time that lysosomal (tritosomal) membranes of rat liver contain enzymes that are responsible for the deacylation-reacylation of phospholipids; their activity optimum lies at pH 7.0. Deacylation of lysosomal membrane phospholipids is controlled by a cascade of enzymatic reactions involving Ca2(+)-dependent phospholipase A1 which exhibits the maximal activity at 2.5 mM Ca2+ and at neutral values of pH, as well as lysophospholipase. Reacylation of lyso-derivatives of phospholipids is catalyzed by Mg2(+)-activated oleoyl-CoA:lysophosphatidylcholine acyltransferase having an activity optimum at pH 7.2.     

Preferential hydrolysis of peroxidized phospholipid by lysosomal phospholipase C

The susceptibility of partially peroxidized liposomes of 2-[1-14C] linoleoylphosphatidylethanolamine ([14C]PE) to hydrolysis by cellular phospholipases was examined. [14C]PE was peroxidized by exposure to air at 37 degrees C, resulting in the formation of more polar derivatives, as determined by thin-layer chromatographic analysis. Hydrolysis of these partially peroxidized liposomes by lysosomal phospholipase C associated with cardiac sarcoplasmic reticulum, and by rat liver lysosomal phospholipase C, was greater than hydrolysis of non-peroxidized liposomes. By contrast, hydrolysis of liposomes by purified human synovial fluid phospholipase A2 or bacterial phospholipase C was almost completely inhibited by partial peroxidation of PE. Lysosomal phospholipase C preferentially hydrolyzed the peroxidized component of the lipid substrate which had accumulated during autoxidation. The major product recovered under these conditions was 2-monoacylglycerol, indicating sequential degradation by phospholipase C and diacylglycerol lipase. Liposomes peroxidized at pH 7.0 were more susceptible to hydrolysis by lysosomal phospholipases C than were liposomes peroxidized at pH 5.0, in spite of greater production of polar lipid after peroxidation at pH 5.0. Sodium bisulfite, an antioxidant and an inhibitor of lysosomal phospholipases, prevented: (1) lipid autoxidation, (2) hydrolysis of both non-peroxidized and peroxidized liposomes by sarcoplasmic reticulum and (3) loss of lipid phosphorus from endogenous lipids when sarcoplasmic reticulum was incubated at pH 5.0. These studies show that lipid peroxidation may modulate the susceptibility of phospholipid to attack by specific phospholipases, and may therefore be an important determinant in membrane dysfunction during injury. Preservation of membrane structural and functional integrity by antioxidants may result from inhibition of lipid peroxidation, which in turn may modulate cellular phospholipase activity.     

The phospholipase activity of sheep pancreatic juice.

Sheep pancreatic juice was found to contain at least two enzymes which hydrolysed biliary lecithin. One enzyme was heat and acid labile and hydrolysed the fatty acid from position 1 (phospholipase A1); the other was heat and acid stable hydrolysing the fatty acid at position 2 (phospholipase A2). Lysophospholipase activity was also present. The phospholipases were active at pH values greater than 4.2, and would therefore function in the acid conditions (pH 3-6) of the sheep small intestine. The activity of the pancreatic phospholipases, and A2 in particular, was dramatically stimulated by the presence of the secretions of Brunner's glands which could be important in accelerating the hydrolysis of biliary lecithin in the lumen of the intestine. Phospholipase A1 was sensitive to acid in the range pH 2.5-3.5 and could therefore be partially inactivated by abomasal digesta; but phospholipase A2 was resistent to acid treatment.     

Purification and characterization of a lysophospholipase from a macrophage-like cell line P388D1

Two lysophospholipase activities (designated I and II) were identified in the macrophage-like cell line P388D1. Lysophospholipase I was purified (8,500-fold) to homogeneity by DEAE-Sephacel, Sephadex G-75, Blue-Sepharose, and chromatofocusing chromatography. Lysophospholipase II was separated from the lysophospholipase I in the Blue-Sepharose step. The apparent molecular mass of lysophospholipase I and II are 27,000 and 28,000 daltons, respectively, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their pI values were 4.4 and 6.1 respectively, as determined by isoelectric focusing. Lysophospholipase I exhibited a broad pH optimum between 7.5-9.0. The double-reciprocal plot of the substrate dependence curve of the purified lysophospholipase I showed a break around the critical micelle concentration of the substrate (1-palmitoyl-sn-glycerol-3-phosphorylcholine). The apparent Km, determined from substrate concentrations above 10 microM was 22 microM, and the apparent Vmax was 1.3 mumol min-1mg-1. The purified enzyme did not have phospholipase A1, phospholipase A2, acyltransferase, or lysophospholipase-transacylase activity. No activity was detected toward triacylglycerol, diacylglycerol, p-nitrophenol acetate, p-nitrophenol palmitate, or cholesterol ester. The enzyme did, however, hydrolyze monoacylglycerol although at a rate 20-fold less than lysophospholipid, 0.06 mumol min-1mg-1. The lysophospholipase I was inhibited by fatty acids but not by glycerol-3-phosphorylcholine, glycerol-3-phosphorylethanolamine, or glyc-fjerol-3-phosphorylserine. A synthetic manoalide analogue 3(cis,cis,-7,10)hexadecadienyl-4-hydroxy-2-butenolide inhibited the enzyme with half-inhibition (IC50) at about 160 microM. Triton X-100 decreased the enzymatic activity, although this apparent inhibition can be explained by a "surface dilution" effect. The pure lysophospholipase I was stable for at least 5 months at -20 degrees C in the presence of glycerol and beta-mercaptoethanol. Lysophospholipid also demonstrated a protective effect during the later stage of purification.     

Phospholipase A2 activity of rat stomach

Phospholipase A activity in rat stomach wall and in gastric content was studied using [1-14C]dioleoylphosphatidylcholine as substrate. The optimum activity of the stomach wall was found to take place at pH 7.0. During optimal phospholipase action about 40% of the [1-14C]oleic acid released was due to an active intracellular lysophospholipase. The gastric phospholipase required 5 mM Ca2+ for full activity and is inhibited by EDTA. It specifically hydrolyzed the sn-2 position of the phospholipid molecule. The enzyme was heat labile and inactivated by acidification at pH 3.0. The gastric content enzyme had a lower specific activity and an optimum pH of 8.0. It was heat stable and was not inactivated by acidification. These results indicate that gastric content phospholipase A is of pancreatic origin, via a duodenal reflux. By ligating the stomach we were able to further confirm that the gastric wall phospholipase was different from that of the gastric content. It originated from the stomach mucosa. Subcellular fractionation suggests that the gastric phospholipase A2 is essentially bound to the plasma membrane. About 6% of the activity was found to be soluble. Biopsies of human gastric mucosa displayed a phospholipase A activity which had similar properties to that of rat gastric enzyme. The physiological function of this enzyme is discussed in terms of prostaglandin synthesis via the release of arachidonic acid.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

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.     

Effects of antimalarial drugs on phospholipase A and lysophospholipase activities in plasma membrane, mitochondrial, microsomal and cytosolic subcellular fractions of rat liver

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 x 10(-4) M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.     

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.     

Biomembrane-modulated, lysosomal phospholipase A2 contamination of chromaffin granule ghosts

It was reported that subcellular fractionation of bovine adrenal medulla results in the separation of distinct, non-calcium-dependent phospholipases A2--one associated with chromaffin granule ghosts, another with lysosomes. The basis of this distinction is pH optimum: in routine assays utilizing neat liposomal substrates, the chromaffin granule ghost-associated enzyme is alkaline-active whereas the lysosomal enzyme is acid-active (Husebye, E.S. and Flatmark, T. (1987) Biochim. Biophys. Acta 920, 120-130). We now report that biomembranes after liposomal substrates and/or lysosomal phospholipase A2 such that the enzyme now hydrolyzes them (at low cation concentration) with an alkaline pH optimum. In a lysosomal membrane fraction, phospholipase A2 activity at pH 7.5 relative to activity at pH 5.0 increases as increasing amounts of lysosomal membranes are assayed. The pH optimum of chromaffin granule ghost-associated phospholipase A2 toward liposomal substrates is likewise biomembrane-dependent and, when assayed carefully, is indistinguishable on the basis of optimal pH from the lysosomal enzyme. Although chromaffin granule ghost-associated phospholipase A2 is most likely a lysosomal contaminant, its broad, biomembrane-modulated pH range may still allow it to participate in catecholamine secretion. More importantly, however, sensitivity of adrenal medullary lysosomal phospholipase A2 to biomembranes broadens its potential physiologic pH range and may also play a role in the regulation of this potentially deleterious activity.     

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.     

Isolation and characterization of a phospholipase A2 from an inflammatory exudate.

Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed.