Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Acid phospholipase A activities in rat hepatocytes

Cultured rat hepatocytes exhibit acid phospholipase A activity. On the basis of product formation from stereospecifically radiolabeled phosphatidylethanolamine substrates, phospholipases A1 and A2 have been identified with optimal activities at pH 4.5. According to subcellular fractionation studies, the acid phospholipases in hepatocytes appear to be located in the lysosomal compartment. Application of specific inhibitors of the biosynthesis, glycosylation, and translocation of lysosomal enzymes in hepatocyte cultures suggests a half-life of approx. 1 day for the acid lysosomal phospholipase A1. About the same value for the half-life was obtained for the lysosomal marker enzymes, acid phosphatase and beta-N-acetyl-D-hexosaminidase.     

The effect of calmodulin and chlorpromazine on the activity of phospholipases A2 from various sources

The effects of calmodulin and chlorpromazine on purified phospholipase A2 preparations from snake venoms: cobra (Naja naja oxiana), echis (Ehis multisquamatus) and Agkistrodon halys halys, as well as on phospholipases A2 from rat liver mitochondria and human platelets were studied. It was shown that within the concentration range of 1-5 microM calmodulin stimulates the phospholipase activity. Chlorpromazine inhibits the activity of these enzymes, the degree of inhibition being different for various phospholipases. Calmodulin was shown to interact with the phospholipases in the absence of exogenous Ca2+. The results obtained indicate that all phospholipases tested are calmodulin-dependent enzymes.     

Isolation of a phospholipase-A2-like protein from human fetal intestine. Lysis of erythroid cells from bovine liver by this protein and porcine phospholipase A2

Extracts of human fetal intestine contain factors that can stimulate or inhibit thymidine incorporation into fetal bovine erythroid cells. An inhibitory factor was purified to homogeneity by gel-permeation and reversed-phase high performance liquid chromatography. The inhibitory action was due to cell lysis. The first 25 amino acids of the N-terminal segment were identical to the human lung and pancreatic phospholipase A2. The isolated protein released arachidonic acid from 2-arachidonyl phosphatidylcholine. Porcine phospholipase A2 had the same effects as the intestinal protein, including its tissue-specific lysis of fetal bovine liver erythroid cells. No decrease of thymidine incorporation was seen in fetal bovine intestinal cells, 3T3 cells, or K562 cells incubated with the porcine enzyme. No release of hemoglobin or cell lysis was observed with human erythrocytes or fetal bovine erythrocytes. Porcine and bee phospholipases, which have low sequence homology, are nearly equipotent in inhibiting thymidine incorporation, whereas melittin and beta-bungarotoxin were less active than the pancreatic enzyme. These results support the tissue-specific effects observed with other phospholipases A2. The high sensitivity of liver erythroid cells towards some phospholipases A2 suggest that these enzymes may be involved in the elimination of hepatic erythroid cells at the end of gestation.     

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.     

Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.     

Regulation of sphingomyelinase and phospholipase C activity in liver cell membranes of rats of different ages

The changes in the functional activities of sphingomyelinase and phospholipase C from rat liver cell plasma membranes were studied in postnatal ontogenesis in the presence of thyroxin and mercasolyl. It was found that endogenous phospholipases of plasma membranes control of phospholipid content in rat liver cells. The sphingomyelinase activity is under control of thyroid hormones, whereas that of phospholipase C which is phosphatidyl choline-specific, is unaffected by them. The data obtained testify to the possible involvement of diacylglycerols formed via enzymatic hydrolysis of phosphatidylcholine, in the regulation of the sphingomyelinase activity.     

Mechanisms of lysophosphatidic acid production

Lysophosphatidic acid is one of the most attractive phospholipid mediator with multiple biological functions and is implicated in various human diseases. In the past ten years much has been learned about the physiological roles of LPA through series of studies on LPA actions and its receptors. However, the molecular mechanisms of LPA have been poorly understood. LPA is produced in various conditions both in cells and in biological fluids, where multiple synthetic reactions occur. At least two pathways are postulated. In serum and plasma, LPA is mainly converted from lysophospholipids. By contrast, in platelets and some cancer cells, LPA is converted from phosphatidic acid. In each pathway, at least two phospholipase activities are required: phospholipase A1 (PLA1)/PLA2 plus lysophospholipase D (lysoPLD) activities are involved in the first pathway and phospholipase D (PLD) plus PLA1/PLA2 activities are involved in the second pathway. Now multiple phospholipases are identified that account for PLA1, PLA2, PLD, and lysoPLD activities. In the absence of specific inhibitors and genetically modified animals and individuals, the contribution of each phospholipase to LPA production can not be easily determined. However, apparently certain extracellular phospholipases such as secretory PLA2 (sPLA2-IIA), membrane-associated PA-selective PLA1 (mPA-PLA1), lecithin-cholesterol acyltransferase (LCAT), and lysoPLD are involved in LPA production.     

Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions

Abstract: Enrichment in the base-exchange activities was found in the micro-somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin-sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin-insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane-bound base exchange activities. Some activity was restored in phospholipase C-treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A-treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D-treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.     

Monoclonal antibodies against rat platelet phospholipase A2

Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.     

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.     

Effects of chronic amiodarone treatment on cat myocardial phospholipid content and on in vitro phospholipid catabolism

Amiodarone is used extensively for the chronic treatment of life-threatening arrhythmias caused by ischemic heart disease. However, chronic therapy with this agent results in phospholipidosis in various tissues and it has been suggested that the inhibition of lysosomal phospholipase A by this drug contributes to this abnormality. Exogenous amiodarone has been shown to inhibit purified rat liver lysosomal phospholipase A₁, as well as acid phospholipase activities of alveolar macrophage homogenates and those of snake venom phospholipase A₂ and bacterial phospholipase C. The effects of drug treatment on heart have not been explored. The results described here demonstrate that amiodarone also significantly increases (37%, p < 0.001) phospholipid content in cat hearts. This increase is proportionately distributed to all major phospholipid classes, with the exception of sphingomyelin which appears to increase more than the others. In addition, the data also show that following amiodarone treatment, the endogenous drug levels in the heart were sufficient to reduce in vitro losses of membrane phospholipid at 37°C by inhibiting a variety of endogenous phospholipases at physiological (7.4), ischemic (6.2) and acidic (5.0) pH values. This protection is more pronounced at acidic pH values than at physiological pH. Endogenous amiodarone also affects myocardial phospholipase activities towards exogenous phosphatidylcholine and again the extent of inhibition is more at acidic pH. These results suggest that amiodarone induces phospholipidosis in the heart by inhibiting phospholipid catabolism and that its antiarrhythmic properties may reside in its ability to modulate alkaline, neutral and acid phospholipase activities in ischemia. To what extent amiodarone metabolites (desethylamiodarone and bis-desethylamiodarone) are involved in these actions remains to be determined.     

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix)

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-change chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A₂-type phospholipase A. The medium lethal dose 50% (LD₅₀) values of the two phospholipases A are 0.27 and 0.28 μg/g, respectively, by intravenous injection and 1.05 and 1.00 μg./g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14 000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.     

The effect of retinol and retinoic acid on the testicular phospholipase a activity in retinol-deficient rats.

Both phospholipases A1 and A2 activities (EC 3.1.1.4) at pH 7.4 were found to be significantly decreased in retinol-deficient rat testes supplemented with retinoic acid as compared to retinol-fed controls using 1-acyl-2-[1-(14)C]-oleoyl-sn-glycero-3-phosphocholine as substrate. However, little or no difference was observed in phospholipase A1 activity at pH 3.0 in both groups of rats.     

Solubilization and partial characterization of phospholipase A from rat heart sarcoplasmic reticulum

Phospholipase A has been solubilized from the sarcoplasmic reticulum of rat heart by treatment with Tris buffer, potassium chloride, taurodeoxycholate or octyl glucoside. On HPLC gel permeation, two phospholipases were identified at the void volume of a TSK 3000 column and at an apparent molecular mass of 60 kDa. The two activity peaks exhibited a predominance of phospholipase A1 activity (83-91%) and a lesser phospholipase C activity (4-9%) using sonicated 1-palmitoyl-2[1-14C]oleoylphosphatidylcholine liposomes as substrate. The voiding phospholipase A peak, which represented the bulk of the recovered activity, exhibited a requirement for calcium ions in the 0.3-3 microM range. The heat stability and response to mercuric ions was studied and some similarities were noted between the solubilized sarcoplasmic reticulum phospholipases A and the cytosolic phospholipases A of rat heart. It is speculated that the cytosolic phospholipase A which we reported earlier may represent in part phospholipase A released from sarcoplasmic reticulum during isolation of the subcellular membrane fractions.     

Studies on the acyl-chain selectivity of cellular phospholipases A2

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A2 are discussed.     

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.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

Purification of a cellular (granulocyte) and an extracellular (serum) phospholipase A2 that participate in the destruction of Escherichia coli in a rabbit inflammatory exudate

A granule-associated phospholipase A2 from rabbit polymorphonuclear leukocytes and a closely similar phospholipase A2 from rabbit serum have been purified to near homogeneity by ion-exchange and reverse-phase chromatography. The cellular (polymorphonuclear leukocyte) phospholipase A2 has been purified greater than 100,000-fold and the extracellular (serum) phospholipase A2 approximately 60,000-fold. The NH2-terminal amino acid sequence of the ascitic fluid phospholipase A2 that we have recently purified from inflammatory exudates produced in rabbits is nearly identical (15 of 16 residues) to that of the polymorphonuclear leukocyte phospholipase A2 and completely identical (19 of 19 residues) to that of the purified serum phospholipase A2. The functional properties of these three phospholipases A2 are indistinguishable. Each enzyme is active against Escherichia coli killed by the bactericidal/permeability-increasing protein of polymorphonuclear leukocyte, a property shared only by a subset of phospholipases A2. The presence of structurally and functionally very closely similar phospholipases A2 in the cellular and extracellular compartments of an inflammatory exudate is consistent with the apparent role of these enzymes in the destruction of certain microbial invaders during the acute inflammatory response.     

Monoclonal antibodies against human synovial phospholipase A2

Four monoclonal antibodies (HP-1, HP-2, HP-3 and HP-4) with differing reactivities were raised against human synovial fluid phospholipase A2. None of them bound to exocrine phospholipases A2, such as those from pancreas or snake venom. However, antibodies HP-1 and HP-3 showed cross-reactivity with rabbit and rat platelet secretory phospholipases A2, which share common enzymatic and structural features with the human synovial enzyme. Antibodies HP-1, HP-2 and HP-3 inhibited the activity of human synovial phospholipase A2. The antibodies were used to develop a rapid immunoaffinity column chromatographic procedure for enzyme purification. In some preparations, the recovery of total activity after immunoaffinity column chromatography was more than 100% suggesting the existence of endogenous inhibitory factors of phospholipase A2 in human synovial fluid.