Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.     

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.     

Secretory phospholipase A2 enzymes in atherogenesis

PURPOSE OF REVIEW: Immunohistochemistry studies have confirmed the presence of group IIA, group V and group X secretory phospholipase A2 in human or mouse atherosclerotic lesions. The possibility that secretory phospholipase A2 plays a role in the pathophysiology of atherosclerosis (and is not merely a marker for localized inflammation) has been substantiated by a number of recent in-vitro and in-vivo studies. RECENT FINDINGS: A mouse strain with a targeted deletion of group V secretory phospholipase A2 has been developed. Peritoneal macrophages from these mice have significantly blunted eicosanoid generation in response to zymosan, providing the first direct evidence that a secretory phospholipase A2 plays a role in stimulation-induced arachidonic acid production in vivo. A recent in-vitro study indicated that de novo synthesized groups IIA and X secretory phospholipase A2 can mediate arachidonic acid release intracellularly, without the requirement for previous secretion from cells, as was previously thought. Several studies support the previously proposed model that secretory phospholipase A2 hydrolysis generates pro-atherogenic LDL. These data, coupled with the finding that macrophage-specific expression of human group IIA secretory phospholipase A2 promotes atherosclerotic lipid deposition in mice, draw attention to secretory phospholipase A2 as an attractive target for the treatment of atherosclerotic disease. SUMMARY: Secretory phospholipase A2 activity in the arterial intima has the potential to amplify atherogenic processes by liberating potent pro-inflammatory lipid mediators and by generating pro-atherogenic LDL. Future in-vivo studies will aid in defining the mechanism(s) that underlie the pro-atherosclerotic effects of secretory phospholipase A2.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.     

A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2

Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.     

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

Spin-label assay for phospholipase A2

A rapid and continuous method for measuring phospholipase A2 activity using electron spin resonance spectroscopy and a spin-labeled phospholipid as a substrate has been developed. The substrate, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine, gives rise principally to a broad ESR line in aqueous solution due to strong spin-spin interactions, probably resulting from its micellar formation. Upon addition of bee venom phospholipase A2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A2 activity can be followed by monitoring the increase in the ESR signal amplitude of the three-line spectrum, which is linearly proportional to the amount of 4-doxylpentanoic acid produced; no separation of the product from the substrate is needed during the measurement. The rate of hydrolysis of 1 nmol min-1 can be accurately measured within a 5-min period of time in a sample volume of 100 microliters. This new method should be useful for assaying phospholipase A2 activities in various biological systems.     

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.     

Purification and properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

Vitamin E inhibits platelet phospholipase A2

One of the most important functions of phospholipase A2 is the release of arachidonic acid from membrane phospholipids for the synthesis of biologically active eicosanoids. We have demonstrated in our laboratory that vitamin E inhibits platelet phospholipase A2 in a dose-dependent manner. Rats fed a 100 ppm or a 1000 ppm vitamin E diet exhibit diminished phospholipase A2 activity compared to those fed a vitamin E-free diet. Addition of vitamin E to a sonicated platelet suspension resulted in further suppression of the phospholipase A2 activity in all groups of rats. In order to gain insight into the mechanism of vitamin E inhibition of platelet phospholipase A2, we partially purified this enzyme by gel filtration chromatography. Enzyme activity was localized in the soluble supernatant fraction of a high-speed spin. This partially purified rat platelet phospholipase A2 had an absolute requirement for Ca2+ and was inhibited by various forms of tocopherol. Tocol inhibited the enzyme to a greater extent than either D- or DL-alpha-tocopherol, while there was little or no effect from DL-alpha-tocopherol acetate. These results emphasize the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action, compared to that of the methyl groups which are absent in tocol. This inhibitory action of vitamin E on platelet phospholipase A2 suggests a crucial function for vitamin E in regulating arachidonate release from the membrane phospholipids and its subsequent metabolism.     

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 beta-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 of [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.     

Inhibition of phospholipase A2 by protein I

The 36 kDa substrate of several tyrosine protein kinases has been shown to exist in monomeric and oligomeric (362102) forms. Partial sequence data has suggested that the oligomer, referred to as protein I, is homologous to a group of phospholipase A2 inhibitory proteins, collectively called lipocortins. In the present communication we demonstrate that protein I inhibits bovine pancreas phospholipase A2 with similar potency to that of lipocortin. Approximately 44 pmol protein I was required to produce 50% inhibition of 7.2 pmol of phospholipase A2. Inhibition of phospholipase A2 activity by calmodulin, S-100, calregulin, parvalbumin, troponin-C, or CAB-48 was not observed. These results indicate that protein I is a potent and specific inhibitor of phospholipase A2 activity, and thus shares functional homology with the lipocortin proteins. We therefore propose that this protein be named lipocortin-85.     

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A2

Hydrolysis by pancreatic and snake venom (Crotalus atrox) phospholipase A2 of fluorescent monolayers of pyrene-labelled phosphatidylglycerol on solid support was studied. We used a fluorescence microscope equipped with video camera, video recorder and an image analyzer to monitor changes in fluorescence. Decrease in pyrene excimer emission was evident when pyrene phosphatidylglycerol monolayers transferred onto quartz glass slides (at a surface pressure of 15 mN m-1) were subjected to enzymatic hydrolysis. Snake venom phospholipase A2 could hydrolyze the monolayers almost completely while pancreatic phospholipase A2 could cause only 50% decrease in fluorescence intensity. EDTA totally inhibited the action of both A2 phospholipases. When monolayers were transferred onto solid supports at a surface pressure of 31 mN m-1 C. atrox phospholipase A2 could still exert activity whereas porcine pancreatic phospholipase A2 was inactive.     

Calcium-independent phospholipase A2 and apoptosis

Apoptosis or programmed cell death is associated with changes in glycerophospholipid metabolism. Cells undergoing apoptosis generally release free fatty acids including arachidonic acid, which parallels the reduction in cell viability. The involvement of cytosolic group IVA phospholipase A(2)alpha (cPLA(2)alpha) in apoptosis has been the subject of numerous studies but a clear picture of the role(s) played by this enzyme is yet to emerge. More recently, the importance of lipid products generated by the action of a second phospholipase A(2), the group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) in apoptosis has begun to be unveiled. Current evidence suggests that iPLA(2)-VIA-derived lysophosphatidylcholine may play a prominent role in mediating the chemoattractant and recognition/engulfment signals that accompany the process of apoptotic cell death, and gives possibility to the efficient clearance of dying cells by circulating phagocytes. Other lines of evidence suggest that perturbations in the control of free arachidonic acid levels within the cells, a process that may implicate iPLA(2)-VIA as well, may provide important cellular signals for the onset of apoptosis.     

Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1

A membrane-associated phospholipase A2 was purified from rat spleen. The phospholipase A2 was solubilized from the 108,000 x g pellet fraction with 0.3% lithium dodecyl sulfate and then purified to homogeneity by successive DEAE-Cellulofine AM, octyl-Sepharose, Cellulofine GCL 300-m, S-Sepharose, and Bio-Gel P-30 chromatographies in the presence of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate. The apparent Mr of the enzyme, estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, was about 13,600. The purified enzyme had a pH optimum in the range of pH 8.0-9.5 and required the presence of Ca2+ (4 mM) for its maximal activity. The enzyme preferentially hydrolyzed the 2-acyl ester bonds of phosphatidylglycerol in the presence and absence of sodium cholate or sodium deoxycholate. Unlike the phospholipase A2 of rat spleen supernatant, no immunocross-reactivity was observed between the purified enzyme and anti-rat pancreatic phospholipase A2 antibody. The N-terminal amino acid sequence of the enzyme was determined and found to be homologous to that of viperid and crotalid venom phospholipases A2. The results in this and the preceding report (Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731) demonstrate that rat spleen contains two genetically distinct phospholipase A2 isoenzymes.