Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.     

Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.     

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.     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Isolation of homogeneous phospholipase A2 from human platelets

Phospholipase A₂ from human blood platelets has been purified to homogeneity by treatment of the platelet homogenate with H₂SO₄, followed by affinity chromatography of the extract. The platelet phospholipase is a soluble, heat-stable protein of MW 44000. The purified enzyme inhibits ADP-induced platelet aggregation.     

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.     

Isolation and characterization of a gamma-type phosphoinositide-specific phospholipase C (PLC-gamma 2).

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).     

A series of annexins from human placenta and their characterization by use of an endogenous phospholipase A2.

Membranes from human placenta contain proteins which inhibit the activity of phospholipases A2 by binding to phospholipid thus impeding substrate availability. We used unilamellar mixed liposomes and a partially purified cytosolic phospholipase A2 from placenta for characterizing this substrate-depleting activity. A major portion of these inhibitory proteins was released by extracting washed membranes with a Ca+(+)-chelator. Biochemical fractionation and systematic analysis resulted in the unequivocal identification of a series of annexin proteins. We describe a straightforward procedure which allows to obtain 8 annexins from placenta either in pure form or as a mixture of two annexins. One of them was obtained in two forms which had the same molecular mass of 68 kDa but differed in charge. We also present suggestive evidence for a novel annexin I-related polypeptide of Mr 45,000 which is an excellent in vitro substrate for protein kinase C. We estimate that about 2% of the total placental membrane proteins are annexins. For achieving half inhibition of phospholipase A2 activity with pure annexins, up to a 6.5-fold difference in the amounts of protein was observed when calculated on a molar basis. This suggests specificity of individual annexin species.     

Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.     

Purification and characterization of phospholipase A2 from rat stomach

Phospholipase A2, which is localized in the mucosal part of the corpus of rat stomach (Hirohara et al. (1987) Biochim. Biophys. Acta 919, 231-238), was purified 990-fold from the supernatant of a tissue homogenate by heat treatment at acidic pH, ammonium sulfate fractionation, ion-exchange chromatography, gel-filtration and reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The purified enzyme gave a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with a molecular mass of approx. 17 kDa. The enzyme had a pH optimum of 8.0 and hydrolyzed the 2-arachidonoyl residue of phosphatidylcholine preferentially to the 2-oleoyl residue, the Vmax and Km values for the two being 227 and 29 mumol/min per mg protein and 0.037 and 0.019 mM, respectively. The activity was calcium-dependent and was markedly increased by SDS and dimethyl sulfoxide (DMSO). The enzyme showed typical product inhibition. Free unsaturated fatty acids (oleic, arachidonic and docosahexaenoic acids), which are supposedly the main enzymatic products in vivo, inhibited the activity. Arachidonic acid caused noncompetitive inhibition and its concentration for its maximal inhibition (50% inhibition) was 5 x 10(-5) M. Lysophosphatidylcholine, free saturated fatty acids (palmitic and stearic acids) and arachidonic acid metabolites (leukotrienes and prostaglandins) had no effect on the activity.     

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.     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Purification and characterization of the second Streptomyces phospholipase A2 refolded from an inclusion body

A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.