Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2)

An intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca(2+)-dependent phospholipase A(2) (PLA(2)) activity (about 70% of the total PLA(2) activity). The soluble PLA(2) activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA(2) was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW3000 column developed with a buffer containing 0.1-0.2 M salt. When the gel-filtration column was developed in the presence of 1 M salt, almost all the PLA(2) activity was eluted near the total available volume. The soluble PLA(2) was purified to near homogeneity. A Ca(2+)-dependent PLA(2) was also purified from the fractions extracted with 1 M KBr from nucleus pulposus. For comparison, we purified a Ca(2+)-dependent PLA(2) from the KBr fraction of spleen. The splenic PLA(2) was identical to a group IIa PLA(2), as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15. 7-kDa band at the same position as splenic group IIa PLA(2). These results suggest that group IIa PLA(2) is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Trichomonas vaginalis: identification of soluble and membrane-associated phospholipase A1 and A2 activities with direct and indirect hemolytic effects

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.     

Calcium binds to and is required for biological activity of the 104-kilodalton hemolysin produced by Actinobacillus pleuropneumoniae serotype 1

Actinobacillus pleuropneumoniae serotype 1, strain Shope 4074, was grown on agar medium containing 10 mM Ca2+ and under Ca2+ limiting conditions by addition of 2 and 5 mM EGTA to the growth medium. Hemolysis of washed bovine erythrocytes was observed from the culture grown in Ca2+ excess but not from the two cultures where Ca2+ was chelated from the growth medium by using EGTA. However, the hemolytic activity of these latter two cultures could be restored if 10 mM Ca2+ was added to the dilution buffer. This restored hemolytic activity could be neutralized with a rabbit homologous polyclonal antiserum specific for the 104-kDa hemolysin of serotype 1 but not with preimmune serum from the same rabbit. Stained SDS-PAGE gels and immunoblots showed the 104-kDa protein hemolysin in fractions from each of the three growth conditions. Thus, the restored hemolytic activity was associated with the 104-kDa protein in the three cultures. In addition, the 104-kDa protein, when electroblotted onto nylon membranes, bound 45Ca, indicating that the molecule has binding sites for Ca2+. The results indicate that Ca2+ is required for the biological activity of the 104-kDa hemolysin of A. pleuropneumoniae serotype 1.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: a possible signal for IL 1 action

We examined the effects of Interleukin 1 (IL 1) on rabbit articular chondrocytes with particular emphasis on arachidonic acid metabolism in these cells. Articular chondrocytes were isolated from the knee joints of normal New Zealand white rabbits and were cultured in vitro until confluent. Addition of 5 U/ml of purified IL 1 to chondrocytes led to an early increase in cell-associated phospholipase A2 (PLA2; measured by hydrolysis of [14C]arachidonic acid-labeled E. coli). Within 1 hr after IL 1 addition, cell-associated PLA2 activity was increased by more than threefold relative to basal PLA2 activity, and further increases in cellular enzyme activity were observed up to 48 hr of IL 1 treatment. IL 1 stimulation also led to a time- and dose-related release of extracellular PLA2 and PGE2, but IL 1-induced PLA2 and PGE2 secretion occurred after the initial burst of intracellular PLA2 activity. Similar PLA2 and PGE2 responses were also observed when purified human IL 1 or IL 1-containing conditioned medium from LPS-stimulated human monocytes were used, but recombinant IL 2 or IL 3 were inactive. IL 1-induced chondrocyte PLA2 did not release radiolabeled free fatty acid from phosphatidylethanolamine labeled at the C-1 position with [14C]stearic acid, confirming the identity of this enzyme as PLA2. These data, therefore, provide the first direct evidence that IL 1 activates cellular PLA2, and we propose that PLA2 activation may be an early signal that initiates the inflammatory actions of IL 1.     

Characterization of a phospholipase A2 in hamster lung and in vitro and in vivo effects of bleomycin on this enzyme

Phospholipase A2 (PLA2) activity from adult hamster lung was characterized using L-alpha-1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylcholine as the substrate. The released [14C]-arachidonic acid was separated by TLC. The enzyme activity increased with increasing incubation time (0-120 minutes), calcium ion concentration (0-25.0 mM) and protein (0-2.0 mg). The optimum pH was 8.0. Deoxycholate had a concentration dependent (0.1 to 0.5 mM) inhibitory effect on the activity. PLA2 specific activity was the highest in mitochondrial fraction. PLA2 activity following incubation with bleomycin was increased in a dose related fashion. In vivo study showed that both PLA2 activity and collagen content in hamster lung were significantly elevated at 14 days followed intratracheal instillation of bleomycin. The activation of PLA2 may play an important role in bleomycin-induced pulmonary toxicity.     

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.     

Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation

Anandamide and other bioactive N-acylethanolamines (NAEs) are a class of lipid mediators and are produced from glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs). Although the generation of NAPE by N-acylation of phosphatidylethanolamine is thought to be the rate-limiting step of NAE biosynthesis, the enzyme responsible, N-acyltransferase, remains poorly characterized. Recently, we found that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess phospholipid-metabolizing activities including NAPE-forming N-acyltransferase activity, and proposed to call HRASLS1–5 phospholipase A/acyltransferase (PLA/AT)-1–5, respectively. Among the five members, PLA/AT-1 attracts attention because of its relatively high N-acyltransferase activity and predominant expression in testis, skeletal muscle, brain and heart of human, mouse and rat. Here, we examined the formation of NAPE by PLA/AT-1 in living cells. As analyzed by metabolic labeling with [¹⁴C]ethanolamine or [¹⁴C]palmitic acid, the transient expression of human, mouse and rat PLA/AT-1s in COS-7 cells as well as the stable expression of human PLA/AT-1 in HEK293 cells significantly increased the generation of NAPE and NAE. Liquid chromatography–tandem mass spectrometry also exhibited that the stable expression of PLA/AT-1 enhanced endogenous levels of NAPE, N-acylplasmenylethanolamine, NAE and glycerophospho-NAE. Furthermore, the knockdown of endogenous PLA/AT-1 in mouse ATDC5 cells lowered NAPE levels. Interestingly, the dysfunction of peroxisomes, which was caused by PLA/AT-2 and -3, was not observed in the PLA/AT-1-expressing HEK293 cells. Altogether, these results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

Spatio-temporal expression of patatin-like lipid acyl hydrolases and accumulation of jasmonates in elicitor-treated tobacco leaves are not affected by endogenous levels of salicylic acid

We have previously isolated three tobacco genes (NtPat) encoding patatin-like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation. NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins. Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses. PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl-jasmonate (MJ) whereas the ethylene (ET) precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), provoked a moderate induction. In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with beta-megaspermin, a cell death-inducing protein elicitor. A simplified system based on the infiltration of beta-megaspermin into leaves was used to dissect the spatio-temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA. NtPat-encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells. A massive accumulation of 12-oxo-phytodienoic and jasmonic acids occurred in the elicitor-infiltrated zone, but only low levels were detectable outside this area. A similar picture was found in SA-deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR-induced build-up of endogenous SA. Finally, ET-insensitive plants showed a weakened induction of PLA2 activity outside the elicitor-infiltrated tissue.     

Role of secretory and cytosolic phospholipase A(2) enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA(2)) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA(2) enzymes and AA release was strongly reduced by cellular pretreatment with different PLA(2) inhibitors and by pertussis toxin, an inhibitor of G(i)-protein activation. This indicates that both cytosolic and secretory PLA(2) enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.     

Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells

A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.