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PubMed1990

Activation of human neutrophil phospholipase D by three separable mechanisms.

Activation of human neutrophils by receptor-mediated agonists, the Ca2+ ionophore A23187, or the protein kinase C activator phorbol myristate acetate all stimulated phospholipase D activity. This was demonstrated by the increased formation of phosphatidic acid, and in the presence of ethanol, phosphatidylethanol (PEt) accumulation. EGTA completely inhibited A23187-induced PEt formation, but only one-half of the fMLP-induced PEt accumulation. Staurosporin, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but actually stimulated the formation of PEt in response to fMLP by several-fold. Thus, increased cytosolic Ca2+ and activated protein kinase C can each lead to activation of phospholipase D, but neither is required for receptor-mediated activation of phospholipase D activity. Wortmannin is an irreversible inhibitor of the oxidative burst, but does not inhibit NADPH oxidase or known components of signal transduction. Wortmannin inhibited activation of phospholipase D in response to fMPL. It did not directly inhibit phospholipase D, as the response to A23187 was unaffected. Wortmannin did not inhibit other fMPL-stimulated events, such as aggregation or adherence. We conclude that inhibition by wortmannin defines a third pathway to activation of phospholipase D. Further, its effect on phospholipase D correlates with its effect on the respiratory burst.

Androstadienes↗

PubMed1994

Carbodiimide modification enhances activity of pig pancreatic phospholipase A2.

Pig phospholipase A2, pig iso-phospholipase A2 and bovine pancreatic phospholipase A2 were reacted in solution with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of N-hydroxysulfosuccinimide, at pH 7. The influence of micellar protectants was analyzed. In the presence of n-hexadecylphosphocholine, the losses of activity in micellar diheptanoyl-lecithin were 80, 35, and 10% in bovine phospholipase A2, pig iso-phospholipase A2, and pig phospholipase A2, respectively. With 1-oleoylglycerophosphocholine, the bovine enzyme lost 40% activity, but the pig enzyme was activated sevenfold. The modified pig enzyme showed pre-micellar activation on monomeric diheptanoyl-lecithin, and either reduced or increased activities on mixed micelles of bile salt with egg phosphatidylcholine, depending on the composition of the micelles. This activation is consistent with previous protein-engineering studies of pig pancreatic phospholipase A2. In this study, we present new information concerning the specificity and interfacial recognition behaviour of this enzyme in relation to this activation.

Animals↗

PubMed1992

Differential effects of presynaptic phospholipase A2 neurotoxins on Torpedo synaptosomes.

The effects of several phospholipase A2 neurotoxins from snake venoms were examined on purely cholinergic synaptosomes from Torpedo electric organ. The noncatalytic component A of crotoxin had no effect, whereas its phospholipase component B, used alone or complexed to component A, elicited a rapid and dose-dependent acetylcholine (ACh) release and a depolarization of the preparation. Subsequent ACh release evoked by high K+ levels or calcium ionophore was identical to the control after the action of component A but reduced after the action of crotoxin or of component B. These effects were not observed when the phospholipase A2 activity of the toxin was blocked either by replacing Ca2+ by Ba2+ (respectively, activator and inhibitor of phospholipase A2) or by alkylation of component B with p-bromophenacyl bromide. beta-Bungarotoxin, another very potent phospholipase A2 neurotoxin, induced release of little ACh, did not affect ionophore-evoked ACh release, but significantly reduced depolarization-induced ACh release. The single-chain phospholipase A2 neurotoxin agkistrodotoxin behaved like crotoxin component B. A nonneurotoxic phospholipase A2 from mammalian pancrease induced release of an amount of ACh similar to that released by crotoxin but did not affect the evoked responses. The obvious differences in effect of the various neurotoxins suggest that they exert their specific actions on the excitation-secretion coupling process at different sites or by different mechanisms.

Acetylcholine↗

PubMed1984

Activated complex of L-cells and Rickettsia prowazekii with N-ethylmaleimide-insensitive phospholipase A.

The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release of free fatty acids and lysophosphatides from the phospholipids of the L-cell. About 50% of rickettsiae present in the suspension that was centrifuged onto an L-cell monolayer at 0 degree C to effect this interaction formed a tight L-cell-rickettsiae association from which the rickettsiae could not be removed by simple washing. Both the L-cell-rickettsiae association and the rickettsiae before association with L-cells interact with N-ethylmaleimide, so that the subsequent expression of the phospholipase A activity was inhibited (treatment of the L-cells with N-ethylmaleimide before centrifugation does not inhibit phospholipase activity). However, treatment of this association with 2,4-dinitrophenol and KCN caused much less inhibition of this phospholipase A activity than did treatment of the rickettsiae with these agents before centrifugation onto the L-cells. Incubation of the L-cell-rickettsiae association for a short time at 35 degrees C resulted in a very low level of free fatty acid formation and changed this association to an activated complex in which the phospholipase A activity was no longer sensitive to the inhibitory effects of N-ethylmaleimide. The characteristics of the association and activated complex were stable: after a 2-h incubation at 0 degrees C, the association and the activated complex retained both their basal phospholipase A activities and their characteristic responses to N-ethylmaleimide treatment. In scanning electron micrographs of the activated complexes, the rickettsiae that were initially attached were no longer visible after 45 min at 35 degrees C, and the surface of the L-cell appeared to have been etched away. These activated complexes provide a system in which modulators of the phospholipase A can be investigated without the confusion caused by the first-step receptor interaction between rickettsiae and their host cells.

Animals↗

PubMed2001

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

Culture Media↗

PubMed1992

Identification and characterization of human myocardial phospholipase A2 from transplant recipients suffering from end-stage ischemic heart disease.

Although numerous studies have implicated accelerated phospholipid catabolism during myocardial ischemia as an important contributor to ischemic membrane dysfunction, no information is currently available on the subcellular distribution, physical properties, or kinetic characteristics of human myocardial phospholipase A2. In this report, we demonstrate that the overwhelming majority (98%) of total phospholipase A2 activity in human myocardium (obtained from transplant recipients) is calcium independent, plasmalogen selective, and is distributed between the microsomal (60-70% of total activity) and cytosolic (30-40% of total activity) fractions. Both human myocardial microsomal and cytosolic phospholipase A2 enzymes 1) preferentially hydrolyze plasmalogen molecular species containing arachidonic acid at the sn-2 position, 2) are recalcitrant to chemical inactivation by the indole-reactive agent parabromophenacyl bromide, 3) are irreversibly inhibited by covalent modification of an essential thiol residue by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and 4) are exquisitely sensitive to mechanism-based inhibition by (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone). In sharp contrast, human mitochondrial phospholipase A2 1) accounts for only a diminutive amount of total myocardial phospholipase A2 activity (1-2%), 2) is augmented by calcium ion, 3) exhibits a higher reaction velocity using phosphatidylcholine in comparison with plasmenylcholine substrate, and 4) is not substantially inhibited by either DTNB or bromoenol lactone. Collectively, these results demonstrate that the majority of phospholipase A2 activity in human myocardium is catalyzed by a novel class of calcium-independent plasmalogen-selective phospholipases A2 and underscore the potential importance of this class of enzymes in mediating membrane dysfunction during myocardial infarction in humans.

Calcium↗

PubMed1995

Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells.

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. In this study we have investigated the localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrated that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling complex of its receptor, phospholipase C-gamma 1 and filamentous actin which is primarily localized at membrane ruffles.

Actins↗

PubMed1993

Determination of the catalytic activity of phospholipase A2: E. coli-based assay compared to a photometric micelle assay.

Phospholipase A2 activity in human sera was determined of the basis of the E. coli assay and compared to a photometric micelle assay. The E. coli assay is based on the hydrolysis of phospholipids from [1-14C]oleic acid-labelled E. coli biomembranes. In the photometric assay the phospholipase A2 acts on mixed phospholipid micelles. The amount of fatty acid produced is quantitated in a subsequent photometric assay by coupling in the reaction to the coenzyme A metabolism. The E. coli membranes are essentially resistant to other lipases in human sera, i.e. lipoprotein lipases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A2 of human serum. The photometric assay, though, is susceptible to other lipases in human serum. The ratio of [1-14C]oleic acid to released total fatty acids served as the basis for the calculation of the true enzymatic activity. The assay closely correlated with the photometric assay based on mixed micelles in the higher ranges of phospholipase A2 activity, but not in the normal range. The sensitivity is higher by at least two powers of 10. The human serum phospholipase A2 strongly preferred E. coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine. In conclusion, the modified phospholipase A2 assay based on E. coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A2 activity in human sera. The photometric assay suffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation.

Acute-Phase Proteins↗

PubMed1993

Group I and group II phospholipases A2 in serum in uraemia.

Time-resolved fluoroimmunoassays were used for the detection of pancreatic group I and synovial-type group II phospholipases A2 in sera of patients suffering from chronic renal failure before and after haemodialysis. The concentration of group I phospholipase A2 was ten-fold higher in sera of uraemic patients than in healthy controls. There was no significant difference in the concentrations of group I phospholipase A2 in serum before and after haemodialysis. The concentration of group II phospholipase A2 was only marginally increased in sera of uraemic patients, compared with healthy controls. There was no significant difference in the concentrations of group II phospholipase A2 before and after haemodialysis. The results indicate that the metabolism of group I phospholipase A2 differs from that of group II phospholipase A2 in chronic renal failure.

Adult↗

PubMed1998

Elevated group II phospholipase A2 mass concentration in serum and colonic mucosa in Crohn's disease.

Group II phospholipase A2 has been proposed to play an important role in the pathophysiology of inflammatory bowel diseases. This enzyme has also been linked to host defence mechanisms against bacteria. The current study aimed at measuring the mass concentrations of group II phospholipase A2 in serum and colonic mucosa of patients with Crohn's disease of different severity and of appropriate control patients without any inflammatory disease. The activity of the disease was determined by clinical factors (the simple index score) and endoscopic and histological scoring. The mass concentration of group II phospholipase A2 was measured by a time-resolved fluoroimmunoassay. The mass concentrations of group II phospholipase A2 in serum and colonic mucosa were significantly higher both in patients with active and inactive Crohn's disease when compared with controls. There was statistically significant difference in the mass concentration of group II phospholipase A2 in colonic mucosa but not in serum between inactive and active Crohn's disease. The current results indicate that the mass concentration of group II phospholipase A2 is increased in serum and colonic mucosa of patients with Crohn's disease and that the latter is associated with the degree of the inflammatory activity in the intestinal wall. These results support the idea that group II phospholipase A2 is involved in the local and generalised pathological processes of Crohn's disease.

Adolescent↗

PubMed1992

A novel type of phospholipase A2 inhibitor, thielocin A1 beta, and mechanism of action.

Thielocin A1 beta, a novel phospholipase A2 inhibitor, was isolated from Thielavia terricola RF-143. It inhibited various phospholipase A2s in a dose-dependent manner. Among these, group II phospholipase A2 from rat was most sensitive to thielocin A1 beta (IC50 = 0.0033 microM). The inhibition of phospholipase A2 by thielocin A1 beta was independent of Ca2+ and substrate concentration. In addition, the inhibition of rat group II phospholipase A2 was noncompetitive (Ki = 0.0068 microM) and reversible. Furthermore, thielocin A1 beta quenched the relative fluorescent intensity of Naja naja venom phospholipase A2 and in a dose-dependent manner; 50% quench was noted with a molar ratio of thielocin A1 beta/enzyme of 2.2. These observations indicated that inhibition of phospholipase A2 by thielocin A1 beta may result from direct interaction with the enzyme.

Animals↗

PubMedDate not supplied

[Conserved and variable segments of the snake phospholipase A2 amino acid domain].

Homologous amino acid sequences of phospholipases A2 of snakes belonging to families Elapidae, Viperidae and Colubridae were considered in order to study the location of conservative and variable regions. To identify significant conservative and variable regions a comparison between two groups of aligned sequences of snake phospholipases A2 was successfully applied. The phospholipases A2 sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair distance matrix. Results of the comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown, that the results of the comparison between two phylogenetic groups of snake phospholipases A2 didn't depend much on the number of each group representatives, and the location of conservative and variable regions didn't significantly change if one of the groups was represented by the single sequence. It should be mentioned, that the more the phylogenetic difference between groups of phospholipases A2 the more was the number of significant conservative and variable regions. The knowledge of the number and location of conservative and variable regions and their dependence on phylogenetic relations between the compared taxons can be used to predict the synthetic peptide structure to obtain antibodies of various specificity. These antibodies may have either a wide range of cross-reactivity against all of phospholipases A2 or a limited range of cross-reactivity against phospholipases A2 of one taxon.

Amino Acid Sequence↗

PubMed1991

Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate.

Recently, the prototype of a novel class of calcium-independent plasmalogen-selective phospholipase A2 activities was identified in the cytosolic fraction of canine myocardium (Wolf, R.A., and Gross, R.W. (1985) J. Biol. Chem. 260, 7295-7303) and subsequently purified and characterized (Hazen, S.L., Stuppy, R.J., and Gross, R.W. (1990) J. Biol. Chem. 265, 10622-10630). We now demonstrate that 15 min of myocardial ischemia utilizing a rabbit Langendorf perfused heart model results in a 10-fold increase in membrane-associated calcium-independent phospholipase A2 activity whose detection is entirely dependent upon utilization of plasmalogen substrate. Ischemia-induced phospholipase activity was identified as a membrane bound member of this class of phospholipases A2 by demonstration of: 1) concomitant production of lysoplasmenylcholine and sn-2 fatty acid from plasmenylcholine substrate; 2) maximal enzymatic activity in the absence of calcium ion; and 3) a 16-fold higher maximum reaction velocity utilizing plasmenylcholine compared to phosphatidylcholine substrate at multiple surface concentrations. Ischemia-induced phospholipase A2 activity was specifically localized to the microsomal fraction and could not be solubilized by sonication, salt treatment, exposure to chelators, or utilization of submicellar concentrations of detergent. The appearance of microsomal phospholipase A2 activity did not require ischemia-induced transcription or translation since identical increases in enzymic activity were obtained in hearts previously treated with actinomycin D and cycloheximide. Collectively, these results demonstrate that a membrane-associated calcium-independent phospholipase A2 that selectively hydrolyzes plasmalogen molecular species is the likely enzymic mediator of accelerated phospholipid catabolism during early myocardial ischemia.

Animals↗

PubMed1989

Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain.

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1989

Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction.

We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.

Animals↗

PubMed1993

Inhibition of tyrosine phosphorylation by wortmannin in human neutrophils. Dissociation from its inhibitory effects on phospholipase D.

BACKGROUND: Recent studies have indicated that the regulation of the activation of human neutrophils depends on tyrosine phosphorylation and on phospholipase D. Furthermore, a tentative causal relationship between these two signalling pathways has been indirectly implied derived through the use of inhibitors of tyrosine kinases. The fungal metabolite, wortmannin is at present the only compound known to inhibit the receptor-mediated activation of phospholipase D in human neutrophils. Its mechanism of action is presently unknown. EXPERIMENTAL DESIGN: The ability of peripheral blood neutrophils to respond to various agonists with an increase in activity of phospholipase D and an enhancement of tyrosine phosphorylation in the absence or presence of wortmannin was monitored. RESULTS: Wortmannin was found to inhibit the stimulation of tyrosine phosphorylation by fMet-Leu-Phe, and by the inflammatory microcrystals monosodium urate and calcium pyrophosphate dihydrate. This effect of wortmannin was not secondary to inhibition of phospholipase D as U73122, a previously described phospholipase C inhibitor, was also found to inhibit phospholipase D without affecting tyrosine phosphorylation. CONCLUSIONS: The results make it likely that one of the earliest sites of action of wortmannin in human neutrophils is at the level of tyrosine phosphorylation which then exerts a modulatory influence on the activation of phospholipase D.

Androstadienes↗

PubMed1986

Enhanced phospholipase activity in peripheral blood monocytes from patients with rheumatoid arthritis.

Peripheral blood monocytes (PBM) from patients with rheumatoid arthritis (RA) produce greater amounts of prostaglandins (PG) than do control cells. To further explore the reasons for the increased PG production, we assessed the phospholipase activities in these cells. We found that PBM from patients with severe RA expressed greater phospholipase A2 (PLA2) and phospholipase C (PLC) activities than did the control cells. Enhanced PLA2 activities were observed in RA patient cells when phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates. Enhanced PLC activities also were seen when PC, PE, and phosphatidylinositol (PI) were used as labeled substrates. Increased PLC activity was observed whether linoleic acid or arachidonic acid was esterified to the 2 position of the phospholipid substrate used. Because all patients with RA were treated with nonsteroidal antiinflammatory drugs, we examined the effects of aspirin ingestion on phospholipase activities. Aspirin had no consistent effect on PLA2 activities but markedly inhibited PLC activities against PC, PI, and PE with arachidonic acid in the R2 position. That aspirin enhanced PLC activities against PC and PI with linoleic acid in the R2 position, suggests that PLC activity may be regulated in part by the R2 fatty acid. Our results indicate that increased phospholipase activities exhibited by PBM from RA patients may help explain the increased PG production by these cells. The increased phospholipase activities in PBM from RA patients do not appear to be due solely to nonsteroidal antiinflammatory drug therapy.

Arthritis, Rheumatoid↗

PubMed1998

Identification of a new high-affinity binding protein for neurotoxic phospholipases A2.

Ammodytoxin C is a neurotoxic phospholipase A2 which blocks the release of neurotransmitter from the nerve terminal. Using a radioiodinated derivative of the toxin, we located its specific high-affinity binding site in the demyelinated P2 fraction of porcine cerebral cortex (Kd = 15 nM; Bmax = 1.5 pmol/mg membrane protein). In cross-linking experiments on a membrane preparation, 125I-ammodytoxin C labeled a protein of 25 kDa. The formation of a specific adduct was not inhibited by nontoxic phospholipases A2 or even by neurotoxic phospholipases A2 which have practically identical pathophysiological activities to ammodytoxin C: agkistrodotoxin, Oxyuranus scutellatus 2 phospholipase A2, taipoxin, beta-bungarotoxin, notexin, and crotoxin. 125I-ammodytoxin C specific cross-linking was inhibited, however, by mannosylated BSA, suggesting the presence of a carbohydrate-recognition domain in the acceptor structure. According to the pharmacological and structural properties, the ammodytoxin acceptor from porcine cerebral cortex differs from other so far identified as phospholipase A2 acceptors and represents a new type of a high-affinity binding protein for neurotoxic phospholipases A2.

Affinity Labels↗