Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors

Lucas, Karin; Dennis, Edward A.

doi:10.1016/j.prostaglandins.2005.02.004

Cited by 61 publications

(53 citation statements)

References 25 publications

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“…However, we find that even unstimulated cells produce PAF when PAF degradation is blocked. The nalkyl fluorophosphonates MAFP and MLnFP are potent irreversible inhibitors of plasma (55) (see above) and type I PAF acetylhydrolases, but they also inhibit group IV Ca 21 -dependent and group VI Ca 21 -independent phospholipases A 2 (48). The latter two enzymes supply lyso-PAF for stimulated PAF synthesis (49,56) and phospholipid remodeling (57).…”

Section: Discussionmentioning

confidence: 99%

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Chen

Yang

Foulks

et al. 2007

Journal of Lipid Research

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Stimulated inflammatory cells synthesize plateletactivating factor (PAF), but lysates of these cells show little enhancement in PAF synthase activity. We show that human neutrophils contain intracellular plasma PAF acetylhydrolase (PLA2G7), an enzyme normally secreted by monocytes. The esterase inhibitors methyl arachidonoylfluorophosphonate (MAFP), its linoleoyl homolog, and Pefabloc inhibit plasma PAF acetylhydrolase. All of these inhibitors induced PAF accumulation by quiescent neutrophils and monocytes that was equivalent to agonist stimulation. Agonist stimulation after esterase inhibition did not further increase PAF accumulation. PAF acetylhydrolase activity in intact neutrophils was reduced, but not abolished, by agonist stimulation. Erythrocytes, which do not participate in the acute inflammatory response, inexplicably express the type I PAF acetylhydrolase, whose only known substrate is PAF. Inhibition of this enzyme by MAFP caused PAF accumulation by erythrocytes, which was hemolytic in the absence of PAF acetylhydrolase activity. We propose that PAF is continuously synthesized by a nonselective acyltransferase activity(ies) found even in noninflammatory cells as a component of membrane remodeling, which is then selectively and continually degraded by intracellular PAF acetylhydrolase activity to modulate PAF production.-Chen, J., L. Yang, J. M. Foulks, A. S. Weyrich, G. K. Marathe, and T. M. McIntyre. Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis. J. Lipid Res.

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Section: Discussionmentioning

confidence: 99%

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Chen

Yang

Foulks

et al. 2007

Journal of Lipid Research

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“…The protein in the native state was eluted in the "protein buffer" (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 125 mM imidazole, and 2 mM dithiothreitol). The protein concentration was measured using the Bradford assay, and the activity was assayed using mixed micelles in the modified Dole assay (31,32). Purified GIVA PLA 2 (2 mg/ml) was stored in the protein buffer on ice for DXMS experiments (16).…”

Section: Methodsmentioning

confidence: 99%

Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A2 as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

Hsu

Burke

Stephens

et al. 2008

Journal of Biological Chemistry

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The GIVA phospholipase A 2 (PLA 2 ) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA 2 activity is Ca 2؉ -dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA 2 activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA 2 . We also analyzed the changes in H/D exchange of the intact GIVA PLA 2 upon Ca 2؉ binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca 2؉ binding on GIVA PLA 2 are localized in the C2 domain and suggest that binding of two distinct Ca 2؉ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA 2 activation.The cytosolic group IVA (GIVA) 3 phospholipase A 2 (PLA 2 ), also known as cPLA 2 , was purified and cloned in 1991 (1, 2). It is one of the few phospholipases in the phospholipase A 2 superfamily shown to be important in lipid mediator biosynthesis (3, 4). GIVA PLA 2 hydrolyzes membrane phospholipids at the sn-2 position to release free arachidonic acid, which is the precursor of numerous eicosanoids including the prostaglandins and leukotrienes (5, 6) involved in the inflammatory and pain response (7-9). Understanding the regulation of the catalytic activity of GIVA PLA 2 is crucial for understanding eicosanoid metabolism.The activity of the 85-kDa GIVA PLA 2 has been suggested to be regulated by several factors including the intracellular Ca 2ϩ concentration, its phosphorylation state, and the binding of various activators. Since the GIVA PLA 2 was discovered in 1986, the activity of this enzyme has been known to be Ca 2ϩ -dependent (10, 11). The Ca 2ϩ -dependent lipid-binding domain (C2 domain) at the N terminus, which is linked to a catalytic domain where phospholipid hydrolysis occurs, was later identified (12). Further studies of the GIVA PLA 2 activity showed up-regulation by p38 protein kinase mainly through Ser-505 phosphorylation (13-15). Various membrane-associated activators have been shown to bind to GIVA PLA 2 and upregulate its activity. In particular, we have shown that the membrane-associated phosphatidylinositol 4,5-bisphosphate can bind to the "lysine pocket" of GIVA PLA 2 with high affinity and specificity to ac...

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“…The inhibition of ∼50% was in the same range as the inhibition by the irreversible PLA 2 inhibitors MAFP and BEL. The relatively low efficacy of the latter two may be due to the preincubation scheme used (62,63) or more likely residual calcium-and phospholipaseindependent hydrolytic activities. Nevertheless, the assay employed was robust enough to detect i/cPLA 2 inhibitors and to compare potencies of individual compounds.…”

Section: Setup Of Whole Blood Assay and Immunopharmacological Profilimentioning

confidence: 99%

Ginger Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

Nievergelt

Marazzi

Schoop³

et al. 2011

The Journal of Immunology

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The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A2 (iPLA2)-triggered maturation and the cytosolic phospholipase A2 (cPLA2)-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA2 assay, most major ginger phenylpropanoids directly inhibited i/cPLA2 from U937 macrophages, but not hog pancreas secretory phospholipase A2. The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA2 and methyl arachidonyl fluorophosphonate for cPLA2, with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE2 and thromboxane B2 (>50%) and partially also leukotriene B4 in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA2 and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA2, the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

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Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors

Cited by 61 publications

References 25 publications

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A2 as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

Ginger Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

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