Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

Wittenauer, Laura A.; Shirai, Kohji; Jackson, Richard L.; Johnson, J D

doi:10.1016/0006-291x(84)91479-7

Cited by 60 publications

(18 citation statements)

References 6 publications

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“…To further confirm that the acyl chain at position sn-2 was removed, we treated PnLTA with two additional PLA2 enzymes (porcine pancreas and bee venom PLA2) and examined the reaction products with a MALDI-TOF mass spectrometer (Fig. 2) (44). Untreated PnLTA showed the three major peaks, as before, although the peak position shifted down by about 10 m/z units compared to that shown in Fig.…”

Section: Methodsmentioning

confidence: 70%

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Seo

Kim²,

Nahm

2006

Clin Vaccine Immunol

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Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells.

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Section: Methodsmentioning

confidence: 70%

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Seo

Kim²,

Nahm

2006

Clin Vaccine Immunol

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“…All other chemicals were of reagent grade. Activity of PLA 2 was assayed using C6-NBD-PC as substrate according to a previous report (Wittenauer et al, 1984), with some modifications. Briefly, that substrate (41 nM) was dissolved in 50 mM Tris-HCl buffer at pH 8.0 containing 5 mM CaCl 2 .…”

Section: Chemicalsmentioning

confidence: 99%

Activation of phospholipase PLA2 actvity in Ricinus communis leaves in response to mechanical wounding

Domingues

Souza

Soares

et al. 2007

Braz. J. Plant Physiol.

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In order to investigate the defense response in castor bean (Ricinus communis) against predators, we analyzed the effect of mechanical wounding upon the phospholipase A 2 (PLA 2 ) activity of leaf extracts. Time course experiments revealed that the highest levels of increased PLA 2 activity (ca. two fold) occurred 15 min and 60 min after injury. The induced activities demonstrated high sensitivity towards aristolochic acid (10 mM), a PLA 2 inhibitor. Based on SDS-PAGE analysis, the PLA 2 activity induced 15 min after wounding migrated with a molecular mass of 40 kDa and was denoted RcPLA 2 I. The protein activity induced 60 min after wounding, RcPLA 2 II, migrated with a molecular weight of 14 kDa. Furthermore its N-terminal sequence shared homology with PLA 2 from elm and rice. The PLA 2 enzymes were purified to near homogeneity by a combination of gel filtration and electro-elution of protein bands after native PAGE. Key words: cellular defense, castor bean, plant signaling, phospholipase A 2 , Ricinus communis Com o objetivo de avaliar a resposta de defesa da mamona (Ricinus communis) contra predadores, analisaram-se as alterações nas atividades de fosfolipases A 2 (PLA 2 ) em extratos de folhas de plantas, submetidas à injúria mecânica após diferentes intervalos de tempo. Observou-se que os níveis dessa enzima aumentaram de forma significativa (cerca de duas vezes) 15 e 60 min após o ferimento. As atividades que foram induzidas foram sensíveis ao ácido aristolóquico (10 mM), um inibidor específico para PLA 2 . A enzima induzida após 15 min foi denominada Rc-PLA 2 I e apresentou uma massa molecular aparente, determinada por SDS-PAGE, de 40 kDa. A proteína induzida após 60 min, RcPLA 2 II, apresentou uma massa molecular de 14 kDa. A seqüência N-terminal parcial da Rc-PLAII mostrou homologia com PLA 2 de arroz e olmo. As fosfolipases foram parcialmente purificadas por associação da cromatografia de filtração em gel, seguida por eluição das bandas que apresentaram atividade enzimática após separação por PAGE não desnaturante.

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“…This difference in phospholipid hydrolysis cannot be attributed to the differences in aggregation states of the fluorescent phospholipids [13,14] because the hydrolysis has been carried out in mixed micelles containing a large excess of Triton X-100 [5], under identical conditions, and monitored only after extracting the reaction mixture with organic solvents and quantifying the amounts of unhydrolysed phospholipid by HPLC. In using this assay we observed complete linearity at least up to 10 mg of I or II even in the presence of fluorescent derivatives of C 6 or C 12 fatty acids.…”

Section: Discussionmentioning

confidence: 99%

Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues

Puri

Arora

Gupta

1999

European Journal of Biochemistry

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To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A 2 ±glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A 2 . Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A 2 ± phospholipid interactions.Keywords: phospholipase A 2 ; phosphatidylcholine; phospholipid conformation; butanetriol analogue.Cobra venom phospholipase A 2 is a Ca 2+ -dependent enzyme that catalyses the hydrolysis of the fatty acyl group at the sn-2 position of membrane phospholipids. It is composed of a single polypeptide chain of 119 amino acids which contains seven disulphide bonds [1]. The crystal structures of phospholipase A 2 from several sources have been determined [1±3], and the amino acids that form the enzyme catalytic site have been reported to be highly conserved [1]. Also, the structure of the catalytic surface has been shown to be exactly identical regardless of the degree of evolutionary divergence, state of oligomerization, ionic strength, or pH of the crystallization conditions [3,4].Binding of phospholipase A 2 with phosphatidylcholines involves interactions of the enzyme active site calcium with the phospholipid phosphate oxygen and the sn-2 carbonyl oxygen followed by the hydrophobic interactions between the phospholipid fatty acyl chains and the enzyme catalytic site [2±5]. Apart from the sn-2 acyl chain [5], the acyl chain at the sn-1 position also plays an important role in phospholipase A 2 ±glycerophos-pholipid interactions [6,7]. It has been reported that an increase in the hydrophobicity of the sn-1 acyl residue increases the affinity between the phospholipid molecule and the enzyme [6]. However, it is not yet known whether the ester moiety (-CO-O-) of the sn-1 acyl chain plays any role in phospholipase A 2 ± glycerophospholipid interactions. To investigate this we introduced one additional methylene residue between the glycerol C1 and C2 carbon atoms and then studied the interactions of the resulting modified phospholipids (Fig. 1) with Naja naja phospholipase A 2 . Results of these studies indicate that the sn-1 ester moiety plays an important role in binding of the enzyme with the substrate. MATERIALS AND METHODS MaterialsPhospholipase A 2 from N. naja snake venom, sn-glycero-3-phosphocholine, 6-N-(7-nitrobe...

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Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

Cited by 60 publications

References 6 publications

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Activation of phospholipase PLA2 actvity in Ricinus communis leaves in response to mechanical wounding

Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues

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Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

Cited by 60 publications

References 6 publications

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Activation of phospholipase PLA2 actvity in Ricinus communis leaves in response to mechanical wounding

Probing the role of C‐1 ester group in Naja naja phospholipase A2–phospholipid interactions using butanetriol‐containing phosphatidylcholine analogues

Contact Info

Product

Resources

About

Probing the role of C‐1 ester group in Naja naja phospholipase  A₂–phospholipid interactions using butanetriol‐containing  phosphatidylcholine analogues