The use of C6-NBD-PC for assaying phospholipase A2-activity: Scope and limitations

Meyuhas, Dina; Yedgar, Shaul; Rotenberg, Michal; Reisfeld, Nurit; Lichtenberg, Dov

doi:10.1016/0005-2760(92)90133-g

Cited by 15 publications

(4 citation statements)

References 26 publications

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“…NBD-ceramide was completely recovered in the organic phase, whereas over 95% of NBD-caproic acid was recovered in the aqueous phase (Fig. 1A) as reported previously (29,30). Although NBD-ceramide and NBD-caproic acid showed similar Rf values (about 0.9), the two compounds could be separated because of the different solubility in organic and aqueous solutions.…”

Section: Detection Of Nbd-caproic Acid a Counterpart Of Ceramidase-msupporting

confidence: 82%

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Tada¹,

Toyomura²,

Nakamura³

et al. 2010

J Pharmacol Sci

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Abstract. Ceramide, a key molecule in the metabolism of sphingolipids, is converted by ceramidase to sphingosine, and phosphorylated by ceramide kinase to form ceramide-1-phosphate (C1P). In this study, we improved on a method of thin-layer chromatography using a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide) by adding another step for separation of extracted ceramide metabolites by lipophilicity, and determined levels of C1P, caproic acid, sphingomyelin, and glucosylceramide simultaneously. Also we found that 1) treatment of NBD-ceramide-labeled cells (human lung adenocarcinoma A549 cells and Chinese hamster ovary cells) with Na 3 VO 4 increased the amount of NBD-C1P formed within 30 min, 2) the treatment increased production of NBD-caproic acid, a counterpart of sphingosine, by ceramidase within 2 h, 3) expression of ceramide kinase enhanced the Na 3 VO 4 -induced formation of NBD-C1P, and tyrosine kinase inhibitors (herbimycin and genistein) decreased the response, 4) the production of NBD-caproic acid in A549 cells was inhibited by genistein, and 5) the responses for 2 h after Na 3 VO 4 treatment were accompanied by a decrease in the production of NBD-sphingomyelin, not a loss of NBD-ceramide. The improved thin-layer chromatography method was useful for the simultaneous determination of enzymatic activities for ceramide metabolism in cells.

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Section: Detection Of Nbd-caproic Acid a Counterpart Of Ceramidase-msupporting

confidence: 82%

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Tada¹,

Toyomura²,

Nakamura³

et al. 2010

J Pharmacol Sci

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“…MC540 binds to the outer leaflet of cell membranes (Sieber, 1987) and detects subtle differences in membrane packing revealed by a 20-nm red-shift in emission maximum as lipids become less tightly packed (Langner and Hui, 1993;Stillwell et al, 1993;Yu and Hui, 1992). NBD-PC has been used extensively to study the behavior of membrane phospholipids (reviewed in Chattopadhyay, 1990) and functions as a substrate for sPLA 2 (Meyuhas et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Mechanisms Governing the Level of Susceptibility of Erythrocyte Membranes to Secretory Phospholipase A2

Jensen

Burgess

Gonda

et al. 2005

Biophysical Journal

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Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.

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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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The use of C6-NBD-PC for assaying phospholipase A2-activity: Scope and limitations

Cited by 15 publications

References 26 publications

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Mechanisms Governing the Level of Susceptibility of Erythrocyte Membranes to Secretory Phospholipase A2

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

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The use of C6-NBD-PC for assaying phospholipase A2-activity: Scope and limitations

Cited by 15 publications

References 26 publications

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Mechanisms Governing the Level of Susceptibility of Erythrocyte Membranes to Secretory Phospholipase A2

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

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