The substrate specificities of sunflower and soybean phospholipases D using transphosphatidylation reaction
© Abdelkafi and Abousalham; licensee BioMed Central Ltd. 2011
Received: 25 September 2011
Accepted: 1 November 2011
Published: 1 November 2011
Phospholipase D (PLD) belongs to a lipolytic enzyme subclass which catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond.
In this work, we have studied the substrate specificity of PLDs from germinating sunflower seeds and cultured-soybean cells, using their capacity of transphosphatidylation. In the presence of a nucleophilic acceptor, such as [14C]ethanol, PLD catalyzes the production of phosphatidyl-[14C]-ethanol. The resulting product is easily identified since it is well separated from the other lipids by thin-layer chromatography. The main advantage of this assay is that the phospholipid used as substrate does not need to be radiolabelled and thus allow us a large choice of polar heads and fatty acids. In vitro, we observed that sunflower and soybean cell PLD show the following decreasing order of specificity: phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol; while phosphatidylserine and phosphatidylinositol are utilized much less efficiently.
The substrate specificity is modulated by the fatty acid composition of the phosphatidylcholine used as well as by the presence of other charged phospholipids.
Phospholipase D (PLD) (phosphatidylcholine phosphatidohydrolase, [EC220.127.116.11]) is a ubiquitous enzyme present in mammals, plants and bacteria [1–3]. PLD catalyses two reactions (i) hydrolysis of phospholipids to produce phosphatidic acid (PA) and a free polar head group such as choline in the case of phosphatidylcholine (PC), and (ii) transphosphatidylation reaction which, in the presence of a primary alcohol, PLD leads to the formation of the corresponding phosphatidyl alcohol . The latter product is less subject than PA to further metabolism, and can easily be separated from the substrate and other hydrolytic products by performing thin-layer chromatography (TLC). Transphosphatidylation is a useful reaction synthesizing natural phospholipids, such as phosphatidylserine (PS) and phosphatidylglycerol (PG), and novel artificial phospholipids . These phospholipids have been used for pharmaceuticals, foods, cosmetics, and other industries. Transphosphatidylation is usually carried out in a biphasic system consisting of water and water-insoluble organic solvents. The reaction is usually accompanied with various amounts of the hydrolysis product PA [5, 6].
PLD was first identified in plants where an isoform called PLDα is widespread. The plant PLDα has been purified for the first time to homogeneity in 1993 in two different laboratories from the cabbage leaves  and castor bean endosperm . Twelve PLD isoforms grouped into five types (PLDα, PLDβ, PLDγ, PLDδ and PLDζ) could be identified in Arabidopsis thaliana . Subsequently, it has been described in mammals, bacteria and yeasts. PLD activity and PA levels increase rapidly in plant tissues under various stress conditions . Increasing studies indicate that PLD and PA are involved in regulating plant growth, development, and response to environmental and biotic stresses [5, 10–12]. The function of PLD and PA has been linked to the survival, proliferation, and reproduction of cells and organisms. Latest results indicate that specific PLD- and PA-mediated signalling play important roles in plant biomass production and response to water deficits and nutrient deficiency [13, 14].
PLDs from different sources share some characteristics in their molecular organization and constitute, together with some other evolutionary related proteins, the PLD superfamily . Mammalian PLD activities have been found to preferentially catalyze platelet-activating factor and PC phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PI-glycan [16–18].
Work from our laboratory have examined previously the fatty acid specificity of PLD purified from germinating sunflower seeds using wide range of PC compounds with various fatty acid contents . We have shown that sunflower PLD is most active on medium-chain fatty PC compounds. In general, PLDs hydrolyses a broad range of phospholipids with different head groups including PC, PE, PG, PS, PI, lyso-PC, cardiolipin, and plasmalogens with preferences depending on the enzyme source and isoform . Despite these informations, the study of substrate specificity of pure PLD using the transphosphatidylation reaction is limited. In order to systematically investigate the substrate specificity of purified PLD from germinating sunflower seeds and cultured-soybean cell, we used the transphosphatidylation reaction, with [14C]-ethanol as the acceptor. The resulting phosphatidyl-[14C]-ethanol could be easily identified by performing TLC, since it was clearly separated from the other compounds. During this reaction, the various phospholipids used do not need to be radiolabelled, so that a wide choice of possible phospholipids is available.
Materials and methods
Purification of sunflower and soybean PLDs
Acetone sunflower seeds powder was prepared from germinating sunflower seeds using the standards procedures developed at our laboratory [19–21]. The delipidated powder was extracted with 50 mM Tris/HCI, pH 7.5, and the purification was carried out using the procedure developed by Abousalham et al.  for use with the cabbage enzyme. Soybean PLD was purified from Soybean (Glycine max L.) suspension-culture cells as described previously by Abousalham et al. . Pure PLDs were aliquoted and stored at - 80°C until use.
Protein quantification and gel electrophoresis
Protein concentrations were determined routinely using the Bradford procedure  with Bio-Rad Dye Reagent and bovine serum albumin as the standard. Samples were separated by 12% sodium dodecyl sulfate (SDS) polyacrylamide gel (PAGE) as described by Laemmli . The apparent molecular masses of proteins were estimated by co-electrophoresis of marker proteins (Biorad, Hercules, CA, USA) with masses ranging from 14.4 to 116 kDa. The protein in sample buffer (0.9 g glycerol, 0.1 ml 1% bromo-phenol blue, 1 mL 10% (w/v) SDS, and 0.1 mL mercaptoethanol) was heated for 5 min in boiling water and applied to the gel. The proteins separated on the SDS-PAGE were stained with Coomassie Brilliant Blue R-250.
PLD activity was assayed spectrophotometrically by measuring the free choline released upon PC hydrolysis, using a continuous method  adapted for microplates (96 wells) from Takamara and Taylor . Choline was continuously transformed into betain by means of choline oxidase, which simultaneously yielded H2O2. In the presence of 4-aminoantipyrine and sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate, the H2O2 was used instantaneously by the added peroxidase to form a colored product absorbing light at 500 nm. Optical density measurements were performed using a microplate scanning spectrophotometer (PowerWave, Bio-Tek instruments). The egg PC substrate was prepared by dispersing egg PC in an equimolar mixture (0.83 mM) of SDS and Triton X-100. The assay mixture contained 50 mM Tris/HCl, pH 8.0, 20 mM CaCl2, 1.7 mM 4-aminoantipyrine, 9 mM sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate, 0.5 U choline oxidase and 0.5 U peroxidase. The reaction was initiated by adding PLD and the substrate (egg PC mixed micelles, 0.26 mM, final concentration) and absorbance measurements were carried out every 30 s for 5 min. The amounts of free choline released were quantified, based on a standard curve obtained with pure choline. One unit of PLD activity was defined as the amount of enzyme releasing 1 μmol of choline/min under the experimental conditions specified above.
TLC assay for transphosphatidylation activity
The reaction mixture (0.5 mL final volume) was composed of 0.26 mM phospholipid dispersed in an equimolar mixture of SDS and Triton X-100, 50 mM Tris-HCl (pH 8), 20 mM CaCl2 and 2% ethanol (final concentration). After incubating this mixture with the appropriate amount of enzyme for 10 min at 37°C, the phospholipids were extracted with 0.8 mL of chloroform-methanol (2:1, v/v) under vigorous shaking. Phase separation was facilitated by centrifugation for 10 min at 2, 000 rpm. The lower organic phase was collected and transferred to a 5-mL test tube, where it was dried over anhydrous magnesium sulfate (MgSO4). Once MgSO4 had precipitated, 0.4 ml of the clear organic phase was transferred to a 2-ml vial with a screw-cap and dried under a stream of nitrogen. The samples were dissolved in 40 μL of chloroform methanol (2:1) and applied along with PEt standard to aluminum TLC plates. Plates were developed in a solvent system consisting of chloroform/methanol/acetic acid (65:25:4 v/v/v). In this solvent system, PEt (Rf = 0.54) migrates faster than PA (Rf = 0.3) and the other phospholipid substrates (Rf = 0-0.2). The phospholipids were revealed by iodine vapour, and the radioactive spots corresponding to PEt were cut out from the plates and the radioactivity was quantitated by liquid scintillation count in a Beckman LS 5000 TD beta counter after the disappearance of the iodine vapour .
Results and discussion
One might have expected PLD, acting upon the polar heads of phospholipids, to be less dependent on the "quality" of the interface. Like many other lipolytic enzymes , PLD activity seems to depend however upon the physico-chemical properties characteristic of the lipid/water interface, in addition to the chemical nature of the phospholipid polar head group. In this context, research by Abousalham et al. , using phospholipid monomolecular films as a substrate, is of particular interest. These authors have shown that 12:0/12:0 PC are optimally hydrolyzed at a surface pressure of 10 mN/m. Another family of lipolytic enzymes, phospholipase C, also acting upon the polar heads of phospholipids, shows the same type of behavior. For instance Moreau et al.  have studied the hydrolysis of PC films by phospholipase C and the optimal surface pressure was around 15 mN/m, using 10:0/10:0 PC and 12:0/12:0 PC films as substrates. However, 10:0/10:0 PC was hydrolyzed at about twice the rate of 12:0/12:0 PC.
A detailed description of phospholipid specificity is necessary to be able to interpret the physiological action of PLD on natural membranes. Our results show clearly that the best substrate for plant PLD is the PC species and that the substrate specificity of these enzymes is also determined by the fatty acid composition and by the phospholipid environment.
- Abousalham A, Nari J, Noat G: Phospholipase D from plants: purification, enzymology and structural analysis. Current Topics in Plant Biology. 1999, 1: 123-132.Google Scholar
- Exton JH: Phospholipase D-structure, regulation and function. Review in Physiology, Biochemistry and Pharmacology. 2002, 144: 1-94.View ArticleGoogle Scholar
- Mansfeld J, Ulbrich-Hofmann R: Modulation of phospholipase D activity in vitro. Biochimica and Biophysica Acta. 2009, 1791: 913-926.View ArticleGoogle Scholar
- Kobayashi M, Kanfer JN: Phosphatidyl ethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D. Journal of Neurochemistry. 1987, 48: 1597-1603.View ArticlePubMedGoogle Scholar
- Li M, Hong Y, Wang X: Phospholipase D- and phosphatidic acid-mediated signaling in plants. Biochimica and Biophysica Acta. 2009, 1791: 927-935.View ArticleGoogle Scholar
- Uesugi Y, Hatanaka T: Phospholipase D mechanism using Streptomyces PLD. Biochimica and Biophysica Acta. 2009, 1791: 962-969.View ArticleGoogle Scholar
- Abousalham A, Riviere M, Teissere M, Verger R: Improved purification and biochemical characterization of phospholipase D from cabbage. Biochimica and Biophysica Acta. 1993, 1158: 1-7.View ArticleGoogle Scholar
- Wang X, Dyer JH, Zheng L: Purification and immunological analysis of phospholipase D from castor bean endosperm. Archives in Biochemistry Biophysics. 1993, 306: 486-494.View ArticleGoogle Scholar
- Wang X: Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions. Progress in Lipid Research. 2000, 39: 109-149.View ArticlePubMedGoogle Scholar
- Wang X: Regulatory functions of phospholipase D and phosphatidic acid in plant growth, development, and stress responses. Plant Physiology. 2005, 139: 566-573.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, Devaiah SP, Zhang W, Welti R: Signaling functions of phosphatidic acid. Progress in Lipid Research. 2006, 45: 250-278.View ArticlePubMedGoogle Scholar
- Xue H, Chen X, Li G: Involvement of phospholipid signaling in plant growth and hormone effects. Current Opinion in Plant Biology. 2007, 10: 483-489.View ArticlePubMedGoogle Scholar
- Hong Y, Devaiah SP, Bahn SC, Thamasandra BN, Li M, Welti R, Wang X: Phospholipase D epsilon and phosphatidic acid enhance Arabidopsis nitrogen signaling and growth. Plant Journal. 2009, 58: 376-387.PubMed CentralView ArticlePubMedGoogle Scholar
- Hong Y, Pan X, Welti R, Wang : Phospholipase Dalpha3 is involved in the hyperosmotic response in Arabidopsis. Plant Cell. 2008, 20: 803-816.PubMed CentralView ArticlePubMedGoogle Scholar
- Ponting CP, Kerr ID: A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. Protein Science. 1996, 5: 914-922.PubMed CentralView ArticlePubMedGoogle Scholar
- Kater LA, Goetzl EJ, Austen KF: Isolation of human eosinophil phospholipase D. Journal of Clinical Investigation. 1976, 57: 1173-1180.PubMed CentralView ArticlePubMedGoogle Scholar
- Panagia V, Ou C, Taira Y, Dai J, Dhalla NS: Phospholipase D activity in subcellular membranes of rat ventricular myocardium. Biochimica and Biophysica Acta. 1991, 1064: 242-250.View ArticleGoogle Scholar
- Scallon BJ, Fung W-JC, Tsang TC, Li S, Kado-Fong H, Huang K-S, Kochan JP: Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D. Science. 1991, 252: 446-448.View ArticlePubMedGoogle Scholar
- Abousalham A, Nari J, Teissere M, Ferte N, Noat G, Verger R: Study of fatty acid specificity of sunflower phospholipase D using detergent/phospholipid micelles. European Journal of Biochemistry. 1997, 248: 374-379.View ArticlePubMedGoogle Scholar
- Abdelkafi S, Fouquet B, Barouh N, Durner S, Pina M, Scheirlinckx F, Villeneuve P, Carrière F: In vitro comparisons between Carica papaya and pancreatic lipases during test meal lipolysis: potential use of CPL in enzyme replacement therapy. Food Chemistry. 2009, 115: 100-106.View ArticleGoogle Scholar
- Abdelkafi S, Abousalham A: Kinetic study of sunflower phospholipase Dα: interactions with micellar substrate, detergents and metals. Plant Physiology and Biochemistry. 2011, 49: 752-757.View ArticlePubMedGoogle Scholar
- Abousalham A, Teissere M, Gardies AM, Verger R, Noat G: Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties. Plant Cell Physiology. 1995, 36: 989-996.PubMedGoogle Scholar
- Bradford MM: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-deye binding. Analitycal Biochemistry. 1976, 72: 248-254.View ArticleGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.View ArticlePubMedGoogle Scholar
- Takrama JF, Taylor KE: A continuous spectrophotometric method for monitoting phospholipase D-catalyzed reactions of physiological substrates. Journal of Biochemical and Biophysical Methods. 1991, 23: 217-226.View ArticlePubMedGoogle Scholar
- Abdelkafi S, Ogata H, Barouh N, Fouquet B, Lebrun R, Pina M, Scheirlinckx F, Villeneuve P, Carriere F: Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex. Biochimica and Biophysica Acta. 2009, 1791: 1048-1056.View ArticleGoogle Scholar
- Moreno-Perez AJ, Martinez-Force E, Garces R, Salas JJ: Phospholipase Dalpha from sunflower (Helianthus annuus): cloning and functional characterization. Journal of Plant Physiology. 2010, 167: 503-511.View ArticlePubMedGoogle Scholar
- Allgyer TT, Wells MA: Phospholipase D from savoy cabbage: purification and preliminary kinetic characterization. Biochemistry. 1979, 18: 5348-5353.View ArticlePubMedGoogle Scholar
- Horwitz J, Davies LL: The substrat specificity of brain microsomal phospholipase D. Biochemical Journal. 1993, 295: 793-798.PubMed CentralView ArticlePubMedGoogle Scholar
- Heller M: Phospholipase D. Advance in Lipid Research. 1978, 16: 267-326.Google Scholar
- Pessin JE, Salter DW, Glaser M: Use of a fluorescent probe to compare the plasma membrane properties in normal and transformed cells. Evaluation of the interference by triacylglycerols and alkyldiacylglycerols. Biochemistry. 1978, 17: 1997-2004.View ArticlePubMedGoogle Scholar
- Bugaut M, Kuksis A, Myher JJ: Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1, 2-diacylglycero-3-phosphocholine. Biochimica and Biophysica Acta. 1985, 835: 304-314.View ArticleGoogle Scholar
- Shimbo K, Iwasaki Y, Yamane T, Ina K: Purification and properties of phospholipase D from Streptomyces antibioticus. Bioscience. Biotechnology and Biochemistry. 1993, 57: 1946-1948.View ArticleGoogle Scholar
- Verger R, de Haas GH: Interfacial enzyme kinetics of lipolysis. Annuar Review of Biophysics and Bioengineering. 1976, 5: 77-117.View ArticleGoogle Scholar
- Abousalham A, Ivanova M, Douchet I, Verger R: A new method for determining phospholipase D activity using the monomolecular film technique. Chemistry and Physics of Lipids. 1996, 79: 107-112.View ArticlePubMedGoogle Scholar
- Moreau H, Piéroni G, Jolivet-Reynaud C, Alouf JE, Verger R: A new kinetic approach for studying phospholipase C (Clostridium perfringens toxin) activity on phospholipid monolayers. Biochemistry. 1988, 27: 2319-2323.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.