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Biochimica et Biophysica Acta 1760 (2006) 1202 1209

http://www.elsevier.com/locate/bba

Purification and biochemical characterization of phospholipase A2


from dromedary pancreas
Abir Ben Bacha a , Youssef Gargouri a,, Sofiane Bezzine a , Hafedh Mejdoub a,b
a
Laboratoire de Biochimie et de Gnie Enzymatique des Lipases, ENIS route de Soukra, 3038 Sfax, Tunisia
b
USCR Squenceur de Protines, Facult des Sciences de Sfax, route de Soukra, 3038 Sfax, Tunisia
Received 23 December 2005; received in revised form 9 March 2006; accepted 13 March 2006
Available online 19 April 2006

Abstract

Dromedary pancreatic PLA2 (DrPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat and acidic treatment
(70 C; pH 3.0), precipitation by ammonium sulphate and ethanol respectively, followed by sequential column chromatographies on Sephadex G-50,
MonoS Sepharose, MonoQ Sepharose and C-8 reverse phase high pressure liquid chromatography. Purified DrPLA2, which is not glycosylated
protein, was found to be monomeric protein with a molecular mass of 13748.55 Da. A specific activity of 600 U/mg for purified DrPLA2 was
measured at optimal conditions (pH 8.0 and 37 C) in the presence of 3 mM NaTDC and 7 mM CaCl2 using PC as substrate. The sequence of the first
fourteen amino-acid residues at the N-terminal extremity of DrPLA2 was determined by automatic Edman degradation. One single sequence was
obtained and shows a close similarity with all other known pancreatic secreted phospholipases A2.
2006 Elsevier B.V. All rights reserved.

Keywords: Pancreatic phospholipase; PC; Bile salts; Ca2+; Sequence

1. Introduction the oxidized lipids, called PAF acetylhydrolases (PAF-AH)


[1,2].
Phospholipases A2 (PLA2, EC 3.1.1.4) catalyse specifically To date, there are 11 forms of mammal sPLA2 classified into
the cleavage of the fatty acid residue at the sn-2 position of groups IB, IIA, IIC, IID, IIE, IIF, V, X, III, XIIA and XIIB according
phospholipids that liberate free fatty acid and lysophospholipid. to their origin, sequence similarity and molecular mass as well as
Several new PLA2 have been identified. The diversity of these substrate specificity [3,4]. sPLA2 exhibit tissue and species specific
enzymes called for the reevaluation of PLA2 classification expression which suggests that their cellular behaviours and
scheme that had been used for many years [1]. PLA2 were functions are different. It is well established that some sPLA2
classified mainly into three groups: (i) cytosolic PLA2 (cPLA2); participate in a variety of pathological processes by releasing
(ii) Ca2+-independent intracellular PLA2 (iPLA2) and (iii) Ca2+- arachidonic acid from membrane phospholipids, leading to the
dependent secreted PLA2 (sPLA2). There is also a PLA2 production of various types of proinflammatory lipid mediators such
class which hydrolyses the platelet-activating factor (PAF) and as prostaglandins, thromboxanes and leukotrienes [5]. The sPLA2
do not manifest significant fatty acid selectivity in vitro. They are
Ca2+ dependant lipolytic enzymes with a conserved Ca2+ binding
Abbreviations: DrPLA2, dromedary pancreatic phospholipaseA2; sPLA2, loop and HisAsp dyad at the catalytic site [3,4]. Moreover, these
secreted pancreatic phospholipase A2; TPP, turkey pancreatic phospholipase;
PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; RP-HPLC, reverse
enzymes are low-molecular mass proteins (14 kDa) containing 6
phasehigh pressure liquid chromatography; AU, absorbance unit; DTT, 8 conserved disulfide bridges with a rigid tertiary structure, which
dithiothreitol; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene confers both stability against proteolysis and resistance to
glycol-bis (-aminoethylether) N,N,N,N-tetraacetic acid; BSA, bovine serum denaturation [1,2]. Only sPLA2-IB and X, have an N-terminal
albumine; NaDC, sodium deoxycholate; NaTDC, sodium taurodeoxycholate; prepropeptide and the proteolytic cleavage of this prepropeptide is
TX-100, triton X-100; PC, phosphatidylcholine; TC4, tributyrin; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis the regulatory step for the generation of an active enzyme [6,7].
Corresponding author. Tel.: +216 74274088; fax: +216 74275595. sPLA2-IB was found in large amounts in the pancreas and its
E-mail address: ytgargouri@yahoo.fr (Y. Gargouri). principal function is the digestion of dietary lipids [8]. This
0304-4165/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2006.03.014
A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209 1203

Table 1 In the present study, we report the purification to homoge-


Pancreatic phospholipase level in some animals neity of the active sPLA2 from the dromedary pancreas. This
Species U/g Fresh pancreas mammalian phospholipase A2 was characterized with respect to
Turkey 200 20 its biochemical properties.
Bovine 110 17
Chicken 70 25 2. Material and methods
Dromedary 20 3.5
Sheep 10 5 2.1. Materials
The determination of the phospholipase content of pancreases from all species
was performed in a homogenate prepared in a Waring Blendor (2 30 s) with Trypsin (treated with L-1-tosylamino 2-phenylethyl chloromethylketone),
10 ml, per gram of fresh tissue, of 10 mM TrisHCl, 150 mM NaCl, pH 8.5 at tributyrin (99%; puriss), benzamidine were from Fluka (Buchs, Switzerland),
room temperature. After centrifugation at 12.000 rpm during 20 min, the amount bovine serum albumine (BSA), sodium deoxycholate (NaDC), sodium
of enzyme was estimated on an aliquot of the supernatant using PC emulsion as taurodeoxycholate (NaTDC), triton X-100 (TX-100) were from Sigma
substrate in the presence of 3 mM NaTDC and 7 mM CaCl2. The phospholipase Chemical (St. Louis, USA); acrylamide and bis-acrylamide electrophoresis
activity was measured titrimetrically at pH 8.0 and at 37 C using a pH-stat. One grade were from BDH (Poole, UK), marker proteins and supports of
phospholipase unit corresponds to one mole of fatty acid released per min chromatography used for phospholipase purification: Sephadex G-50,
using PC emulsion as substrate in the presence of 3 mM NaTDC and 7 mM MonoS, MonoQ were from Pharmacia (Uppsala, Sweden); protein sequencer
CaCl2. For each species, the activity represents the average standard error Procise 492 equipped with 140 C HPLC system provided from Applied
mean of five assays obtained from three different pancreases. Biosystems (Roissy, France); C-8 reverse-phase eurospher 100 column
(250 mm 4.6 mm) from Knauer (Germany); pH-stat was from Metrohm
(Herisau, Switzerland).
enzyme is secreted as an inactive zymogen and it is activated
after pentapeptide bond cleavage of the proform by trypsin into 2.2. Pancreas collections
the mature form [8]. More recently, this enzyme was identified
and cloned in other tissues such as lung, spleen, kidney and Pancreases from dromedary were collected immediately after slaughter and
ovary [9,10], and it has now been proposed to be involved in kept at 20 C. Pancreases were collected from a local slaughterhouse (Sfax,
various physiological and pathophysiological responses such as Tunisia).
cell proliferation [11], cell contraction [12,13], lipid mediator
release [14], acute lung injury [15] and endotoxic shock [16]. 2.3. Delipidation of pancreases
sPLA2-IB have been isolated from pancreases of various After decongelation, pancreases were cut into small pieces (12 cm2) and
species [1721]. The activation mechanism [22] and amino acid delipidated according to the method described previously [30]. After
sequence of these enzymes [23,24] is known and extensive delipidation, about 15 g of delipidated powder of pancreas were obtained
studies on the mechanism of action of these enzymes have been from 100 g of fresh tissue.
reported [2528].
2.4. Determination of lipase activity
In our laboratory, some assays to purify the turkey PLA2
(TPP) were done. The pure enzyme on SDS-PAGE analysis was The lipase activity was measured titrimetrically at pH 8.5 and at 37 C
found totally inactive and the enzymatic properties were with a pH-stat, under the standard assay conditions described previously,
realized with the partially purified TPP [29]. using tributyrin (0.25 ml) in 30 ml of 2.5 mM TrisHCl, 5 mM CaCl2 pH 8.5

Fig. 1. (A) Chromatography of dromedary pancreatic PLA2 on Sephadex G-50. The column (2.6 cm 90 cm) was equilibrated with 10 mM acetate buffer pH 5.0
containing 2 mM benzamidine and 0.05% TX-100. The elution of proteins was performed with the same buffer at a rate of 50 ml/h and 5 ml by fraction. DrPLA2
activity was measured as described in Materials and methods using PC emulsion as substrate. The pooled fractions containing the PLA2 activity were indicated by a
line. (B) Chromatography dromedary pancreatic PLA2 on Mono S Sepharose. The column (2.6 cm 20 cm) was equilibrated with 10 mM acetate buffer pH 5.0
containing 2 mM benzamidine and 0.05% TX-100; a linear salt gradient (0 to 0.4 M NaCl) was applied to the column; gradient chamber 200 ml; 4.2 ml fraction; flow
rate, 50 ml/h. The pooled fractions containing the PLA2 activity were indicated by a line.
1204 A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209

[31] as substrate. One lipase unit corresponds to 1 mole of fatty acid 2.6. Determination of protein concentration
liberated per min.
Protein concentration was determined as described by Bradford et al. [32]
2.5. Determination of phospholipase activity using BSA (E1%1 cm = 6.7) as reference.

The PLA2 activity was measured titrimetrically at pH 8.0 and at 37 C with a


pH-stat, under the standard assay conditions described previously [1], using PC 2.7. Oligosaccharides content
or egg yolk emulsions as substrate in the presence of 3 mM NaTDC and 7 mM
CaCl2. Some assays were performed with NaDC. One unit of phospholipase The presence of glycan chains in the purified enzyme was checked by
activity was defined as 1 mole of fatty acid liberated under standard conditions. anthrone-sulfuric acid method using glucose as a standard [33].

Fig. 2. RP-HPLC and SDS-PAGE (15%) of DrPLA2. (A) RP-HPLC on a eurospher 100, C-8 column, elution was performed at room temperature within 30 min using
a gradient from 0 to 90% solvent B at a flow rate of 0.6 ml/min. [solvent A, water/trifluoroacetic acid (1000:1, v/v)] and solvent B, acetonitrile]. The effluent was
monitored at 220 nm. The gradient is indicated by the dotted line. (B) SDS-PAGE (15%) of pure DrPLA2 of RP-HPLC. lane a, 10 g of DrPLA2 eluted from RP-
HPLC. lane b, 15 g of DrPLA2 obtained after MonoQ chromatography. lane c, 20 g of DrPLA2 obtained after MonoS chromatography. Lane d, molecular mass
markers (Pharmacia). The gel was stained with Coomassie blue.
A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209 1205

2.8. Alkylation of Cys residues 3.2.2. Ammonium sulphate precipitation


The treated supernatant (500 ml, 1470 U) was brought to
The alkylation of Cys residues of phospholipase was realized as the technique of 75% saturation with solid ammonium sulphate under stirring
Okazaki [34]. 100 pmol of DrPLA2 in 1 ml of 10 mM TrisHCl, pH 8.2 were
denatured in 185 l of 8 M guanidine hydrochloride, 65 l of 1 M TrisHCl, 4 mM conditions and maintained during 45 min at 4 C. After
EDTA (pH 8.5) and 80 mM DTT during 30 min at 60 C. S-Pyridylethylation of centrifugation (30 min at 12.000 rpm), the precipitated PLA2
cysteine residues of protein was performed by adding 4 l of vinyl pyridine during 3 h was resuspended in 10 ml of buffer A containing 2 mM
at 25 C. The modified enzyme was dialysed against water for N-terminal sequencing. benzamidine. Insoluble material was removed by centrifugation
during 10 min at 12.000 rpm. Approximately 64% of the
2.9. Analytical methods starting amount of PLA2 was recovered.
Analytical polyacrylamide gel electrophoresis of proteins in the presence of
3.2.3. Ethanol fractionation
sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli
[35]. The proteins were stained with Coomassie brilliant blue. The molecular An equal volume of pure ethanol solution was added to the
mass of purified PLA2 was determined by MALDI-TOF (matrix assisted laser supernatant (11 ml, 1100 U) at 0 C. Precipitated proteins were
desorption ionization-time of flight). removed by centrifugation and the supernatant was added
slowly with four times its volume of ethanol to bring the alcohol
2.10. Amino acid sequencing concentration to 90% (v/v) at 0 C. After centrifugation for
30 min at 12.000 rpm the ethanol precipitated PLA2, which
The N-terminal sequence was determined by automated Edman's degrada- contains about 47% of the enzyme starting amount, was
tion, using an Applied Biosystems Protein Sequencer Procise 492 equipped with
140 C HPLC system [36].
solubilized in 10 mM acetate buffer pH 5.0 containing 0.05%
triton X-100 and 2 mM benzamidine (buffer B). The obtained
sample was subjected to chromatography purification.
3. Results and discussion
3.2.4. Filtration on Sephadex G-50
3.1. Level of expression of DrPLA2 activity The sample containing PLA2 (9 ml, 800 U) was filtered through
a column (95 cm 2.6 cm) of sephadex G-50 equilibrated with
In order to compare the level of dromedary PLA2 activity buffer B. Elution of proteins was performed with the same buffer at
with other species, the rate of hydrolysis of PC by bovine, 50 ml/h. The fractions containing the PLA2 activity eluted between
sheep, chicken, and turkey pancreases were measured under the 1.4 and 2 void volumes were pooled together (Fig. 1A).
same conditions. Results reported in Table 1 show that the
pancreases tested secreted various levels of PLA2 activity. The 3.2.5. Cation exchange chromatography
highest level was observed with turkey (200 U/g). Dromedary The pooled fractions containing PLA2 activity of Sephadex
presented only 20 U/g in its pancreas. G-50 column were applied to a MonoS column (2.6 cm 20 cm)
equilibrated with buffer B. Non fixed proteins were washed out
3.2. Purification of DrPLA2 with buffer B. The elution of the adsorbed proteins was then

48 g of delipidated dromedary pancreas were suspended in Table 2


600 ml of buffer A: 0.01 M TrisHCl, 0.15 mM NaCl, pH 8.5 Flow sheet of the DrPLA2 purification
and ground mechanically twice for 30 s at room temperature Purification step Total Total Specific Yield Purification
using the Waring Blendor system. Then, the mixture was stirred activity protein activity (%) factor
(U) a (mg) b (U/mg)
with a magnetic bar for 20 min at room temperature and
centrifuged during 30 min at 12.000 rpm. The total PLA2 Extraction (pH 8.5) 1720 6000 0.3 100 1
Heat and acidic 1470 2500 0.6 85 2
activity obtained (1720 U) did not increase when trypsin was
treatments (70 C and
added at different ratios to the phospholipase A2 solution. It can pH 3)
be concluded that the endogenous trypsin is sufficient to (NH4)2SO4 1100 600 1.83 64 6.1
achieve the PLA2 activation (data not shown). Precipitation
(3070%)
Ethanol 800 200 4 47 13.33
3.2.1. Heat and acidic treatment
Precipitation
As has been established for many pancreatic PLA2 [1722], (5090%)
dromedary PLA2 present in the homogenate can tolerate the Sephadex G-50 600 90 6.66 34 22.2
incubation at pH 3.0 and at 70 C. The previous PLA2 solution MonoS 500 20 25 29.07 83.33
was incubated 3 min at 70 C. After rapid cooling, insoluble Sepharose
MonoQ 350 1.2 291.6 20.35 972
denatured proteins were removed by centrifugation during
Sepharose
30 min at 12.000 rpm. Afterwards the pH of the supernatant was RP-HPLC 200 0.33 600 11.63 2000
brought to 3.0 by adding 6N HCl under gentle stirring at 0 C. a
1 Unit: mole of fatty acid released per min using PC emulsion as substrate
After centrifugation (30 min at 12.000 rpm), the clear in the presence of 3 mM NaTDC and in the presence of 7 mM CaCl2.
supernatant was adjusted to pH 7.0 with 3 N NaOH. The b
Proteins were estimated by Bradford method [32]. The experiments were
recovery of PLA2 activity was of about 85%. conducted three times.
1206 A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209

pooled and lyophilized. At this stage of purification, the enzyme


presented a specific activity of 290 U/mg. We obtained 350 UT of
phopholipase A2 with a recovery of 70% at this stage which was not
the case of the TPP partially purified in our laboratory. In deed, a
very poor yield was obtained during the elution of the PLA2 enzyme
from DEAE cellulose. This difference was explained by the fact that
TPP strongly interacts with the chromatography supports [29].

3.2.7. RP-HPLC C-8 column


Twenty units of lyophilized sample from Mono-Q column
were applied to RP-HPLC eurospher 100, C-8 column
(250 mm 4.6 mm). PLA2 activity was detected in a fraction
eluted at 65% acetonitrile as a single peak (Fig. 2A) and the
overall recovery of the enzyme activity was 11.63% of the
starting amount. In contrast to TPP, the purified DrPLA2 was
found to be stable in the presence of the hydrophobic solvent.
The purification flow sheet is given in Table 2 and shows that
Fig. 3. Effect of the concentration of Ca2+ on DrPLA2 activity. Enzyme activity the specific activity of pure dromedary PLA2 reached 600 U/mg
was measured at various concentrations of Ca2+ using PC emulsion as substrate using PC emulsion as substrate at pH 8.0 and at 37 C in the
at pH 8.0 and at 37 C in the presence of 3 mM NaTDC. The star indicates the presence 3 mM NaTDC and 7 mM CaCl2.
phospholipase activity measured in the absence of CaCl2 and in the presence of The results of SDS-PAGE analysis of the purified DrPLA2
10 mM EDTA or EGTA.
are given in Fig. 2B. As can be seen, the purified DrPLA2
exhibited one band corresponding to an apparent molecular
performed with a linear gradient of NaCl (0 to 0.4 M). As shown mass of about 14 kDa. Mass spectrometry analysis indicated
in the elution diagram, dromedary PLA2 activity emerged in a that DrPLA2 has a molecular mass of 13748.55 Da (data not
single peak (Fig. 1B) at 0.15 M NaCl. The fractions of this peak shown). Pure PLA2 was lyophilized and conserved at 20 C.
were pooled and dialysed against 10 mM TrisHCl buffer pH Glycan chains in pure PLA2, analyzed as described in
8.5 containing 10 mM NaCl and 2 mM benzamidine (buffer C). Materials and methods, indicate that the DrPLA2 is not a
The recovery of PLA2 from MonoS column was of about 30% glycosylated molecule (data not shown). Similar result was
of the starting amount of the enzyme. obtained with all purified pancreatic PLA2 .

3.2.6. MonoQ chromatography 3.3. Enzymatic properties of the purified DrPLA2


A Mono-Q column (1.5 cm 10 cm) was prepared and
equilibrated with buffer C. The dialysed sample from MonoS 3.3.1. Ca2+ dependence
purification was poured into this column and non fixed proteins It is well established that Ca 2+ is essential for both, catalysis and
were removed with the equilibrium buffer. Then, linear salt gradient enzyme binding to the substrate [37,38]. In order to investigate the
with a total volume of 150 ml, 0 to 0.25 M NaCl was applied. effect of Ca2+ on DrPLA2 activity, we studied the variation of
Fractions containing DrPLA2 activity were eluted at 0.1 M NaCl, hydrolysis rates of PC emulsion by pure DrPLA2 in presence of

Fig. 4. Effect of increasing concentration of bile salts: NaTDC (A) and NaDC (B) on DrPLA2 activity. PLA2 activity was measured using PC emulsion as substrate at
pH 8.0 and at 37 C in the presence of 7 mM Ca2+.
A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209 1207

Fig. 5. Effects of pH (A) and temperature (B) on DrPLA2 activity. The enzyme activity was tested at various pHs and different temperatures using PC emulsion as
substrate in the presence of 7 mM Ca2+ and 3 mM NaTDC.

various Ca2+ concentrations. Our results showed that no PLA2 emulsion as a substrate in the presence of 7 mM Ca2+ and 3 mM
activity can be detected in the absence of Ca2+ and in the presence NaTDC. The pH-optimum for dromedary PLA2 activity was
of 10 mM EDTA or EGTA when using PC or egg yolk emulsion as similar to those of PLA2 described so far [1721,36]. The
substrate. In the absence of chelators, the specific activity of purified enzyme was found to be stable between pH 3.0 and 9.0.
purified PLA2 increases to reach 600 U/mg at 7 mM CaCl2 (Fig. 3). In contrast to TPP, pure DrPLA2 maintained about 75% of its
Similar results were obtained with other pancreatic PLA2 like activity after 5 min incubation at 70 C (data not shown).
porcine, bovine, turkey and horse [21,36,39,40]. However, TPP was found to lose its full activity when incubated
at pH lesser than 5. Comparable results were obtained using egg
3.3.2. Bile salts dependence yolk emulsion as substrate in the presence of 7 mM Ca2+ and
Several studies have provided evidence that the bile salts are 3 mM NaTDC.
tensioactive agents ensuring in micellar form, the dispersion of
the products of hydrolysis [21,41]. In the same direction, De 3.3.4. N-terminal sequence of DrPLA2
Haas et al., reported that micellar forms of the substrate were Purified DrPLA2 was alkylated as described in Material and
hydrolysed at a much higher rate than substrates molecularly methods and dialysed against distilled water. The NH2-terminal
dispersed by PLA2 [39]. In this study, we measured the sequencing of the DrPLA2 allowed unambiguously the
DrPLA2 activity at pH 8.0 and at 37 C using PC emulsion as a identification of the first fourteen residues of pure enzyme.
substrate in the presence of increasing concentrations of bile Table 3 shows the N-terminal sequence of DrPLA2, compared
salts. As shown in Fig. 4, sodium taurodeoxycholate (NaTDC) to those of bovine, pig, horse, human and turkey. Pure DrPLA2
(Fig. 4A) and sodium deoxycholate NaDC (Fig. 4B) were exhibits a high degree of homology with both mammalian and
specifically required for DrPLA2 activity. The maximal avian pancreatic PLA2.
phospholipase activity was observed in the presence of 3 mM
NaTDC or 6 mM NaDC. These observations corroborate with 4. Conclusion
previous findings with porcine pancreatic PLA2 [39].
DrPLA2 was purified to homogeneity from delipidated
3.3.3. Effects of pH and temperature on DrPLA2 activity pancreases. The pure enzyme was not a glycosylated protein
As shown in Fig. 5 the maximal activity of DrPLA2 was with a molecular mass of 13748.55 Da. The maximal PLA2
measured at pH 8.0 (Fig. 5A) and at 37 C (Fig. 5B) using PC activity was at pH 8.0 and at 37 C. Pure dromedary pancreatic

Table 3
Alignment of the N-terminal sequence of DrPLA2 with bovine, pig, horse, human and turkey pancreatic phospholipases
1 6 12 18 24 30 36
DrPLA2 :ALWQFR DMIKCK IPDSSP LLDFNN YGCYCG LGGSST PVDE this study
Bovine :ALWQFN GMIKCK IPSSEP LLDFNN YGCYCG LGGSGT PVDD [42]
Pig :ALWQFR SMIKCT IPGSDP LLDFNN YGCYCG LGGSGT PVDE [42]
Horse :AVWQFR SMIQCT IPNSKP YLEFND YGCYCG LGGSGT PVDE [42]
Human :AVWQFR KMIKCV IPGSDP FLEYNN YGCYCG LGGSGT PVDE [43]
Turkey :ALFEFR SMIKCT IPGSDP ELD [29]
For comparison, bold, plain, and italic symbols of residues indicate identical, homologous, and different amino acids, respectively.
1208 A.B. Bacha et al. / Biochimica et Biophysica Acta 1760 (2006) 12021209

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