Secretory phospholipase A2-α from Arabidopsis thaliana: functional parameters and substrate preference

Article abstract of DOI:10.1016/j.chemphyslip.2007.07.001

The secretory phospholipase A₂-α from Arabidopsis thaliana (AtsPLA₂-α), being one of the first plant sPLA₂s obtained in purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of pH, temperature, and calcium concentration were similar to the parameters of secretory PLA₂s from animals. However, substrate preferences markedly differed. In contrast to pancreatic PLA₂s, AtsPLA₂-α preferred zwitterionic phospholipids, and showed lower activity toward anionic phospholipids. In substrates with two identical fatty acid chains, AtsPLA₂-α showed optimum activity toward phospholipids with decanoyl groups. In substrates with palmitoyl groups in sn-1 position, acyl chains with higher degree of unsaturation in sn-2 position were preferred, excluding arachidonic acid, showing the evolutionary adaptation of the enzyme to substrate composition in plants. K_m values for short chain phospholipids were comparable to sPLA₂s from animals, whereas k_cat values were much smaller and interfacial activation was less important.

Full text of DOI:10.1016/j.chemphyslip.2007.07.001

Available online at www.sciencedirect.com
Chemistry and Physics of Lipids 150 (2007) 156–166
Secretory phospholipase A -␣ from Arabidopsis thaliana:
2
functional parameters and substrate preference
∗
Johanna Mansfeld , Renate Ulbrich-Hofmann
Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, D-06120 Halle, Germany
Received 4 April 2007; received in revised form 3 July 2007; accepted 4 July 2007
Available online 10 July 2007
Abstract
The secretory phospholipase A
2
2 2
-␣ from Arabidopsis thaliana (AtsPLA -␣), being one of the first plant sPLA s obtained in
purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of
pH, temperature, and calcium concentration were similar to the parameters of secretory PLA s from animals. However, substrate
-␣ preferred zwitterionic phospholipids, and showed lower
2
preferences markedly differed. In contrast to pancreatic PLA
activity toward anionic phospholipids.
2 2
s, AtsPLA
In substrates with two identical fatty acid chains, AtsPLA
groups. In substrates with palmitoyl groups in sn-1 position, acyl chains with higher degree of unsaturation in sn-2 position were
preferred, excluding arachidonic acid, showing the evolutionary adaptation of the enzyme to substrate composition in plants. K
2
-␣ showed optimum activity toward phospholipids with decanoyl
m
values for short chain phospholipids were comparable to sPLA
activation was less important.
2
s from animals, whereas kcat values were much smaller and interfacial
©
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Secretory phospholipase A2; Arabidopsis thaliana; Substrate preference; Interfacial activation; Short chain phospholipids
1. Introduction
Phospholipases A2 (PLA2s; EC 3.1.1.4) hydrolyse
Abbreviations: AtsPLA2-␣, secretory phospholipase A2-␣ from
Arabidopsis thaliana; Bis-Tris-propane, 1,3-Bis[tris(hydroxymethyl)
methylamino]propane; Caps, 3-[cyclohexylamino]-1-propanesulfonic
acid; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC,
stereospecifically the 2-acyl ester bond of 1,2-diacyl-
sn-3-phosphoglycerides and generate fatty acids and
lysophospholipids. The released fatty acid, in ani-
mals most notably arachidonic acid (Burgoyne and
Morgan, 1990), plays an important role in inflamma-
tion processes as a precursor of prostaglandins and
leukotrienes (Soloff et al., 2000; Wijewickrama et
al., 2006). The corresponding lysophospholipids are
involved in the synthesis of platelet-activating factor
and other physiologically important lipid mediators,
such as lysophosphatidic acid which is a com-
pound with potent mitogenic activity (Gardell et al.,
2006).
1
,2-dipalmitoyl-sn-glycero-3-phosphocholine; PA, phosphatidic
acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine;
PG, phosphatidylglycerol; PI, 1,2-diacyl-sn-glycero-3-phospho-
(
1-d-myo-inositol); PLA2, phospholipase A2; POPC, 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-
sn-glycero-3-phospho-rac-(1-glycerol); PS, phosphatidylserine;
sPLA2, secretory phospholipase A2
∗
Corresponding author at: Institute of Biochemistry and Biotech-
nology, Kurt-Mothes-Straße 3, D-06120 Halle, Germany.
Tel.: +49 345 5524865; fax: +49 345 5527303.
E-mail address: mansfeld@biochemtech.uni-halle.de
(
J. Mansfeld).
0
009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2007.07.001

J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
157
In plant cells, the release of linolenic acid proba-
2. Materials and methods
bly plays a similar role as arachidonic acid in animal
cells. Linolenic acid is assumed to be the precursor of
jasmonic acid which is a potent and multifunctional
growth regulator (Ishiguro et al., 2001) that modu-
lates anther dehiscence, fruit ripening, root growth,
tendril coiling, and plant resistance to insects and
pathogens. Whereas the enzymes involved in jasmonic
acid biosynthesis downstream of linolenic acid are well
known, there is a great number of open questions
concerning the source of linolenic acid in the plant
cell.
Secretory PLA2s (sPLA2s)are one ofthe main classes
within the PLA2 superfamily and some of their mem-
bers are very well characterised. Important common
structural features of the small sPLA2s are two well-
conserved central ␣-helices with the catalytic His–Asp
dyad and a hydrogen-bonding network connecting the
interfacial binding site, the catalytic and the calcium-
binding sites (Six and Dennis, 2000). sPLA2s contain
Guanidine hydrochloride was from ICN (Eschwege,
Germany). 1,2-Dioleoyl-sn-glycero-3-phosphocholine
(DOPC), phosphatidic acid (PA) from soybean,
and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-
glycerol) (POPG) were a gift of Lipoid GmbH (Lud-
wigshafen, Germany). 1-Palmitoyl-2-arachidonoyl-sn-
glycero-3-phosphocholine was purchased from Avanti
Polar Lipids (Alabaster, USA). All other phospholipids,
1,4-dithiothreitol (DTT), the Bradford protein assay
reagent, and all buffer substances were from Sigma
(Taufkirchen, Germany). The BCA protein assay kit was
from Pierce (Bonn, Germany). The NEFA C Kit was
obtained from WAKO Chemicals (Neuss, Germany).
The HiPrep 26/10 Desalting, MonoQ columns, and
the low molecular weight marker kit were supplied by
Amersham Biosciences (Freiburg, Germany). The SP
250/10 NUCLEOSIL 500-5 C3 PPN column was pur-
chased from Macherey & Nagel, D u¨ ren, Germany. All
other chemicals used were of the highest quality com-
mercially available.
®
®
5
–8 disulphide bonds and are highly stable. sPLA2s
are widespread in nature and have so far been mainly
found in the venoms of reptiles and insects, pancre-
atic juices and other extracellular fluids. Since recently,
a steadily increasing number of sPLA2s have been
shown to be present in invertebrate animals, fungi, and
plants.
2.1. Expression and isolation of the protein
Expression in BL21(DE3) cells containing the
AtsPLA -␣ gene in the vector pET-26b(+) was per-
2
In comparison to animal sPLA2s, the knowledge on
sPLA2s from plants is still limited, even though within
the last years some heterologously expressed plant
enzymes have been characterised. The plant sPLA2s
belong to groups XIA and XIB (Six and Dennis, 2000;
Mansfeld et al., 2006) which differ significantly in their
primary structures and the requirement of Ca2+ ions.
Despite the identity to sPLA2s from animal sources
of about 15% only, the known plant sPLA2s show a
significant degree of similarity with animal sPLA2s in
the active site and the calcium-binding loop regions.
The plant sPLA2s contain 12 cysteine residues being
potentially able to form 6 disulphide bridges. Computer-
modelling studies on the isoform sPLA2-␣ from A.
thaliana (AtsPLA2-␣) (accession number AY136317,
Mansfeld et al., 2006) provided the prediction of at least
four disulphide bridges.
In this paper, AtsPLA2-␣ (group XIB) which was
produced by heterologous expression in E. coli and rena-
tured from inclusion bodies (Mansfeld et al., 2006) has
beenstudiedwithrespecttosubstratepreferencesinclud-
ing different physical states of the substrates, calcium ion
requirement, pH and temperature optima. The results are
discussed in comparison with the properties of sPLA2s
from animal sources.
formed as described in Mansfeld et al. (2006). After cell
disruption, inclusion bodies were collected, washed and
subsequently solubilised in 100 mM Tris/HCl buffer, pH
8.3, containing 6 M guanidine hydrochloride, 100 mM
DTT, and 1 mM EDTA. After renaturation, the enzyme
®
was applied to a HiPrep 26/10 Desalting column, and
subsequently purified by anion-exchange chromatogra-
®
phy on Mono Q HR5.5 column. The protein was eluted
with a linear NaCl gradient (0–0.5 M) in 50 mM Tris/HCl
buffer, pH 8.5, 10 mM CaCl . Alternatively, the enzyme
2
was purified by reversed-phase chromatography on a SP
250/10 NUCLEOSIL 500-5 C PPN column (Solvent
3
A: 0.1% (v/v) trifluoroacetic acid in H O, 5% acetoni-
2
trile (v/v); solvent B: 0.1% (v/v) trifluoroacetic acid,
70% (v/v) acetonitrile). Peak fractions were assayed for
activity, and purity was proven by SDS-PAGE and mass
spectrometry using a Bruker REFLEX mass spectrom-
eter (Bruker, Daltonik, Bremen, Germany), upgraded
with a gridless delayed extraction ion source (pulsed ion
extraction).
2.2. Protein determination
Protein concentrations were determined by the Brad-
ford (solubilised inclusion bodies) or the BCA protein

1
58
J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
assay (purified protein) with bovine serum albumine as
standard according to the instructions of the suppliers.
Cell ultrasonic disintegrator (Avantec, France) with a
microtip probe of 3 mm. Final concentration of phos-
pholipids in the assay was 13 mM. Assay conditions and
determinationofinitialratesofthefattyacidreleasewere
as described above.
2
.3. Activity measurements
PLA2 activities were routinely determined with
For the preparation of micelles of short chain PCs
DOPC/SDS/Triton X-100 mixed micelles as substrate
in adaptation to the method described by Kasurinen and
Vanha-Perttula (1987). DOPC (10 mg, 12.7 ␮mol), dis-
solved in 200 ␮L of chloroform and 100 ␮L of methanol,
was dried in vacuum. The lipid film was dissolved in
(C –C8), the corresponding lipid film was dissolved
in 50 mM Tris/HCl buffer, pH 8.5, containing 10 mM
CaCl2.
6
All experiments were run in duplicate or triplicate.
6
4
00 ␮L of buffer containing 0.5 M Tris/HCl, pH 8.5,
2 mM Triton X-100, 8 mM SDS, and 42 mM CaCl2
2.4. Determination of kinetic constants
by vortexing for 15 min. The enzyme (0.1 nmol) dis-
solved in 0.1 M Tris/HCl buffer, pH 8.5, was incubated
for 5–30 min at 37 C with 1.27 ␮mol of DOPC in a
For the determination of the kinetic constants, ini-
tial reaction rates (v0) were determined as described
above at substrate concentrations between 1 and 40 mM
and expressed as ␮mol of liberated fatty acid per
minute and mg protein. v0 was plotted versus substrate
◦
total volume of 100 ␮L. Aliquots (10 ␮L) were removed
after defined times of incubation, and the reaction was
stopped by addition of 10 ␮L of 0.2 M EDTA. Initial
rates were determined from the increase of released fatty
acids using the NEFA C Kit according to Hoffmann et
al. (1986). Enzyme activities were linearly dependent on
enzyme concentration up to 4 ␮M.
For the preparation of mixed micelles of substrates
and Triton X-100 without SDS, the lipid film was dis-
solved in 600 ␮L of buffer containing 0.5 M Tris/HCl,
pH 8.5, 84 mM Triton X-100, and 42 mM CaCl2 by vor-
texing for 15 min.
For the determination of head group and acyl chain
preferences, the corresponding phospholipids were used
instead of DOPC. The use of mixed micelles of phospho-
lipids with Triton X-100 and SDS or solely Triton X-100
is indicated in the text and the corresponding tables. All
experiments were run in duplicate or triplicate.
To vary the molar ratio of Triton X-100 to phospho-
lipid from 2:1, 4:1, and 16:1, the corresponding amounts
of the phospholipid and Triton X-100 stock solutions
were used for the preparation of the mixed micelles. In
all cases, the phospholipid concentration in the assay was
varied between 0 and 20 mM.
app
app
concentration, and KM and Vmax were obtained by non-
linear regression using the Michaelis–Menten function
in Sigma-Plot 8.0. The standard deviations of the derived
kinetic constants were ≤15%.
The kinetic constants for 1,2-dihexanoyl-sn-glycero-
3
-phosphocholine were determined according to Berg
and Jain (2002) assuming that above the critical
micelle concentration (CMC) the bulk concentration of
phospholipid present as micelles ([S*] = [ST] − CMC)
determines the concentration of enzyme bound to the
interface. [ST] represents the total substrate concentra-
tion.
2
.5. Inactivation by p-bromophenacylbromide
The enzyme (6.5 ␮M) was incubated with 2 mM p-
◦
bromophenacylbromide at 25 C in 0.05 M Tris/HCl
buffer, pH 8.5, in the absence or presence of 10 mM
CaCl2. Aliquots of the reaction mixture were with-
drawn at appropriate time intervals and immediately
assayed for activity using the routine assay described
above.
For the preparation of small unilamellar vesicles
(
SUVs) of phosphatidylcholine (PC) from soybean
or DOPC containing different amounts of other
phospholipids, stock solutions of the corresponding
phospholipids (42.3 mM) were prepared in chloroform
or chloroform/methanol (2:1, v/v), respectively. Varying
mol% of other phospholipids in PC were obtained by
using appropriate amounts of the corresponding stock
solutions. Then, the organic solvent was removed by
vacuum. The lipid film was subsequently dissolved in
2.6. Determination of temperature optimum
For the determination of the temperature optimum,
the enzyme (1 ␮M) was incubated with DOPC in mixed
micelles with SDS and Triton X-100, prepared as
◦
described above, at temperatures between 20 and 90 C
for 6 min in 50 mM Tris/HCl buffer, pH 8.5, 10 mM
CaCl2. Product formation was quantified by the NEFA C
kit. Activity is expressed as the amount of released fatty
acid per minute and mg protein.
6
00 ␮L of 50 mM Tris/HCl buffer, pH 8.5, by ultra-
−
1
sonic treatment with 300 Ws mL using a 60 W Vibra

J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
159
2
.7. Determination of pH optimum
For the determination of the pH optimum the follow-
ing buffers were used for the preparation of DOPC in
mixed micelles with SDS and Triton X-100 and dilution
of enzyme solution instead of the usual buffer: 75 mM
acetate buffer (pH 4.5–5.5), 75 mM Mes buffer (pH
5
7
.5–6.5), 75 mM Bis–Tris–propane buffer (pH 6.5–9.5),
5 mM Tris/HCl buffer (pH 7.0–8.5), 75 mM Caps buffer
(
pH 9.5–11). The enzyme (1 ␮M) was incubated with
the substrate in the corresponding buffer and assayed as
described above.
3
. Results
3
.1. Production of AtsPLA2-α
Recombinant AtsPLA2-␣ was obtained by expression
in E. coli, renaturation from inclusion bodies and purifi-
cation by anion-exchange chromatography as described
in Mansfeld et al. (2006). The enzyme could also be
purified by reversed-phase chromatography on a C3 col-
umn without great loss of activity. The enzyme was
homogeneous in SDS-PAGE (Fig. 1) and showed the
correct mass (14,299.6 kDa, expected 14,299.9 kDa) in
mass spectrometry.
Fig. 1. SDS-PAGE analysis of purified AtsPLA2-␣. Lane 1 shows the
molecular weight marker and lane 2 the purified enzyme.
the presence of calcium ions. The fractional ampli-
tudes for the two phases were 0.52 and 0.48 in the
presence of CaCl2; 0.49 and 0.52 in the absence of
CaCl2.
3
.2. Temperature optimum
3.5. Activity of AtsPLA2-α toward phospholipids
The temperature optimum of AtsPLA2-␣, determined
with different head groups
with mixed micelles of DOPC, Triton X-100, and SDS
◦
as substrate, was between 30 and 40 C (Fig. 2A).
As depicted in Table 1, zwitterionic PC with oleoyl
groups in sn-1 and sn-2 position, DOPC, is cleaved
with approximately the same rate as the correspond-
ing phosphatidylethanolamine (PE) when the substrate
was prepared as mixed micelles under standard assay
conditions. The corresponding anionic 1,2-dioleoyl-
sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), was
3
.3. pH optimum
AtsPLA2-␣ has no activity toward mixed micelles of
DOPC, Triton X-100, and SDS in the pH range below
.5 in the presence of 10 mM CaCl2. The pH optimum
is between pH 8.5 and 9 (Fig. 2B).
6
converted with a 40.7% lower activity. AtsPLA -␣ did
2
not accept phosphatidylserine (PS) and PA as sub-
strate but it hydrolysed phosphatidylinositol (PI), with
3
.4. The effect of Ca2+ ions
1
7.2% activity in relation to DOPC. The absence of
2
+
AtsPLA2-␣ requires millimolar amounts of Ca ions
SDS in the mixed micelles did not change activities
significantly (not shown) and also the variation of the
molar ratio of Triton X-100 to phospholipid from 2:1
to 4:1 or 16:1 did not modify the observed trends.
Selected analyses according to the concept of surface
dilution kinetics (Carman et al., 1995) showed that nei-
ther Vmax nor KM(app) are dramatically changed by the
Triton X-100 content. Thus, Vmax for DOPC was 26.7,
and is not active in the absence of Ca2 ions. Enzyme
activity increases up to 10 mM CaCl2 and reaches sat-
uration at this concentration (Fig. 3A). The KD value
was determined to be 0.42 ± 0.06 mM. Calcium ions
+
(
2
10 mM) protect the enzyme partially from alkylation by
mM p-bromophenacylbromide (Fig. 3B). The inactiva-
tionprogresscurvescanbefittedbyadouble-exponential
decay function with half-lives of <1 and 142 min in
the absence of calcium ions and 41 and 827 min in
−
1
−1
in mixed micelles
29.8, and 27.7 ␮mol min mg
with Triton X-100/DOPC ratios of 2:1, 4:1, and 16:1,

1
60
J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
Fig. 3. (A) Hydrolytic activity of AtsPLA2-␣ as a function of Ca2+
ion concentration. The enzyme was incubated with mixed micelles
of 12.7 mM DOPC/25 mM Triton X-100 and 4.8 mM SDS in 50 mM
Tris/HCl buffer, pH 8.5, containing varying amounts of CaCl2 as
described in Section 2. The curve was fitted by a hyperbolic function.
(
B) Inactivation of AtsPLA2-␣ by 2 mM p-bromophenacylbromide [in
Fig. 2. (A) Temperature optimum of AtsPLA2-␣. The enzyme was
incubated with mixed micelles of 12.7 mM DOPC/25 mM Triton X-
the absence (ꢀ) and presence of 10 mM CaCl2 (᭹)]. The enzyme was
incubated with 2 mM p-bromophenacylbromide in 50 mM Tris/HCl
buffer, pH 8.5 [without or with 10 mM CaCl2]. Residual activities were
determined using the routine activity assay. The curves were fitted by
a double-exponential decay function.
1
00 and 4.8 mM SDS in 0.3 M Tris/HCl buffer, pH 8.5, 10 mM CaCl2,
and the amount of released product was determined as described in
Section 2. Initial rates were determined and related to the maximum
◦
−1
−1
activity measured at 40 C (17.1 ␮mol min mg ). (B) pH optimum
of AtsPLA2-␣. The enzyme was incubated with mixed micelles of
1
2.7 mM DOPC/25 mM Triton X-100 and 4.8 mM SDS in the corre-
◦
These values showed the same trends concerning the
head group preferences as the results obtained with
mixed micelles. Thus, oleic acid was released from
phosphatidylglycerol with 58% of the activity toward
PC. The pure anionic PS could also not be converted
when used as SUVs, and PA was not attacked, too.
Surprisingly, however, the use of substrates contain-
ing an equimolar mixture of PC and PS resulted in an
increased activity (1.3-fold) compared to PC alone. The
sponding buffer, containing 10 mM CaCl2, at 37 C and the amount
of released product was determined as described in Section 2. Initial
rates were determined and related to the maximum activity measured
−1
−1
at pH 8.5 (16.7 ␮mol min mg ).
app
while the corresponding KM values were 5.4, 5.7, and
3
.1 mM.
Activities toward phospholipids with different head
groups were also determined in detergent-free SUVs.

J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
161
Table 1
Specific activities of AtsPLA2-␣ toward glycerophospholipids with
different head groups
−
1
−1
)
Substrate
Activity (␮mol min mg
1
1
1
1
1
,2-Dioleoyl-sn-glycero-3-
phosphocholine
16.7 ± 1.1
,2-Dioleoyl-sn-glycero-3-
phosphoethanolaminea
,2-Dioleoyl-sn-glycero-3-phospho-
rac-(1-glycerol)
,2-Dioleoyl-sn-glycero-3-
phosphoserine
,2-Diacyl-sn-glycero-3-
phosphocholine from
soybean
15.3 ± 1.6
9.9 ± 0.5
0.0 ± 0.2
11.6 ± 1.4
1
1
,2-Diacyl-sn-glycero-3-phospho-(1-
d-myo-inositol) from
soybean
,2-Diacyl-sn-glycero-3-phosphate
from soybean
2.0 ± 0.3
0.0 ± 0.2
Activities were determined in 0.3 M Tris/HCl, pH 8.5, 10 mM CaCl2,
using mixed micelles of the corresponding phospholipid (12.7 mM)
with 25 mM Triton X-100 and 4.8 mM SDS as described in Section
2. The standard deviation was determined from at least 3 independent
assays.
a
Triton X-100 concentration was 50 mM due to poor solubility of
the substrate.
activity increase observed with equimolar amounts of
PC and PG in the SUVs was less pronounced (1.1-
fold), whereas mixing of PC and PE had no effect in
comparison to the pure phospholipids. Because of the
activating effect of the anionic PS on PC hydrolysis,
also the anionic PA was included into the measure-
ments. In Fig. 4, the activation effects of PG, being
itself a good substrate, and PA, being no substrate
of AtsPLA2␣, are compared as function of their per-
centages in PC-SUVs. In both cases, reaction rates
were increased considerably up to a maximum mole-
percentage of 12.5%. At higher fractions of PG or PA,
activities decreased again. In all cases, the reaction
progress curves for the hydrolysis of PC showed no
latency phase.
Fig. 4. Influence of anionic phospholipids on activity of AtsPLA2-
␣
toward PC in form of SUVs. (A) Influence of PG percentage in
substrate preparation. Varying mol% of PG and PC were obtained by
using appropriate amounts of the corresponding stock solutions. The
phospholipid concentration in the assay was 12.7 mM. Activities were
determined as described in Section 2. (B) Influence of PA percent-
age. Varying mol% of PA and PC were obtained by using appropriate
amounts of the corresponding stock solutions. The phospholipid con-
centration in the assay was 12.7 mM. Activities were determined as
described in Section 2.
3
.6. Activity of AtsPLA2-α toward
phosphatidylcholine with different acyl chains
in sn-2 position fatty acid residues with the sequence
linoleoyl > palmitoyl > oleoyl (Table 2). PC with arachi-
donic acid in sn-2 position was a very poor substrate
(Table 2) and also in PE arachidonic acid could not be
released (results not shown). With oleoyl chains in sn-1
position, no differences were found between PCs with
palmitoyl, stearoyl and oleoyl groups in sn-2 position
(Table 2). For PCs with identical acyl chains in sn-1
The specific activities of AtsPLA2-␣ toward mixed
micelles of PCs with homogeneous and heterogeneous
fatty acid composition and with different chain lengths of
the fatty acids (C –C22) in sn-1 and sn-2 position have
6
been determined (Table 2). When palmitic acid chains
were present in sn-1 position, AtsPLA2-␣ preferred

1
62
J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
Table 2
3.7. Kinetic constants for substrates with short acyl
chains in sn-1 and sn-2 position
Specific activities of AtsPLA2-␣ toward different glycerophospho-
cholines in form of mixed micelles
Substrates with palmitoyl chains in
sn-1 position
Activity
(␮mol min mg
Short chain phospholipids have found wide applica-
tion as substrates for sPLA2s (De Haas et al., 1971)
because their critical micelle concentration is in the
millimolar range in contrast to CMCs in the sub-
nanomolar range for phospholipids with long acyl
−
1
−1
)
1
1
1
1
-Palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine
,2-Dipalmitoyl-sn-glycero-3-
phosphocholine
16.5 ± 1.4
20.6 ± 0.7
25.3 ± 1.6
0.04 ± 0.03
−
10
chains (e.g. 10
M for 1,2-dipalmitoyl-sn-glycero-3-
-Palmitoyl-2-linoleoyl-sn-glycero-
phosphocholine (Smith and Tanford, 1972)). Therefore,
they can also be used as momomers and do not need sur-
factants to form micelles. Hence, we studied the activity
of AtsPLA2-␣ toward 1,2-dioctanoyl-, 1,2-diheptanoyl-,
and 1,2-dihexanoyl-sn-glycero-3-phosphocholine as a
function of their concentrations.
In the case of 1,2-dihexanoyl-sn-glycero-3-
phosphocholine, a significant increase of activity
was observed, when the CMC (10 mM, Table 3) was
reached showing a preference of the enzyme toward
the substrate in micellar form (Fig. 5). The complex
kinetics can be described by the sum of two hyperbolic
functions, below and above the CMC, yielding two
3
-phosphocholine
-Palmitoyl-2-arachidonoyl-sn-
glycero-3-phosphocholine
Substrates with oleoyl chains in sn-1 position
1
1
1
-Oleoyl-2-palmitoyl-sn-glycero-3-
18.0 ± 0.6
17.8 ± 0.9
16.7 ± 1.4
phosphocholine
-Oleoyl-2-stearoyl-sn-glycero-3-
phosphocholine
,2-Dioleoyl-sn-glycero-3-
phosphocholine
Substrates with identical acyl chains in sn-1 and sn-2 position
1
1
1
1
1
1
1
1
1
,2-Dioleoyl-sn-glycero-3-
phosphocholine
,2-Dilinoleoyl
sn-glycero-3-phosphocholine
,2-Dipalmitoyl-sn-glycero-3-
phosphocholine
,2-dimyristoyl-sn-glycero-3-
phosphocholine
,2-Dilauroyl-sn-glycero-3-
phosphocholine
,2-Didecanoyl-sn-glycero-3-
phosphocholine
,2-Dioctanoyl-sn-glycero-3-
phosphocholine
,2-diheptanoyl-sn-glycero-3-
phosphocholine
16.7 ± 1.4
19.5 ± 1.1
20.6 ± 0.7
27.3 ± 1.2
29.3 ± 1.4
35.5 ± 1.7
14.4 ± 0.8
5.9 ± 0.2
apparent kcat and K values (Table 3). Addition of NaCl,
M
which drastically lowers the CMC (Berg et al., 1997)
resulted in a significant change of the kinetics which
could be described by a normal Michaelis–Menten fit
(Fig. 5, Table 3).
In contrast, the initial rate of the hydrolysis of 1,2-
dioctanoyl-sn-glycero-3-phosphocholinebyAtsPLA2-␣
as function of its concentration showed a normal
Michaelis–Menten kinetics even in the absence of NaCl
and was not influenced by the addition of 2 M NaCl.
Table 3 shows the corresponding catalytic parameters
and CMCs.
,2-Dihexanoyl-sn-glycero-3-
phosphocholine
1.1 ± 0.03
Activities were determined in 0.3 M Tris/HCl, pH 8.5, 10 mM CaCl2,
using mixed micelles of the corresponding phospholipid (12.7 mM)
with 25 mM Triton X-100 and 4.8 mM SDS as described in Section 2.
The standard deviation was determined from at least three independent
assays.
and sn-2 position, a strong dependence on the chain
length of the fatty acids was found (Table 2). Maximal
activity of AtsPLA2-␣ was found for PC with decanoic
acid showing that 10 carbon atoms in the acyl chains
are required for optimal activity. With increasing chain
length, activity decreases significantly. Activities toward
substrates with short acyl chains, such as hexanoyl, hep-
tanoyl, and octanoyl chains were considerably lower
than those toward substrates with longer acyl chains
Fig. 5. Initial rates of the hydrolysis of 1,2-dihexanoyl-sn-glycero-3-
phosphocholine by AtsPLA2-␣ in the absence (᭹) and presence of 2 M
NaCl (ꢀ) as a function of the substrate concentration.
(Table 2).

J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
163
Table 3
Kinetic constants of phospholipids with short chains in sn-1 and sn-2 position
app −1
app
Substrate
CMC (mM)
0.17a
k
(s
)
Km (mM)
0.12 ± 0.2
3.92 ± 0.32
cat
1
1
1
,2-Dioctanoyl-sn-glycero-3-phosphocholine
4.38 ± 0.20
3.86 ± 0.18
,2-Diheptanoyl-sn-glycero-3-phosphocholine
2b
,2-Dihexanoyl-sn-glycero-3-phosphocholine
10c
Below CMC
Above CMC
In 2 M NaCl
0.53 ± 0.06
1.71 ± 0.21
1.71 ± 0.18
0.94 ± 0.15
13.85 ± 1.88
3.85 ± 0.36
Kinetic constants were determined in 50 mM Tris/HCl, pH 8.5, 10 mM CaCl2, as described in Section 2.
a
Wells, 1974.
Van Eijk et al., 1983.
Pl u¨ ckthun and Dennis, 1981.
b
c
4
. Discussion
used for micelle formation, apparent kinetic parame-
ters are obtained, which are the result of the interplay
of interface recognition and active-site specificity. Sev-
eral approaches such as the model of the surface dilution
kinetics by the group of Dennis (Carman et al., 1995)
were developed to separate effects of the physical char-
acteristics of the interface from true effects of enzyme
kinetics. Although most of the results presented here
are restricted to measurements of apparent activities and
renounce a laborious accurate kinetic analysis, several
general conclusions can be drawn.
AtsPLA2-␣ (group XIB) (Lee et al., 2005; Mansfeld
et al., 2006) is one of the first plant PLA2s which were
produced in amounts sufficient for biochemical charac-
terisation. Despite low similarities between the amino
acid sequences of AtsPLA2-␣ and the well-known ani-
mal sPLA2s, if considering the whole sequences, the
cores of the proteins are similar. On the basis of the crys-
tal structures of sPLA2 from bovine or porcine pancreas
(
group I) and bee venom (group III), we have shown
by homology modelling that the catalytic region with
its two ␣-helices stabilised by two disulphide bonds as
4.1. Functional parameters
2+
well as the Ca -binding loop of AtsPLA2-␣ are widely
congruent with the corresponding regions in the animal
sPLA2s (Mansfeld et al., 2006). Interestingly, however,
the catalytic His–Asp dyad typical of PLA2 from ani-
mal sources does not exist in AtsPLA2-␣ and other plant
PLA2s. In AtsPLA2-␣ the Asp residue of the dyad is
replaced by a Ser residue which is obviously involved in
the catalytic mechanism (Mansfeld et al., 2006). How-
ever, the Ser residue does not represent a nucleophile in
the sense of cytosolic PLA2s and patatins because it is
not part of a GXSXG(S) consensus sequence being typ-
ical of serine hydrolases. In the present study, we have
focussed on a screening of similarities and differences
in the enzymatic properties, particularly in substrate
preferences, of AtsPLA2-␣ and the well-known animal
sPLA2s. In nature, plant and animal sPLA2s meet very
different membrane compositions and fulfil different
physiological functions. Hence, remarkable differences
were expected.
The temperature and pH optima of AtsPLA -␣ being
between 30 and 40 C and pH 8.5 and 9, respectively,
2
◦
under standard assay conditions (Fig. 2) show no strik-
ing differences neither in comparison with other plant
sPLA s nor with animal sPLA s. The enzymes from N.
2
2
tabacum (Fujikawa et al., 2005) and elm (Stahl et al.,
1999) had pH optima in the more alkaline range (8–10
and 8–9, respectively) with 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC) as substrates,
while the highest activity of the enzyme from N. tabacum
was found between pH 6 and 7 with the poorer substrate
POPG (Fujikawa et al., 2005). Similarly, the pH optima
of sPLA s from porcine pancreas (de Haas et al., 1968)
2
or bee venom (Shipolini et al., 1971) are reported to be
around 8.0.
Temperature optima of sPLA s are seldom reported.
2
In our laboratory, a similar temperature optimum
◦
Because phospholipases act on interfaces, their
kinetic characterisation is not trivial. As the activity of
PLA2s strongly depends on the supramolecular structure
of the substrate aggregates including charge and molec-
ular dimensions of the phospholipids and surfactants
(30–40 C) was found for recombinant sPLA from bee
2
venom (Markert et al., 2007).
Like most sPLA s from animal sources, AtsPLA -
2
2
␣ requires calcium ions in the millimolar range and is
inactive in the absence of calcium ions (Fig. 3A). The

1
64
J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
homology model (Mansfeld et al., 2006) shows that the
calcium-binding loop in AtsPLA2-␣ has a structure sim-
ilar to sPLA2s from animal sources. Correspondingly,
the KD value of AtsPLA2-␣ for the dissociation of cal-
cium ions (0.42 mM) is in the range of the KD values of
sPLA2s from bovine (0.15 mM, Huang et al., 1996) and
porcine (0.35 mM, Yu etal., 1993)pancreas. Like inmost
sPLA2s from animal sources (Bayburt et al., 1993, Jain
et al., 1991b), calcium ions are able to protect the enzyme
from inactivation by p-bromophenacylbromide. Interest-
ingly, AtsPLA2-␣ contains only two histidine residues
pholipids than to zwitterionic ones (Jain et al., 1991a,
Tatulian, 2001, Pande et al., 2006). This might reflect the
evolutionary adaptation of the pancreatic enzymes to the
micromilieu in the digestive system where they mainly
degrade phospholipids codispersed with bile salts.
PA which was not hydrolysed by AtsPLA2-␣ and
other anionic phospholipids such as PG or PS enhanced
the initial reaction rates of hydrolysis of zwitterionic
phospholipids (Fig. 4). However, enhancement was less
pronounced for AtsPLA2-␣ (2.3 with PA, 2.2 with PG,
and 1.3-fold with PS, in comparison to 50-fold with
PA for porcine pancreatic sPLA2, Ghomashchi et al.,
1991). The reaction progress curves for the hydroly-
sis of the zwitterionic PC showed no latency phase as
observed with the enzymes from bee venom (Upreti and
Jain, 1978), porcine pancreas (Apitz-Castro et al., 1982),
and the human non-pancreatic enzyme (Bayburt et al.,
1993). Altogether, these results point to a weaker elec-
trostatic component of membrane binding of AtsPLA2-␣
in comparison to most enzymes from animal sources.
(
His9 in the N-terminal region, His62 in the active site).
The two-step inactivation (Fig. 3B) and the correspond-
ing fractional amplitudes of about 0.5 each suggest that
the fast step (half-life <1 min), which is decelerated by
a factor of more than two orders of magnitude in the
presence of Ca2 ions, results from the alkylation of the
catalytic His62, while the slower step (half-life 41 min)
is caused by alkylation of His9 influencing the activity
to a lower degree.
+
4
.2. Head group preferences
4.3. Fatty acid preference
The use of different substrate forms (mixed micelles
AtsPLA2-␣ has been shown to release fatty acids in
sn-2 position while the sn-1 ester bond in glycerophos-
pholipids is not attacked (Mansfeld et al., 2006). With
respect to the acyl chain specificity, however, the type of
fatty acid in sn-1 position was found to be important.
With palmitic acid chains in sn-1 position, a prefer-
ence of linoleoyl over palmitoyl and palmitoyl over
oleoyl chains for the sn-2 position was found, whereas
no differences were observed when an oleic acid chain
was present in sn-1 position (Table 2). Most interest-
ingly, PCs or PEs with arachidonic acid in sn-2 position
which are extremely rare in plants (arachidonic acid
is found for instance in mosses, ferns, liverworts, but
in higher plants it is only known for Agathis robusta
(http://www.lipidlibrary.co.uk)) were not cleaved. These
resultsareinstrongcontrasttothefattyacidspecificityof
animal sPLA2s, which easily hydrolyse arachidonic acid
containing phospholipids, and suggest an evolutionary
adaptation of enzyme specificity to the natural phospho-
lipid composition. Phospholipids in A. thaliana contain
mainly fatty acid chains with 16 carbon atoms in sn-1 and
preferably unsaturated fatty acid chains with 18 carbon
atoms in sn-2 position (linoleic and linolenic acid). Phos-
pholipids with identical fatty acid chains in sn-1 and sn-2
position are also good substrates. Comparable to pan-
creatic PLA2s which preferentially hydrolyse substrates
with nine carbon atoms in the acyl chains (Pattus et al.,
1979), AtsPLA2-␣ has an optimum activity toward sub-
strates with 10 carbon atoms in the acyl chains (Table 2).
containing Triton X-100 of different concentrations or
in the presence of SDS; SUVs) allows to extract general
trends for the acceptance of phospholipid head groups by
AtsPLA2-␣. The results show that zwitterionic PCs are
the best substrates (Table 1), independent of the aggre-
gate type. Zwitterionic PEs are accepted as well, while
AtsPLA2-␣ shows a lower activity toward PG, a still
lower activity to PI, and no activity to PS and PA, all of
them being anionic. These trends were independent of
the addition of neutral or anionic surfactants. Therefore,
the results suggest that the active-site specificity plays a
greater role than interfacial recognition. Moreover, the
results lead to the presumption that the head group speci-
ficity is linked to the biological function of AtsPLA2-␣.
Thus, the acceptance of PI as substrate might imply a
possible involvement of AtsPLA2-␣ in signal transduc-
tion pathways. On the other hand, the preference of PC is
plausible because PC is the most abundant phospholipid
in plants (http://www.lipidlibrary.co.uk).
Comparable studies on other plant PLA2s, as far
as available, showed similar results. Thus, recombinant
sPLA2 from N. tabacum was shown to prefer PC over PG
and PE in mixed micelles with sodium cholate (Fujikawa
et al., 2005) while a slight preference of PE over PC for
AtsPLA2-␣ and -␤ in mixed micelles with Triton X-100
was reported by Lee et al. (2005).
In contrast, the pancreatic PLA2s analysed so far
are characterised by a tighter binding to anionic phos-

J. Mansfeld, R. Ulbrich-Hofmann / Chemistry and Physics of Lipids 150 (2007) 156–166
165
4
.4. Interfacial activation on short chain
acids in sn-2 position of PC. The less abundant anionic
phospholipids seem to be poorer substrates than the most
abundant PC. Also the interfacial activation and the cat-
alytic efficiency is presumably adapted to the enzyme
function in plants.
phospholipids
With AtsPLA2-␣, an activation above the CMC as
typical for sPLA2s could be obtained for 1,2-dihexanoyl-
sn-glycero-3-phosphocholine(Fig. 5, Table3). However,
the activation was abolished by the addition of 2 M NaCl
which decreases the CMC (Berg et al., 1997). Normal
hyperbolic curves were obtained for 1,2-dioctanoyl- and
Acknowledgement
The authors thank Mrs. M. Sonntag for excellent
technical assistance and Dr. A. Schierhorn for mass
spectrometric controls. The donation of samples of phos-
pholipids by Lipoid GmbH, Ludwigshafen, Germany is
gratefully acknowledged.
1
,2-diheptanoyl-sn-glycero-3-phosphocholine. In con-
trast, with porcine pancreatic and cobra venom PLA2,
dramatic activity increases were observed when the con-
centration of zwitterionic substrates reached the CMC
and aggregation into micelles occurred (Pieterson et
al., 1974). Obviously, AtsPLA2-␣ is less influenced by
interfacial activation than these enzymes. Similarly, also
sPLA2 frombeevenomislesssensitivetowardtheaggre-
gation state of the substrate (Lin et al., 1988).
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