|
Plant Physiol. (1998) 117: 197-205
Purification and Characterization of a Low-Molecular-Weight
Phospholipase A2 from Developing Seeds of Elm1
Ulf Ståhl*,
Bo Ek, and
Sten Stymne
Department of Plant Biology, P.O. Box 7080, Swedish University of
Agricultural Sciences, S-750 07 Uppsala, Sweden (U.S., B.E.); and Department of Plant Breeding Research, Swedish University of
Agricultural Sciences, Herman von Ehles v. 4-6, S-268 31 Svalöv,
Sweden (S.S.)
 |
ABSTRACT |
Phospholipase A2
(PLA2) was purified about 180,000 times compared with the
starting soluble-protein extract from developing elm (Ulmus
glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel
electrophoresis the purified fraction showed a single protein band with
a mobility that corresponded to 15 kD, from which activity could be
recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced
mass of 13,900 D. A 53-amino acid-long N-terminal sequence was
determined and aligned with other sequences, giving 62% identity to
the deduced amino acid sequence of some rice (Oryza
sativa) expressed sequence tag clones. The purified enzyme had
an alkaline pH optimum and required Ca2+ for activity. It
was unusually stable with regard to heat, acidity, and organic solvents
but was sensitive to disulfide bond-reducing agents. The enzyme is a
true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and
amino acid sequence alignments indicate that the enzyme is related
to the well-characterized family of animal secretory
PLA2s and, to our knowledge, is the first plant enzyme of
this type to be described.
| |
INTRODUCTION |
PLA2 (phosphatide 2-acylhydrolase, EC
3.1.1.4) specifically hydrolyzes glycerophospholipids at the
sn-2 position to yield free fatty acids and
lysophospholipids. Little is known about plant
PLA2s; no plant PLA2 has
been purified to any extent and no plant gene or cDNA encoding such an
enzyme has been identified. The animal PLA2s,
however, are a diverse family of well-studied enzymes that are known to
play important roles in a number of basic cellular processes (Waite,
1987 ; Dennis, 1994). They are divided into several distinct groups,
groups I, II, III, and V, which belong to a class of small (13-18 kD),
secretory, and extremely heat-stable enzymes having between five and
eight disulfide bonds and requiring millimolar concentrations of
Ca2+ for maximum activity. This type of
PLA2 is best known from snake venom, bee venom,
and pancreatic juice, where it occurs abundantly and has a digestive
role. More recently, PLA2 has been found in high levels in
rheumatoid arthritic synovial fluid (Vadas et al., 1981; Seilhamer et
al., 1989) and also at low levels in many mammalian tissues
(Tischfield, 1997). The group IV enzymes are intracellular, high-Mr PLA2s that are
translocated to membranes in the presence of micromolar concentrations
of Ca2+. This type of PLA2
has a preference for phospholipids, with arachidonate at the
sn-2 position, and has been suggested to be involved in signal transduction and in the initiation of eicosanoid synthesis (Dennis, 1994). In addition to these groups there is also a group of
40kD, Ca2+-independent, intracellular
PLA2s specific for arachidonyl-plasmenylcholine (Dennis, 1994).
In plant tissues phospholipid-degrading enzymes other than
PLA2 have been studied in detail, such as
phospholipase D (Pappan et al., 1997a, 1997b), phospholipase C
(Yotsushima et al., 1993; Hirayama et al., 1995), and acyl hydrolases
(Huang 1987; Sahsah et al., 1994; Teissère et al., 1995). With
regard to PLA2 several studies of plant enzyme
activities, in more or less crude preparations, have been reported
(Moreau and Morgan, 1988; Kawakita et al., 1993; Kim et al., 1994; Roy
et al., 1995). The two products of PLA2 activity,
fatty acids and LPC, have also been shown to stimulate several plasma
membrane enzymes, such as H+-ATPase (Palmgren et
al., 1988), NADH oxidases (Brigthman et al., 1991) and protein kinases
(Klucis and Polya, 1987; Martiny-Baron and Scherer, 1989). These have
been proposed to serve as second messengers in signal transduction.
Linolenic acid, the precursor of the plant-signal substance jasmonic
acid (Creelman and Mullet, 1997), is thought to be liberated from
phospholipids by phospholipase A or an acyl hydrolase for further
metabolism into jasmonic acid. This is analogous to the synthesis of
the structurally related prostaglandins from arachidonic acid in
animals (Bergey et al., 1996).
Previously, we studied the formation of hydroxylated and epoxygenated
fatty acids in microsomal preparations from developing oil seeds that
accumulate these acids in their triacylglycerols. Both ricinoleic
(12-hydroxy-octadeca-9-enoic) acid in castor bean (Ricinus
communis) seeds and vernolic (12-epoxy-octadeca-9-enoic) acid in
Euphorbia lagascae seeds were found to be synthesized by
oxygenation of acyl chains attached to phospholipids, after which the
oxygenated fatty acids were selectively removed by microsomal PLA2 activities (Bafor et al., 1991, 1993; Ståhl
et al., 1995). It was further shown that microsomal fractions from
developing seeds of Cuphea procumbens and elm (Ulmus
glabra), which accumulate mainly capric (decanoic) acid in their
triacylglycerols, had PLA2 activities with
pronounced selectivities for
C8-C12 fatty acids (Ståhl
et al., 1995). The developing seed PLA2
activities were proposed to be involved in the channeling of these
uncommon fatty acids from the membrane lipids into the storage lipids.
We have also found PLA2 activities specific for
phospholipids with oxygenated acyl groups in microsomal preparations
from plant tissues that are not involved in the accumulation of such
fatty acids, and we proposed that such activities are part of a general
repair mechanism after oxidative damage to membranes (Banas et al.,
1992; Bafor et al., 1993).
With the intention to characterize and purify one of the acyl-selective
PLA2s in microsomal preparations of developing
oil seeds that accumulate uncommon fatty acyl groups, we initiated the
purification of the medium-chain-selective PLA2
activity found in microsomal fractions of developing elm seed. We
achieved a 30,000-fold purification of the main
PLA2 activity found in detergent-solubilized microsomes, as communicated in a preliminary report (Ståhl et al.,
1997). However, the low abundance of the enzyme and limitations in the
collection of material prevented us from further purification to
homogeneity of this low Mr
PLA2. From the present study we report the
purification to homogeneity and the characterization of a biochemically
related, soluble PLA2 from the same tissue and
show that this enzyme is related to the low-Mr
secretory PLA2s found in various animal tissues.
| |
MATERIALS AND METHODS |
[1-14C]Palmitic acid,
[1-14C]oleic acid, and
1,2-[1-14C]dipalmitoyl-PC were purchased from
Amersham. [1-14C]Capric acid, 1-palmitoyl-LPC,
palmitic acid, oleic acid, capric acid, PLA2
(from cobra [Naja naja kaouthia] venom), and phospholipase C (from Bacillus cereus) were obtained from Sigma. Lubrol PX
(Thesit) was obtained from Boehringer Mannheim. Q-Sepharose Fast-Flow, Superose 12 (10/30 column) and µRPC SC
C2/C18 (2.1/10 column) were
from Pharmacia.
Radioactive fatty acids were adjusted to a specific radioactivity of
167 Bq/nmol by dilution with nonradioactive fatty acids before used in
[14C]acyl-PC synthesis.
1-Palmitoyl-2-[14C]acyl-PC substrates were
synthesized by acylating TFA anhydrides of the
14C-labeled fatty acids to palmitoyl-LPC by the
method described by Kanda and Wells (1981).
1-Palmitoyl-2-[14C]palmitoyl-DAG was prepared
by treatment of 1-palmitoyl-2-[14C]palmitoyl-PC
with commercial phospholipase C. The products were purified on
silica-gel TLC plates before used in assays.
Seeds from elm (Ulmus glabra) trees were collected from
local trees at the mid to late stage of seed development (i.e. when the
seed coats were partly filled and the seed wings were still green). The
seed coats were removed and the white embryos were immediately frozen
in liquid N2 and stored at  80°C.
Assays of PLA2 Activity
PLA2 assays were done using radioactive
phospholipids dispersed in mixed micelles of the nonionic detergent
lubrol PX as follows: 14C-Acyl substrate (1-50
nmol per assay) dissolved in chloroform was taken to dryness under a
stream of N2 and solubilized in assay buffer (50 mm Tris-HCl, pH 8.0, 10 mm
CaCl2, and 0.06% [w/v] lubrol PX) by
incubation at 70°C for 5 to 10 min and mixed thoroughly. In standard
assays, enzyme fractions (0.5-10 µL) were incubated at 30°C for 5 to 30 min with 5 or 10 nmol of
1-palmitoyl-2-[14C]palmitoyl-PC in a total
volume of 50 µL. The reaction was stopped by the addition of 400 µL
of chloroform:methanol:acetic acid (50:50:1, v/v) followed by 150 µL
of water to yield a two-phase system, according to the method of Bligh
and Dyer (1959). The samples were mixed thoroughly and centrifuged at
10,000g for 1 min.
Assays of PLA2 Activity from SDS-PAGE
Chromatographed SDS-PAGE (ExelGel 8-18%, Pharmacia) lanes (not
fixed) were divided in 2- to 3-mm-wide pieces and placed in Eppendorf
tubes. Proteins were eluted from the gel pieces in 400 µL of 20 mm Tris, pH 8.0, containing 0.5% (w/v) SDS by rotating the
tubes end over end for 16 h at 37°C. The eluates were
concentrated to 100 µL in a SpeedVac concentrator (Savant,
Farmingdale, NY) and the proteins were precipitated with
ethanol/chloroform (Wessel and Flügge, 1984) to remove the SDS.
Air-dried pellets were solubilized in 150 µL of assay buffer, and
assays were initiated by adding 5 nmol of
1-palmitoyl-2-[14C]palmitoyl-PC solubilized in
50 µL of assay buffer. The assays were performed for 3 h at
30°C and terminated and extracted as described above.
Analytical Procedures
The lipid-containing chloroform phases from
PLA2 assays were applied to minicolumns of silica
gel (about 40 mg of silica gel) and the columns were rinsed with an
additional 400 µL of chloroform. The eluates from the silica columns,
which contained the free 14C-fatty acids, were
collected into scintillation vials. The radioactivity was determined by
liquid-scintillation counting after addition of 6 mL of toluene:ethanol
(2:1, v/v) containing 0.4% (w/v) of 2(1-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole as a scintillant.
When, in addition to free fatty acids, radioactive complex lipids were
to be monitored, the chloroform-soluble lipids were separated on
silica-gel TLC plates (Silica 60, Merck, Darmstadt, Germany). The
plates were developed with chloroform:methanol:acetic acid:water
(170:30:20:7, v/v) when PC or LPC was used as the substrate and with
hexane:diethylether:acetic acid (70:30:1, v/v) when DAG was the
substrate. Unsaturated lipids were located by staining with
I2 vapor, whereas saturated lipids were
visualized by spraying with water. Lipid areas and the rest of the
lanes were removed from the plates and assayed for radioactivity by
scintillation counting as described above.
Purification of the Elm PLA2
Frozen, developing elm embryos, 60 g, were homogenized with a
mortal and pestle and subsequently with an Ultra-Turrax (Janke and
Kunkel, Staufen, Germany) in 600 mL of ice-cold 100 mm
KH2PO4 buffer, pH 7.2, containing 0.33 m Suc. The homogenate was filtered through two layers of
Miracloth (Calbiochem) and centrifuged at 10,000g for 12 min. The supernatant was filtered through one layer of Miracloth and
centrifuged at 100,000g for 90 min. The 100,000g supernatant was either used immediately or stored at 20°C.
The 100,000g supernatant was brought to 55% (w/v) with
(NH4)2SO4
and stirred at 4°C for 1 h. Precipitated proteins were pelleted by centrifugation at 10,000g for 10 min and resuspended in
130 mL of 50 mm diethanolamine buffer, pH 8.5. Ice-cold
acetone was added to a final concentration of 45% (v/v) after which
the extract was left at 4°C for 30 min. Precipitated proteins were
removed by centrifugation for 10 min at 10,000g. The
resulting supernatant was dialyzed against 20 volumes of 20 mm piperidine, pH 11.0, overnight and then applied to a
Q-Sepharose Fast-Flow column (1 × 10 cm) equilibrated in 20 mm piperidine, pH 11.0. The column was eluted with a linear
NaCl gradient from 100 to 500 mm in 20 mm
piperidine, pH 11.0, at a flow rate of 2 mL/min. Three-milliliter fractions were collected and assayed for PLA2
activity. A single broad peak of activity was eluted at an NaCl
concentration of 200 to 300 mm.
Peak fractions were pooled, concentrated on Centricon-10 to 0.6 mL,
and chromatographed in three separate runs on a Superose 12 gel-filtration column (1.0 × 30.0 cm) in 20 mm
Tris-HCl, pH 8.0, with 150 mm NaCl at a flow rate of 0.4 mL/min. Fractions (0.5 mL) were collected and assayed for
PLA2 activity. Peak fractions from all three runs
were pooled, and PLA2 was further purified using
a C4 reverse-phase HPLC column (0.46 × 10.0 cm, Vydac, Hesperia, CA) equilibrated with 0.1% TFA. The column was
developed at 0.4 mL/min with a 30-min gradient (40-55% of
acetonitrile in 0.1% TFA) and peaks, monitored at 214 and 280 nm, were
collected manually. Collected fractions from four separate runs were
assayed for PLA2 activity. Peak fractions were
pooled and the acetonitrile content was reduced by evaporation in a
SpeedVac concentrator. The PLA2 was then purified
to apparent homogeneity on a reverse-phase
C2/C18 column (0.21 × 10.0 cm) equilibrated in 0.1% TFA and developed at 100 µL/min with a
60-min gradient (30-60% acetonitrile in 0.1% TFA) using a SMART
system (Pharmacia). Peaks monitored at 214 nm were automatically
collected and then assayed for PLA2 activity. The
PLA2 activity eluted as a discrete peak in the
gradient at about 48% acetonitrile.
SDS-PAGE
Protein fractions were, if necessary, concentrated by evaporation
in a SpeedVac and precipitated with ethanol/chloroform according to the
method of Wessel and Flügge (1984). Samples (final volume 20 µL) in 50 mm Tris-acetate, pH 7.5, containing 1% SDS
(w/v), with or without 10 mm DTT, were heated to 95°C for
5 min, centrifuged for 5 min at 13,000g, and then loaded
onto a horizontal 8 to 18% gradient polyacrylamide gel (ExelGel SDS,
Pharmacia) with a 33-mm stacking zone and a 77-mm separating zone.
Electrophoresis was performed in a Multiphor II unit (Pharmacia) at
15°C for 80 min at 600 V and stained with colloidal Coomassie blue
(Neuhoff et al., 1988) or with silver according to the manufacturer's
instructions.
Molecular Mass Determination by MALDI-TOF MS
Native or reduced and iodoacetamide-alkylated-purified
PLA2 was mixed with matrix 2,5-dihydroxybenzoic
acid (14 mg/mL) in a 1:1 ratio (v/v). From each sample 1-µL aliquots
were deposited on gold-covered steel sample mesa and dried in a vacuum.
The molecular masses of native or reduced and alkylated
PLA2 were analyzed in the positive mode on an
LDI-1700XP instrument (Linear Scientific, Inc., Reno, NV) equipped with
an N2 laser (339 nm) using 7 µJ of energy.
N-Terminal Sequence Analysis and Similarity Searches in
Databases
About 1.8 µg of purified PLA2 was reduced
by incubation with 0.1 m Tris-HCl, pH 8.5, containing 8 m guanidine-HCL, 10 mm EDTA, and 20 mm DTT at 56°C for 30 min and then the Cys residues were alkylated with 20 mm iodoacetamide for 60 min at room
temperature. Both reduction and alkylation were performed under an Ar
atmosphere. The reduced and alkylated PLA2 was
desalted on a reverse-phase C2/C18 column (0.21 × 10 cm) equilibrated with 0.1% TFA in water and eluted at 100 µL/min
with a 30-min acetonitrile gradient (30-60%) using a SMART
chromatographic station. Peaks were monitored at 214 and 280 nm, and
automatic peak collection was used. Amino acid sequencing was performed
on a sequencer (model ABI 476A, Applied Biosystems/Perkin-Elmer)
according to the manufacturer's instructions. The N-terminal amino
acid sequence was used as a query for the Basic Local Alignment Search
Tool (BLAST) at the National Center for Biotechnology Information
(Bethesda, MD), and the "blastp" search program was used against
the Nonredundant GenBank CDS
translations+PDB+SwissProt+SPupdate+PIR, and the "tblastn" search program against nonredundant GenBank+EMBL+DDBJ+ PDB sequences and the Nonredundant Database of GenBank EST Division.
Determination of Protein Concentration
The protein concentrations in fractions of the purification scheme
were determined by the method of Bradford (1976), except in the
fractions from the two last purification steps, the determination was
done by comparing eluted peak areas monitored at 214 nm with peak areas
of standard proteins (Buck et al., 1989), and the staining intensities
of the sample protein band were compared with known standards in
colloidal Coomassie blue-stained SDS-PAGE gels.
| |
RESULTS |
Purification of PLA2
Eighty percent of the total PLA2 activity in
a crude extract from developing seed embryos was recovered in the
100,000g supernatant after centrifugation (data not shown).
This fraction was used for further purification according to the
procedure summarized in Table I. The
procedure included
(NH4)2SO4
fractionation, acetone precipitation, and column chromatography on
Q-Sepharose Fast Flow (anion-exchange), Superose 12 (gel filtration),
and C4 and
C2/C18 reverse-phase
columns (Fig. 1). Several of the
purification steps were achievable because of the extreme stability of
the PLA2 in organic solvents at a low pH. Both
the precipitation with acetone 45% (v/v) and reverse-phase
chromatography with 0.1% (v/v) TFA and elution with an acetonitrile
gradient are extremely harsh conditions that denature most enzymes.
These steps were very useful for the PLA2
purification, giving a high degree of purification and little loss in
activity. The final purification, to apparent homogeneity, was achieved
with a C2/C18 column;
PLA2 activity coincided with a discrete protein
peak eluted at a acetonitrile concentration of about 48% (Fig. 1D).
Based on specific activity the PLA2 was purified
about 180,000-fold compared with the 100,000g supernatant,
with a total recovery of 16% (Table I).
View larger version (23K):
[in this window]
[in a new window]
| Figure 1.
Elution profiles of protein ( ) and
PLA2 activity (- - - - -) from the different
chromatographic steps in the purification of the elm PLA2:
Q-Sepharose Fast Flow (A), Superose 12 (B), C4 reverse-phase HPLC (C), and C2/C18
reverse-phase SMART (D).
|
|
Molecular Mass of Purified PLA2
The purified PLA2 fraction showed one band
with a molecular mass of about 15 kD when subjected to SDS-PAGE on an 8 to 18% gradient gel under reducing conditions and stained with
colloidal Coomassie blue (Fig. 2). After
electrophoresis and under nonreducing conditions,
PLA2 activity could be recovered and was found to coincide with the 15-kD band (Fig. 3). A
similar amount of snake venom PLA2 was included
in adjacent lanes for a comparison and gave about the same recovery of
activity (Fig. 3). For a more precise molecular mass determination a
MALDI-TOF MS analysis was performed. The purified fraction gave a major
peak with a molecular mass of 13,900 D and two minor peaks with masses
of 13,200 and 12,700 D (Fig. 4). With
alkylation of Cys residues in the purified fraction all three peaks
gained about 700 to 800 mass units, indicating the presence of 12 to 14 Cys residues (data not shown).
View larger version (103K):
[in this window]
[in a new window]
| Figure 2.
SDS-PAGE analysis of fractions from the three last
purification steps of the elm PLA2 purification, along with
a commercial PLA2 from cobra venom. Lane 1, Twenty-five
micrograms of Superose 12 eluate; lane 2, 2 µg of
C4-reverse phase eluate; lane 3, 0.1 µg of
C2/C18 reverse-phase eluate; lane 4, molecular
size markers; and lane 5, 0.1 µg of cobra venom PLA2.
Samples were reduced with DTT and separated on an 8 to 18% gradient
gel.
|
|
View larger version (52K):
[in this window]
[in a new window]
| Figure 3.
Recovery of activity from elm and cobra
PLA2 from a SDS-PAGE gel. Duplicate samples of 50 ng of
PLA2 from cobra venom and of purified elm-seed
PLA2, unreduced, were separated on an 8 to 18% gradient
gel. One lane with cobra venom and one lane with elm PLA2
were immediately sliced into 2- to 3-mm-wide pieces, from which
proteins were eluted, precipitated, and assayed for PLA2
activity, as described in ``Materials and Methods''. Remaining lanes
were stained overnight with colloidal Coomassie blue. Lane 1, Cobra
venom PLA2; lane 2, elm seed PLA2.
|
|
Characterization of Purified PLA2 Activity
The enzyme exhibited optimal activity at a pH between 8.0 and 9.0 and had no activity below pH 5.0 (Fig.
5A). EGTA (1 mm) totally
abolished activity (data not shown), indicating an absolute requirement
for Ca2+. Optimal concentration of
CaCl2 for activity was as high as 10 to 15 mm, although 0.5 mm was sufficient to achieve
50% of maximal activity (Fig. 5B).
View larger version (27K):
[in this window]
[in a new window]
| Figure 5.
A, The effect of pH. Incubations were carried out
with 1 ng of purified PLA2, 0.2 mm
1-palmitoyl-2-[14C]palmitoyl-PC, 1 mm lubrol
PX, and 10 mm CaCl2 in 75 mm buffer at 30°C for 15 min in a final volume of 50 µL. Buffers used were: acetic acid, pH 4.5 to 5.5; Mes, pH 5.5 to 6.5; Bis-Tris propane, pH
6.5 to 9.5, and Caps, pH 9.5 to 11. Bis-Tris propane,
1,3-bis(Tris[hydroxymethyl]methylamino)propane; Caps,
3-(cyclohexylamino)-1-propanesulfonic acid. B, The effect of
Ca2+ concentration on PLA2 activity.
Incubations were carried out with 1 ng of purified PLA2 in
50 mm Tris-HCl buffer, pH 8.0, in the presence of 0.2 mm 1-palmitoyl-2-[14C]palmitoyl-PC, 1 mm lubrol PX, and with Ca2+ concentrations as
indicated in the figure in a final volume of 50 µL at 30°C for 15 min. C, Time-course incubations of purified PLA2 with and
without BSA. Purified PLA2, 0.5 ng, was incubated in the
presence of 0.2 mm
1-palmitoyl-2-[14C]palmitoyl-PC, 1 mm lubrol
PX, 10 mm CaCl2, 50 mm Tris-HCl, pH 8.0, in the absence of BSA ( ), with 50 µg of BSA ( ), or with 250 µg of BSA (×), in a final volume of 50 µL for the indicated times at 30°C. D, Effect of substrate concentration at various lubrol
PX/phosphatidylcholine molar ratios. Purified PLA2, 1 ng, was incubated in the presence of 10 mm CaCl2,
50 mm Tris-HCl, pH 8.0, 0.02 to 1 mm
1-palmitoyl-2-[14C]palmitoyl-PC without detergent ()
or with a lubrol PX/phosphatidylcholine molar ratio of 2 () or 6 (×) in a final volume of 50 µL for 10 min at 30°C. conc.,
Concentration.
|
|
The purified enzyme was tested for its specificity with respect to the
sn position in PC by incubation with
1-palmitoyl-2-[14C]palmitoyl-PC and
1,2-[14C]dipalmitoyl-PC for an extended
incubation time, followed by an analysis of
14C-labeled hydrolysis products on TLC plates. No
radioactive LPC was formed when the enzyme was incubated with
1-palmitoyl-2-[14C]palmitoyl-PC, indicating
that the enzyme was not active against the sn-1 position of
PC (Table II). With
1,2-[14C]dipalmitoyl-PC as a substrate about
equimolar amounts of radioactive fatty acids and LPC were formed (Table
II), confirming that the purified enzyme does not possess phospholipase
B activity. The purified PLA2 had no activity
toward the neutral acyl lipid
1-palmitoyl-2-[14C]palmitoyl-DAG or the
lysophospholipid 1-[14C]oleoyl-LPC (Table II).
View this table:
[in this window]
[in a new window]
|
Table II.
Specificity of purified elm seed PLA2
Purified elm PLA2, 2 ng, was incubated for 30 min with 5 nmol of radioactive substrate under standard assay conditions.
|
|
The acyl preferences for the purified elm PLA2
were analyzed by measuring activity with
1-palmitoyl-2-14C-acyl-PC, with caproyl,
palmitoyl, and oleoyl groups at the sn-2 position. Similar
assays with cobra PLA2 were included in this experiment for a comparison. The elm enzyme was found to hydrolyze the
caproyl and oleoyl groups at about twice the rate of the palmitoyl groups, whereas the cobra enzyme hydrolyzed all three acyl groups at
about the same rate (Table III). The
specific activity of the purified elm enzyme with the preferred
substrate 1-palmitoyl-2-[14C]caproyl-PC was
found to be 90 µmol min 1
mg1.
View this table:
[in this window]
[in a new window]
|
Table III.
Acyl specificities of the purified elm seed
and cobra PLA2s
Purified elm or cobra PLA2, 1 ng, was incubated for 10 min
with 25 nmol of radioactive substrate under standard assay conditions.
|
|
The elm PLA2 was stable both in organic solvents
and at extreme pH conditions (see above) and was also extremely heat
stable. Incubations with the purified enzyme at 100°C for 5 min did
not affect activity, whereas a 30-min incubation at this temperature caused only a 40% decrease in activity (results not shown). DTT (5 mm) or 1% (v/v)  -mercaptoethanol were, however, found
to completely abolish activity (results not shown), indicating the
likely presence of structurally important disulfide bonds.
Time-course incubation of about 1 ng of purified
PLA2 with
1-palmitoyl-2-[14C]palmitoyl-PC at a
concentration range of 0.02 to 1 mm (with 1 mm
lubrol PX) showed linear activity for about 10 to 15 min (data not
shown). The inclusion of 50 or 250 µg of BSA in the assay cocktail
prolonged the linearity of time-course incubations substantially (Fig.
5C), possibly indicating a decreased product inhibition because of the
scavenging of the produced free fatty acids. Substrate-saturation
curves of the purified PLA2 without detergent present, and at two fixed detergent/substrate molar ratios (2 and 6), showed that the activity is greatly increased by the dispersion
of the substrate in mixed micelles and that the detergent/PC molar
ratio strongly affects activity (Fig. 5D). Under the assay conditions
used, the enzyme is saturated with about 1 mm
substrate.
N-Terminal Sequence Analysis
About 1.8 µg of purified PLA2 (130 pmol)
was reduced and alkylated with iodoacetamide and the N-terminal
sequence was determined by automatic Edman degradation. Fifty-seven
cycles were run, with an initial yield of approximately 17% and a
repetitive yield of 94.3% for the 36 first cycles. The 53 first
cycles gave a clear, single first-choice amino acid, and the amino acid
sequence was read manually to be: 1LNVGVQATGTSISVGKGCSRKCESEFCSVPPFLRYGKYCGLLYSGCPGEKPCD53. A low, double-background signal (about 10% of the main signal) could be read partly at about 25 cycles and was likely to be two degradation products of the main sequence; the two cleavage sites in
the native protein were inferred from this signal to be between residues 7 and 8 and between residues 13 and 14 (data not shown). The
main N-terminal amino acid sequence was used in a BLAST search (see
``Materials and Methods'') and the best aligned sequences found were
EST-sequences from rice (Oryza sativa; accession nos.
C27540, D47320, D47653, and D47724). The C27540 sequence originated
from a callus mRNA and the others originated from 8-d-old shoots. All four rice clones have nearly the same nucleotide sequences. An alignment of the N-terminal amino acid sequence of the elm
PLA2 with the deduced amino acid sequence from
C27540 and D47653 is shown in Figure 6. The overall identity is 62%
and it is noteworthy that the positions of all six Cys residues in the
elm enzyme sequence are conserved in the rice EST clones.
View larger version (11K):
[in this window]
[in a new window]
| Figure 6.
Alignment of the N-terminal amino acid sequence of
the purified elm PLA2 with the deduced amino acid sequences
of the rice EST sequences D47653 and C27540 (accession nos.) and the 49 first N-terminal amino acids of animal secretory group I (cobra) and
group II (human synovial fluid) PLA2. The last amino acid in the predicted signal peptide (certainty 0.76 by PSORT World Wide Web
server) of the D47653 sequence is underlined. The consensus line shows
identical amino acids for the three plant sequences. Stars denote amino
acid residues that are conserved among all animal secretory
PLA2s (Chen et al., 1994b).
|
|
The PSORT World Wide Web server predicted a 25-amino acid-long signal
peptide for export through the Golgi in the rice-shoot EST sequences.
The predicted cleavage site between Gly-25 and Leu-26 coincides with
the start of the aligned elm PLA2 N-terminal amino acid sequence (Fig. 6). The rice
callus EST sequence starts in the middle of the predicted signal
peptide. A region in the amino acid sequence of the animal secretory
PLA2s, called the Ca2+-binding loop, is found in the N-terminal
sequences of both the elm PLA2 and the putative
rice PLA2s (Fig. 6). The active-site region of
animal secretory PLA2s was also found in the
putative rice PLA2s (Fig. 6) but is downstream of
the elm N-terminal sequence. Of the 17 highly conserved amino acids
that are common to all animal secretory PLA2s
(Chen et al., 1994b), 10 are present in the N-terminal part of the
group I and II PLA2s, which has been aligned in
Figure 6. Of these 10, 9 are found in the putative rice
PLA2 sequences.
| |
DISCUSSION |
The purification to homogeneity of a soluble
PLA2 from developing elm seeds is, to our
knowledge, the first purified PLA2 enzyme from a
plant. The amount of this enzyme is extremely low in the plant tissue
and a purification factor of 180,000-fold was needed to obtain a
homogenous protein, as judged by SDS-PAGE. A crucial part of the
purification protocol was the use of reverse-phase chromatography,
which has also been used successfully in the purification of several
secretory PLA2s from animal tissues (Tojo et al.,
1984; Forst et al., 1986; Kramer et al., 1989).
The purified protein appeared homogeneous when analyzed by SDS-PAGE,
and activity recovered from unreduced samples coincided with a single
protein band with a molecular mass of 15 kD. A positive control of
PLA2 from cobra venom showed that the method of
recovering PLA2 activity from unreduced SDS-PAGE
gels also functioned well for animal secretory
PLA2, with a comparable recovery of activity. When analyzed by MALDI-TOF MS, the purified fraction gave a single major peak of 13,900 D and two minor peaks of 13,200 and 12,700 D. All
three proteins gained about the same amount of mass after alkylation of
Cys residues, indicating 12 to 14 Cys residues in each protein. The
fact that all three proteins have a similarly high number of Cys
residues suggests that they are related, which was also strongly
supported by the N-terminal amino acid sequencing, in which a
background signal gave a sequence implying that these minor proteins
were proteolytic products of the major protein. The calculated
reduction in molecular mass from the native protein caused by these
cleavages corresponded to about 690 and 1230 mass units, respectively.
These figures matched the molecular mass differences between the major
and the two minor peaks obtained by MALDI-TOF MS of the purified
fraction. The main signal in the N-terminal amino acid sequencing of
the reduced and alkylated purified protein yielded a 53-residue-long
sequence. When used as a query for the BLAST program at the National
Center for Biotechnology Information, the N-terminal amino acid
sequence gave best matches with the deduced amino acid sequences of
some rice EST clones. The general identity score obtained was 62%,
indicating that they are closely related. The general identity is
highest from residue 32 of the elm N-terminal sequence and downstream.
Only 2 of the 21 last amino acid residues in the elm N-terminal
sequence are different in the rice sequences. This highly conserved
area in the elm and putative rice PLA2s includes
the conserved Ca2+-binding loop motif of animal
secretory PLA2s and a short stretch between the
Ca2+-binding loop and the active-site region of
animal secretory PLA2s. Furthermore, all six Cys
residues that were found in the elm N-terminal sequence are conserved
in the rice sequences. In animal secretory PLA2s,
the Cys residues, in addition to the Ca2+-binding
loop and the active-site region, are known to be conserved within the
different subgroups (Dennis, 1994).
When the elm and putative rice PLA2 sequences are
compared with sequences of animal secretory
PLA2s, the general identity scores are not
very high. However, the catalytically important motifs of animal
secretory PLA2s, the
Ca2+-binding loop and the active-site motifs, are
both found in the rice sequences. The active site is positioned
downstream of the elm N-terminal sequence. Only one of the Cys residues
found outside of the catalytic motif in the elm enzyme is conserved in
animal PLA2 sequences. This might indicate that,
although the catalytic areas of both plant and animal plant
PLA2s are conserved, the overall
three-dimensional structure and the disulfide arrangement outside of
these areas might be quite different. This has been shown to be the
case for the structural relationship between bee venom and animal
secretory group I and II PLA2s (Scott et al., 1990).
The biochemical and catalytic properties of the elm
PLA2 are similar to those of secretory
PLA2s found in animal tissues. Well-established
properties of secretory PLA2s are the following: molecular mass of 13 to 15 kD; absolute requirement for
Ca2+ for activity with an optimal concentration
in the millimolar range; stability to heat, acid conditions, and
organic solvents but sensitivity to disulfide-reducing agents such as
DTT and -mercaptoethanol; and specificity for the sn-2
position of phospholipids with no activity toward neutral lipids. The
animal secretory PLA2s are one of the
best-studied classes of enzymes, and the basis of their catalytic
activities has been explained in great detail.
Ca2+, which is bound to a conserved amino acid
region called the "Ca2+-binding loop," has
been shown to participate in the catalytic site by stabilizing the
transition state (Scott et al., 1990). The extreme stability of the
enzyme is due to the high number of disulfide bonds giving it a
cross-linked architecture. The N-terminal sequence of the elm
PLA2 is rich in Cys residues, and the MS data
indicate the likely presence of six disulfide bridges in the entire
protein. Most of the animal secretory PLA2s have seven disulfide bonds, although recent findings have shown
PLA2s with six or eight (Chen et al., 1994a,
1994b).
The biochemical similarity between the elm PLA2
and the animal PLA2s (the latter being
extracellular enzymes), as well as the homology of the elm enzyme with
the rice putative PLA2s (which have putative
signal peptide sequences for secretion), indicate that the elm enzyme
is likely to be a secreted protein. Our original suggestion for the
physiological role of PLA2 activities in
developing seeds was the removal of unusual fatty acids from membrane
lipids for their subsequent incorporation into triacylglycerols (Ståhl et al., 1995). The triacylglycerol synthesis is believed to take place
on the cytosolic side of ER membranes, and an involvement of
PLA2 in this metabolism would require a cytosolic
or ER membrane-bound localization. However, disulfide bonds are not
expected to occur in a protein that is fully exposed to the reducing
conditions of the cytosol (Davidson and Dennis, 1990). Furthermore, the
pronounced acyl specificity that we found for the
PLA2 activity in microsomal preparations of the
developing elm seeds (Ståhl et al., 1995) was not observed for the
purified enzyme. The purified elm PLA2 hydrolyzes
PC with caproyl groups only 2 times faster than with palmitoyl groups.
The secretion of a protein out to the cell wall would be a
translocation to an acidic environment with a pH of 5.0 to 6.0. The
purified elm PLA2, like most animal secretory
PLA2s, has an alkaline pH optimum and minimal activity
below pH 6.0. The elm enzyme, if it was secreted, would most likely be
inactive in the cell wall. It is therefore difficult to speculate about
the function of this type of enzyme in plant tissues. In animal tissues
secretory, nonpancreatic PLA2s have been studied
intensively during the last decade, but their functions have not been
conclusively established. Nevertheless, the identification of plant
PLA2 enzymes and genes, and studies of their
patterns of occurrence and expression, will yield new information that
certainly will help us to understand their function in plant tissues.
| |
FOOTNOTES |
1
This research was supported by the Swedish
Agricultural and Forestry Research Council, the Swedish Foundation for
Strategic Research, the Swedish Farmers Research Foundation, and
Metapontum Agrobios (Metaponto, Italy).
*
Corresponding author; e-mail ulf.stahl{at}vbiol.slu.se; fax
46-18-673279.
Received December 1, 1997;
accepted February 6, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DAG, 1,2-diacyl-sn-glycerol.
EST, expressed sequence tag.
LPC, lysophosphatidylcholine.
MALDI-TOF, matrix-assisted laser-desorption ionization-time-of-flight.
PC, 3-snphosphatidylcholine.
PLA2, phospholipase A2.
TFA, trifluoroacetic acid.
| |
ACKNOWLEDGMENTS |
Professor Lennart Kenne, Dr. Suresh Gohil, and Susanna Broberg
(Department of Chemistry, Swedish University of Agricultural Sciences) are acknowledged for help with MALDI-TOF MS analysis.
| |
LITERATURE CITED |
Bafor M,
Smith MA,
Jonsson L,
Stobrt AK,
Stymne S
(1991)
Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean endosperm.
Biochem J
280:
507-514
Bafor M,
Smith MA,
Jonsson L,
Stobart AK,
Stymne S
(1993)
Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae.
Arch Biochem Biophys
303:
145-151
[CrossRef][ISI][Medline]
Banas A,
Johansson I,
Stymne S
(1992)
Plant microsomal phospholipases exhibit preference for phosphatidylcholine with oxygenated acyl groups.
Plant Sci
84:
137-144
[CrossRef]
Bergey DR,
Howe GA,
Ryan CA
(1996)
Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.
Proc Natl Acad Sci USA
93:
12053-12058
[Abstract/Free Full Text]
Bligh EG,
Dyer WJ
(1959)
A rapid method of total lipid extraction and purification.
Can J Biochem Physiol
37:
911-917
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Brigthman AO,
Zhu XZ,
Morrè DJ
(1991)
Activation of plasma membrane NADH oxidase activity by products of phospholipase A.
Plant Physiol
96:
1314-1320
[Abstract/Free Full Text]
Buck MA,
Olah TA,
Weitzman CJ,
Cooperman BS
(1989)
Protein estimation by the product of integrated peak area and flow rate.
Anal Biochem
182:
295-299
[CrossRef][Medline]
Chen J,
Engle SJ,
Seilhamer JJ,
Tischfield JA
(1994a)
Cloning and recombinant expression of a novel human low molecular weight Ca2+-dependent phospholipase A2.
J Biol Chem
269:
2365-2368
[Abstract/Free Full Text]
Chen J,
Engle SJ,
Seilhamer JJ,
Tischfield JA
(1994b)
Cloning and characterization of novel rat and mouse low molecular weight Ca2+-dependent phospholipase A2s containing 16 cysteines.
J Biol Chem
269:
23018-23024
[Abstract/Free Full Text]
Creelman RA,
Mullet JE
(1997)
Biosynthesis and action of jasmonates in plants.
Annu Rev Plant Physiol Plant Mol Biol
48:
355-381
[CrossRef][ISI][Medline]
Davidson FF,
Dennis EA
(1990)
Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms.
J Mol Evol
31:
228-238
[CrossRef][ISI][Medline]
Dennis EA
(1994)
Diversity of group types, regulation, and function of phospholipase A2.
J Biol Chem
269:
13057-13060
[Free Full Text]
Forst S,
Weiss J,
Elsbach P
(1986)
Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate.
Biochemistry
25:
8381-8385
[CrossRef][Medline]
Hirayama T,
Ohto C,
Mizoguchi T,
Shinozaki K
(1995)
A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana.
Proc Natl Acad Sci USA
92:
3903-3907
[Abstract/Free Full Text]
Huang AHC (1987) Lipases. In PK Stumpf, ed, The
Biochemistry of Plants, Vol 9. Academic Press, New York, pp 91-119
Kanda P,
Wells MA
(1981)
Facile acylation of glycerophosphocholine catalyzed by trifluoroacetic anhydride.
J Lipid Res
22:
877-879
[Abstract]
Kawakita K,
Senda K,
Doke N
(1993)
Factors, affecting in vitro activation of potato phospholipase A2.
Plant Sci
92:
183-190
[CrossRef]
Kim DK,
Lee HJ,
Lee Y
(1994)
Detection of two phospholipase A2 (PLA2) activities in leaves of higher plant Vicia faba and comparison with mammalian PLA2's.
FEBS Lett
343:
213-218
[CrossRef][ISI][Medline]
Klucis E,
Polya GM
(1987)
Calcium-independent activation of two plant leaf calcium-regulated protein kinases by unsaturated fatty acids.
Biochem Biophys Res Commun
147:
1041-1047
[Medline]
Kramer RM,
Hession C,
Johansen B,
Hayes G,
MacGray P,
Chow EP,
Tizard R,
Pepinsky RB
(1989)
Structure and properties of a human non-pancreatic phospholipase A2.
J Biol Chem
264:
5768-5775
[Abstract/Free Full Text]
Martiny-Baron G,
Scherer GFE
(1989)
Phospholipid-stimulated protein kinase in plants.
J Biol Chem
264:
18052-18059
[Abstract/Free Full Text]
Moreau RA,
Morgan CP
(1988)
Proteolytic activation of a lipolytic enzyme activity in potato leaves.
Plant Sci
55:
205-211
[CrossRef]
Neuhoff V,
Arold N,
Taube D,
Ehrhart W
(1988)
Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.
Electrophoresis
9:
255-262
[CrossRef][ISI][Medline]
Palmgren MG,
Sommarin M,
Ulvskog P,
Jørgensen PL
(1988)
Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2.
Physiol Plant
74:
11-19
[CrossRef]
Pappan K,
Qin W,
Dyer JH,
Zheng L,
Wang X
(1997a)
Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis.
J Biol Chem
272:
7055-7061
[Abstract/Free Full Text]
Pappan K,
Zheng S,
Wang X
(1997b)
J Biol Chem
272:
7048-7054
[Abstract/Free Full Text]
Roy S,
Pouénat ML,
Caumont C,
Cariven C,
Prévost MC,
Esquerré-Tugayé MT
(1995)
Phospholipase activity and phospholipid patterns in tobacco cells treated with fungal elicitor.
Plant Sci
107:
17-25
[CrossRef]
Sahsah Y,
Thi ATP,
Roy-Macauley H,
d'Arcy-Lameta A,
Repellin A,
Zuily-Fodil Y
(1994)
Purification and characterization of a soluble lipolytic acylhydrolase from cowpea (Vigna unguiculata L.) leaves.
Biochim Biophys Acta
1215:
66-73
[Medline]
Scott DL,
White SP,
Otwinowski Z,
Yuan W,
Gelb MH,
Sigler PB
(1990)
Interfacial catalysis: the mechanism of phospholipase A2.
Science
250:
1541-1546
[Abstract/Free Full Text]
Seilhamer JJ,
Pruzanski W,
Vadas P,
Plant S,
Miller JA,
Kloss J,
Johnson LK
(1989)
Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid.
J Biol Chem
264:
5335-5338
[Abstract/Free Full Text]
Ståhl U,
Banas A,
Stymne S
(1995)
Plant microsomal phospholipid acyl hydrolases have selectivities for uncommon fatty acids.
Plant Physiol
107:
953-962
[Abstract]
Ståhl U,
Ek B,
Banas A,
Lenman M,
Sjödahl S,
Stymne S
(1997)
Purification and characterization of a microsomal phospholipase A2 from developing elm seeds.
In
JP Williams,
MU Khan,
NW Lem,
eds, Physiology, Biochemistry and Molecular Biology of Plant Lipids.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 244-246
Teissère M,
Borel M,
Cailoll B,
Nari J,
Gardies AM,
Noat G
(1995)
Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds.
Biochim Biophys Acta
1255:
105-112
[Medline]
Tischfield JA
(1997)
A reassessment of the low molecular weight phospholipase A2 gene family in mammals.
J Biol Chem
272:
17247-17250
[Free Full Text]
Tojo H,
Teramoto T,
Yamano T,
Okamoto M
(1984)
Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography.
Anal Biochem
137:
533-537
[Medline]
Vadas P,
Wasi S,
Movat HZ,
Hay JB
(1981)
Extracellular phospholipase A2 mediates inflammatory hyperaemia.
Nature
293:
583-585
[CrossRef][Medline]
Waite M (1987) The phospholipases. In DJ Hanahan, ed,
Handbook of Lipid Research, Vol 5. Plenum Press, New York, pp 111-133
Wessel D,
Flügge UI
(1984)
A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.
Anal Biochem
138:
141-143
[CrossRef][ISI][Medline]
Yotsushima K,
Mitsui T,
Takaoka T,
Hayakawa T,
Igaue I
(1993)
Purification and characterization of membrane-bound inositol-specific phospholipase C from suspension-cultured rice (Oryza sativa L.) cells.
Plant Physiol
102:
165-172
[Abstract]
This article has been cited by other articles:
|
|
|
|
 
H. Y. Lee, S. C. Bahn, Y.-M. Kang, K. H. Lee, H. J. Kim, E. K. Noh, J. P. Palta, J. S. Shin, and S. B. Ryu
Secretory Low Molecular Weight Phospholipase A2 Plays Important Roles in Cell Elongation and Shoot Gravitropism in Arabidopsis
PLANT CELL,
September 1, 2003;
15(9):
1990 - 2002.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Holk, S. Rietz, M. Zahn, H. Quader, and G. F.E. Scherer
Molecular Identification of Cytosolic, Patatin-Related Phospholipases A from Arabidopsis with Potential Functions in Plant Signal Transduction
Plant Physiology,
September 1, 2002;
130(1):
90 - 101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Jung and D. K. Kim
Purification and Characterization of a Membrane-Associated 48-Kilodalton Phospholipase A2 in Leaves of Broad Bean
Plant Physiology,
July 1, 2000;
123(3):
1057 - 1068.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. Narváez-Vásquez, J. Florin-Christensen, and C. A. Ryan
Positional Specificity of a Phospholipase A Activity Induced by Wounding, Systemin, and Oligosaccharide Elicitors in Tomato Leaves
PLANT CELL,
November 1, 1999;
11(11):
2249 - 2260.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. H. Gelb, E. Valentin, F. Ghomashchi, M. Lazdunski, and G. Lambeau
Cloning and Recombinant Expression of a Structurally Novel Human Secreted Phospholipase A2
J. Biol. Chem.,
December 15, 2000;
275(51):
39823 - 39826.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Dahlqvist, U. Stahl, M. Lenman, A. Banas, M. Lee, L. Sandager, H. Ronne, and S. Stymne
Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants
PNAS,
June 6, 2000;
97(12):
6487 - 6492.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
