Plant Physiol. Biochem.

39 (2001) 623−630
© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S098194280101275X/FLA

Large scale purification of an almond oleosin

using an organic solvent procedure
Frédéric Beissona, Natalie Fertéa, Robert Voultouryb, Vincent Arondela*
a
Laboratoire de lipolyse enzymatique (CNRS UPR 9025), Institut de biologie structurale et microbiologie (CNRS and
université de la Méditerranée, FR2248), 31, chemin Joseph-Aiguier, 13402 Marseille cedex 20, France
b
6, allée Charles-Perrault, 92160 Antony, France

Received 9 February 2001; accepted 19 March 2001

Abstract – Oleosins are small proteins which are specific to oil-bodies, the plant organelles specialized in oil storage. These
proteins are thought to stabilize the structure and to prevent the oil droplets from coalescing. Since oleosins are insoluble in
water, they are usually purified either as aggregates in water or as polypeptides solubilized with SDS. These conditions raise
major problems for performing biophysical studies, however. We have taken advantage of the solubility of almond oleosin in
chloroform to purify it by differential extraction using solvent mixtures. Detergent-free oleosin was purified to apparent
homogeneity with a purification yield of 20 % using almond meal as starting material. An oil-body suspension was reconstituted
by sonicating the solvent-purified oleosin with phospholipids and triacylglycerols. Protease protection experiments suggest that
oleosins are inserted into the reconstituted oil-body as they are in native organelles. This simple purification procedure allows
to obtain a detergent-free solution of oleosin easy to handle which can be used for oil-body reconstitution and biophysical studies
by the Wilhemly plate method. © 2001 Éditions scientifiques et médicales Elsevier SAS

almond / emulsifying agent / emulsion / oil-body / oleosin / oleosome

DDT, dithiothreitol / EDTA, ethylenediaminetetraacetic acid / FFA, free fatty acid / HPL, human pancreatic lipase /
PAGE, polyacrylamide gel electrophoresis / PMSF, phenylmethylsulphonyl fluoride / SD, standard deviation / SDS,
sodium dodecyl sulphate / TAG, triacylglycerol

1. INTRODUCTION molecular mass of 15 to 26 kDa that are specific to

The oil of most plant seeds is stored in spherical
intracellular organelles 0.5–2 µm in diameter called
oil-bodies or oleosomes [8, 10, 20, 23]. The triacyl-
glycerols (TAGs) present in these organelles are
hydrolysed by lipases during post-germination of the
seeds and the free fatty acids released contribute to the
growth of the seedlings [9, 18]. Based on both experi-
mental evidence and theoretical calculations, a struc-
tural model of the oil-body has been proposed [26]. An
oil-body can be depicted as an oil core (94–98 % w/w)
surrounded by a monolayer of phospholipids (0.6–2 %
w/w) containing embedded proteins (0.6–4 % w/w)
which stabilize the whole structure.
The main proteins of oil-bodies are called oleosins
[11]. They constitute a family of small proteins with
*Correspondence and reprints: fax +33 4 91 71 58 57.
E-mail address: arondel@ibsm.cnrs-mrs.fr (V. Arondel).
oil-bodies [2, 7, 21]. Oleosins are insoluble in water
regardless of the pH and the ionic force conditions, but
they can be solubilized with 1.5 % SDS [15]. At least
two isoforms of oleosins that differ in their amino acid
sequences and are each recognized by specific anti-
bodies have been reported to exist in several species
[27, 28].
Based on sequence analyses, three domains were
predicted to exist in oleosins: two amphipathic stretches
at the N-terminus and C-terminus and a highly con-
served central domain inserted in the TAG core. The
last domain is the longest hydrophobic stretch ever
found to exist in a protein since it ranges between 70
and 80 amino acid residues in length [22, 24, 29]. The
secondary structure of each of the three domains,
especially the central one, however remains unclear
[12, 14, 15, 17]. Oleosins are thought to play a
structural role in preventing oil-bodies from coalesc-
ing [26]. It has also been suggested that they might act
624 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
as an anchor for lipases [19, 30]. Although oleosins are
abundant and can be purified to homogeneity [15], the
methods that have been used up to now are based on
the use of ionic detergents. Since protein preparations
with trace amounts of detergents are unsuitable for
biophysical techniques such as the ones based on the
Wilhemly plate method [16], we have developed a
solvent based method to purify oleosins. We describe
here this purification procedure which yields large
amounts of pure oleosins. We show first that oleosins
can be solubilized from oil-bodies with chloroform
and that it can be purified from defatted almond meal.
Then, experiments of reconstitution of oil-bodies were
carried out using solvent-purified oleosins.
2. RESULTS
2.1. Solubilization in chloroform
of the 16-kDa almond oleosin
Almond oil-bodies were washed as described in
Methods in a NaHCO3 buffer to remove the proteins
bound to their surface as the result of electrostatic
interactions (figure 1A). The 16-kDa almond protein
was identified as an oleosin as it cross-reacted with
specific antibodies directed against macadamia ole-
osins (data not shown).
A suspension (1 v) of isolated oil-bodies was
extracted four times with diethylether (5 v) to remove
the neutral lipids. The upper diethylether phase was
discarded each time. The remaining diethylether present
Figure 1. SDS-PAGE of proteins from almond oil-bodies. The
separating gel was 12.5 % acrylamide. A, Isolated oil-bodies. Lanes 1,
molecular mass standards; 2, non-washed oil-bodies; 3, oil-bodies
washed with 0.1 M NaHCO3. B, Chloroform/methanol extraction of
isolated oil-bodies. Lanes 1, molecular mass standards; 2, interphase
and aqueous phase; 3, organic phase.
in the final aqueous phase and interphase was evapo-
rated under a stream of nitrogen. Five millilitres
chloroform/methanol (2/1, v/v) were added. The mix-
ture was vortexed for 30 s and centrifuged at 3 000 × g
for 5 min and the organic phase saved. The solid
interphase material was washed three times with 1 mL
chloroform/methanol/water (8/4/1, v/v/v). All organic
phases were pooled and completely dried. The solid
residue was dissolved in 100 µL SDS-PAGE denatur-
ation buffer. The remaining interfacial material was
also dissolved in 100 µL of this buffer. SDS-PAGE
analysis (figure 1B) clearly indicates that oleosin
partitions to the organic phase and is almost absent
from the interfacial protein material.
2.2. Purification of the 16-kDa oleosin
from almond meal
For large-scale purification of oleosins, we used a
partially defatted almond meal. This starting material
is more convenient since it contains less oil than
oil-bodies do. All procedures were carried out at room
temperature.
Fifty millilitres chloroform/methanol (2/1, v/v) were
added to 5 g almond meal and mixed in a 80-mL
centrifuge glass bottle using an Ultra Turrax at low
speed (8 000 rpm) for 1 min and then at high speed
(24 000 rpm) for 30 s. The bottle was closed and the
mixture was decanted for 1 h. The liquid upper phase
was filtered through two layers of filter paper previ-
ously rinsed with chloroform/methanol (2/1, v/v).
Equal volumes of filtrate were collected in four 80-mL
centrifugation glass bottles and dried under a stream of
air with strong continuous agitation. The chloroform/
methanol extraction step was repeated twice. Diethyl-
ether (50 mL per bottle) was added and the white solid
material stuck on the surface of the glass bottles was
detached with a metal spatula and resuspended in
diethylether. Water (20 mL per bottle) was added and
the bottles were centrifuged at 1 000 × g for 2 min.
The oleosins are contained in the solid material at the
diethylether/water interface. The upper diethylether
layer which contains lipids was removed with a
Pasteur pipette without disturbing the interfacial mate-
rial. The diethylether extraction step was repeated
twice to remove all the lipid material. The white solid
interfacial material was collected with minimum vol-
umes of water and diethylether and it was transferred
to eight 2-mL polypropylene microtubes, which were
centrifuged at 2 000 × g for 2 min. The lower aqueous
and upper diethylether layer were then removed. The
interfacial material was exposed to a stream of nitro-
gen in order to evaporate all the remaining diethyl
 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
ether. This step is critical since all the organic solvent
needs to be removed but some water must be left in the
sample to allow subsequent recovery of oleosins.
Therefore, care must be taken not to overdry the
sample. The next step in the purification procedure
involved extracting the oleosin from the interfacial
material with chloroform/methanol (95/5, v/v). One
millilitre chloroform/methanol (95/5, v/v) was added
to the interfacial material in each microtube and all the
suspensions were quickly vortexed and transferred to a
glass flask. Filtration allows to separate the 16-kDa
oleosin from a contaminant protein, the molecular
mass of which was 24 kDa (figure 2). This step was
performed as follows. Ten millilitres chloroform/
methanol (95/5, v/v) were added to the pooled suspen-
sions and the mixture was filtered through one layer of
filter paper previously rinsed with chloroform/methanol
(95/5, v/v). The filtrate was collected in ten 4.5-mL
pre-weighed flasks and dried under a stream of nitro-
gen. The oleosin formed a light-yellow vitrous mate-
rial adhering to the surface of the glass flask. Dieth-
ylether (4 mL) was added to each flask and removed
after a few minutes without scraping the surface of the
glass. The diethylether rinse was repeated once and the
flasks were dried under a stream of nitrogen and
weighed. After this step of diethylether rinse, the
preparation of oleosins contained less than 1 % (w/w)
of TAGs and between 5 to 10 % (w/w) of phospholip-
ids as estimated by TLC analysis (data not shown).
The dried oleosin was then dissolved in
chloroform/methanol (95/5, v/v) at a concentration of
5 mg·mL–1 and applied to a column (50 × 1 cm) of
Sephadex LH-60 using chloroform/methanol (95/5,
v/v) as solvent. Oleosins were followed by monitoring
the absorbance at 280 nm in the fractions collected.
The presence of oleosins was checked by SDS-PAGE
analysis. This last step of molecular sieving allowed to
separate oleosins from residual phospholipids. No
phospholipids could be detected in a TLC plate analy-
sis when loading 200 µg oleosins (data not shown).
The amount of residual phosphatidylcholine (if any)
was therefore estimated to be inferior to 0.05 % (w/w),
i.e. 1 % (mol·mol–1). A stock solution of purified
oleosins dissolved in chloroform/methanol (95/5, v/v)
at a concentration of 1 mg·mL–1 was kept at –20 °C
until use.
Using this simple purification procedure, as much as
6 mg oleosin purified to apparent homogeneity was
obtained by extracting 5 g almond meal with an
oleosin content of roughly 6 mg·g–1 almond meal.
Based on this value, the purification yield can be
estimated to be approximately 20 %.
625
Figure 2. SDS-PAGE of almond meal proteins solubilized in organic
solvents. The separating gel was 12.5 % acrylamide. Lanes 1, molecu-
lar mass standards; 2, proteins before the filtration step in the
purification procedure; 3, contaminant protein in the retentate; 4,
purified oleosins in the filtrate.
2.3. Characterization of the purified oleosin
The N-terminal sequence of the protein purified as
described above was found to be blocked as it has been
reported for rapeseed [22] and sunflower oleosins [17].
However, when treated by cyanogen bromide, the
purified almond oleosin was cleaved into two polypep-
tides with molecular masses of about 7–8 kDa (data
not shown). The cleavage site position is consistent
with the 149 amino acid sequence deduced from the
almond oleosin cDNA [5] which contains only one
methionine residue, at position 80. After performing
cyanogen bromide cleavage on pure oleosin, an amino
acid sequence consisting of six residues was obtained
(GFLTSG). This sequence was found to be identical to
the stretch following the methionine residue in the
middle of the oleosin sequence. This result shows
clearly that the protein we purified is the 15.7-kDa
almond oleosin. This is consistent with immunological
and amino acid analysis (data not shown).
Functional assays were then carried out on oleosins
purified using this procedure. For this purpose, we
tested the ability of purified oleosins to form stable
oil-bodies. We attempted to reconstitute some oil-
bodies using a mixture of the main ingredients of
almond oil-bodies in their native proportions as
described in Methods. We obtained lipid globules the
size of which was dependent on the sonication time, as
determined by contrast phase microscopy (data not
shown). Sonicating for 30 s yielded globules that were
roughly the same size as the native oil-bodies, that is
between 1–2 µm. We have checked the stability of the
reconstituted emulsion by turbidimetry (figure 3).
626 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
Figure 3. Turbidimetry of suspensions of oil-bodies (circles), oil-
bodies reconstituted with TAGs, phospholipids and oleosins (squares),
or lipid globules consisting of TAGs and phospholipids (triangles).
The absorbance of the suspensions in the lower part of a 2-mL
spectrophotometric cuvette was read at 600 nm at regular intervals.
Each point gives the mean ± SD, based on triplicate experiments.
When oleosins are omitted from the mixture, the
globules coalesced and tended to float. The turbidity of
the suspension therefore decreased faster than with the
reconstituted or native oil-bodies. Moreover, native
oil-bodies and oil-bodies reconstituted with oleosins
showed similar behaviour. Both were more stable than
the globules devoid of oleosins. These results show
that oleosins solubilized in chloroform can be used to
reconstitute an emulsion whose stability is similar to
oil-bodies.
Reconstituted oil-bodies and native oil-bodies could
be hydrolysed by HPL at about the same rate, but only
in the presence of its physiological cofactor, colipase
(table I). In contrast, the globules reconstituted with-
out oleosins could be hydrolysed by HPL in the
absence of colipase.
Finally, reconstituted oil-bodies were subjected to
digestion by a protease. A polypeptide of 8 kDa was
protected from the digestion (figure 4). The same result
Table I. Lipolysis of oleosomes and reconstituted oleosomes by
human pancreatic lipase (HPL). Five micrograms HPL (with or
without 5 µg colipase) were added to the emulsions under continuous
stirring. The free fatty acids released by HPL were continuously
titrated using the pH-stat technique. Results are expressed as
mean ± SD, based on triplicate experiments.
Emulsion Specific activity Colipase requirement
(µkat·mg–1)
TAG + PL 30.8 ± –3.3 No
TAG + PL + oleosin 12.5 ± –1.1 Yes
Oleosomes 13.8 ± –1.3 Yes
Figure 4. SDS-PAGE analysis of proteins associated with oil-bodies
after treatment by proteinase K. Lanes 1 and 2, reconstituted oil-
bodies; 3 and 4, native oil-bodies; 2 and 3, treatment with proteinase
K; 1 and 4, no treatment with proteinase K.
was obtained with native oil-bodies. The size of the
protease-protected fragment was independent of the
time of incubation (data not shown).
In addition to these experiments, a monolayer of
oleosins could be spread at an air-water interface, on a
Wilhemly plate set-up. Compression/decompression
experiments indicated that the protein film was stable
which means that the sample does not contain any
tensioactive molecules such as detergents (data not
shown).
3. DISCUSSION
We observed on performing SDS-PAGE (figure 1B)
that the 16-kDa oleosin was present in the clear
chloroform phase and not in the interphase of a total
lipid extract from oil-bodies. We therefore purified the
almond oleosin by solubilizing it in chloroform instead
of following previously published procedures in which
protein aggregates are used or the oleosin is solubi-
lized in water with SDS. The solubility of oleosins in
chloroform/methanol might not be a general feature
since oleosins from maize, rice, wheat, rapeseed,
soybean and jojoba were isolated as an insoluble
interfacial material in chloroform/methanol extracts of
oil-body suspensions [26]. Moreover, oleosins could
not be extracted from sunflower meal using the pro-
cedure described here (data not shown). The solubility
of the 15.7-kDa almond oleosin might be due to its
high hydrophobic/hydrophilic balance. Solubilizing
proteins in chloroform/methanol has been previously
used to obtain proteins from chloroplast envelope [25],
pulmonary surfactant [1], brain myelin [3] and other
animal tissues [4].
We found that chloroform-purified oleosins could
indeed be used to reconstitute oil-bodies whose char
 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
acteristics were checked using several criteria. First we
found that the size of reconstituted oil-bodies when
sonicating 30 s is similar to that of native oil-bodies.
Another criteria is the stability of reconstituted oil-
bodies which is comparable to that of native oil-bodies
(figure 3). It is known that the stability of the oil-
bodies is dependant on the integrity of oleosins as
shown by Tzen and Huang [26]. We have also used
human pancreatic lipase (HPL) to probe the properties
of oil-bodies as substrates for lipases. Human pancre-
atic lipase (HPL) is able to hydrolyse on its own the oil
droplets from an oil emulsified with gum arabic.
However, it has an absolute requirement for its physi-
ological cofactor, colipase, to hydrolyse lipids in the
presence of some proteins at the oil/water interface [6].
This is the case with native oil-bodies, which can be
hydrolysed by HPL only in the presence of colipase
(table I). As the TAG/PL globules devoid of oleosins
could be hydrolysed without colipase, it seems likely
that the presence of oleosins at the surface of oil-
bodies may prevent HPL from binding to the oil-body
surface unless colipase is present. This absolute need
for colipase was also observed in the case of reconsti-
tuted oil-bodies. In addition, the specific activities of
HPL/colipase recorded on reconstituted and native
oil-bodies were in the same range and were signifi-
cantly lower than those which occurred on the TAG/PL
globules. This finding is consistent with the results
obtained on the relative stability of the suspensions.
However, this does not demonstrate that the reconsti-
tuted oil-bodies are not a simple emulsion with dena-
tured oleosin lying at the surface of oil droplets. So we
have carried out protease protection experiments. The
results indicate that an 8-kDa polypeptide was pro-
tected from proteinase digestion in both reconstituted
and native oil-bodies (figure 4). This protease-protected
fragment of oleosin has also been observed in native
oil-bodies from maize [26], sunflower and safflower
[12]. It has been shown that the protease-protected
fragment corresponded to the central hydrophobic
domain of oleosins [12], which is somehow inserted in
the oil core and therefore protected from the water-
soluble proteinase. The fact that this fragment was also
observed here in reconstituted oil-bodies strongly
suggests that the oleosins are properly inserted at the
surface of the oil droplets after sonication. Taken
together, these results therefore suggest that the recon-
stituted oil-bodies are structurally similar to the native
ones. Chloroform solutions of oleosins are particularly
convenient to handle in oil-body reconstitution experi-
ments from lipids which are also solubilized in chlo-
roform. Well-controlled reconstituted oil-bodies con-
627
taining homogeneous synthetic lipids and mimicking
the natural hydrophobic environment of oleosins could
therefore be made using purified almond oleosins.
Pure oleosin solubilized in chloroform was obtained
in large amounts from an inexpensive starting material
and without any need to isolate the oil-bodies from the
seed. In addition, the purification procedure allows the
obtention of a detergent-free oleosin solubilized in
chloroform which is convenient to handle. The oleosin
purified using the method described here can therefore
be used in oil-body reconstitution studies or can be
easily be spread at the air/water interface, in the same
way as lipids. Studies using the Wilhemly plate method
[16] are now in progress on the rheological, biochemi-
cal and structural characteristics of monomolecular
films consisting either of pure oleosins or of oleosins
mixed with lipids.
4. METHODS
4.1. Plant material
Almond (Prunus amygdalus, Batsch) seeds from the
‘Nonpareil California’ cultivar were used to isolate
oil-bodies. Partially defatted almond meal made by
grinding the residual cakes remaining after seed oil
extraction was obtained from SICTIA (Marseille,
France). The oil extraction process was performed
using the continuous screwpress technique and did not
involve any organic solvent or enzymic treatment.
4.2. Preparation of oil-bodies
Dry almond kernels were ground using a Waring
blendor at 12 500 rpm for 15 s. The powder was then
homogenized at 4 °C in 0.1 M Tris-HCl (pH 8), 1 mM
EDTA (10 mL·g–1) in a Waring blendor at 12 500 rpm
for 30 s. The homogenate was filtered through one
layer of Miracloth (Calbiochem, La Jolla) and centri-
fuged in 30-mL tubes at 30 000 × g for 20 min at 4 °C.
The white fat pad on top of each tube was collected
and resuspended in 30 mL freshly prepared 0.1 M
NaHCO3, 1 mM EDTA, using an Ultra Turrax at a
speed of 8 000 rpm. The tubes were centrifuged under
the same conditions as those described above and the
oil-bodies were resuspended/centrifuged once in the
same buffer. The fat pad was then recovered and
washed twice in 1 mM Tris-HCl (pH 8), 1 mM EDTA.
The oil-bodies were finally resuspended in a solution
of 1 mM Tris-HCl (pH 8), 150 mM NaCl (7 mL·g–1 fat
pad, i.e. lipid final concentration 100 mg·mL–1).
628 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
4.3. Protein analysis
4.3.1. Protein preparation from oil-bodies
Oil-bodies were suspended in 100 mM Tris-HCl
(pH 8), 1 mM EDTA (50 µL buffer per 1 mg TAGs)
and extracted five times with diethylether (1.5 mL
diethylether added to 200 µL oil-body suspension) by
vortexing the preparation for 10 s and centrifuging it
for 1 min at 16 000 × g. The upper organic phase was
then discarded. The final lower aqueous phases and the
interfacial material were mixed and collected.
4.3.2. SDS-PAGE
The protein samples were left to react with the
denaturating buffer (2 % SDS, 50 mM DTT, 62.5 mM
Tris-HCl (pH 6.8), 10 % glycerol, 1 % bromophenol
blue) for 15 min at room temperature. They were not
heated in order to avoid the irreversible aggregation of
the oleosins. The proteins were separated by perform-
ing SDS-PAGE [13] through a 12.5 % polyacrylamide
gel.
4.3.3. Immunoblotting procedures
Specific antibodies and peroxidase-conjugated goat
anti-rabbit IgG were incubated in PBST (10 mM
phosphate buffer (pH 7.4) containing 150 mM NaCl
and 0.05 % (v/v) Tween 20) for 2 h and 1 h 30 min,
respectively. The peroxidase substrates used were
3,3’-diaminobenzidine and H2O2. Rabbit polyclonal
anti-oleosin antibodies were from the Institut Pasteur
(Lille, France). The antigens used for the immuniza-
tion procedure were oleosins extracted from macad-
amia nuts and purified by molecular sieving in the
presence of SDS.
4.3.4. Protein cleavage
A volume (160 µL) of the stock solution of purified
oleosin corresponding to 10 nmoles protein was dried
under a stream of nitrogen in a 1.5-mL polypropylene
microtube. A solution of 0.15 M CNBr in 70 % (v/v)
formic acid was added (150 µL) and the microtube was
kept closed under nitrogen in darkness at 20 °C for
24 h. Water (150 µL) was then added and the mixture
was dried under vacuum using a Speed Vac apparatus.
The solid residue was washed with 1 mL methanol and
centrifuged at 16 000 × g for 5 min. The pellet was
dried under nitrogen.
4.3.5. N-terminal sequencing
A partial sequence of 10 nmoles protein cleaved as
described above was determined using a gas phase
protein sequencer (Applied Biosystems, model 470 A).
4.4. Reconstitution of oil-bodies
The main constituents of the native oil-bodies used
to reconstitute oil-bodies were as follows: TAGs (98 mg
almond oil), phospholipids (1 mg soybean phosphati-
dylcholine) and pure oleosins (1 mg in 1 mL chloro-
form). The oil, the phospholipids dissolved in chloro-
form and the oleosin solution were mixed and the
chloroform was evaporated under a stream of nitrogen.
A 1-mM Tris-HCl (pH 8)/150 mM NaCl buffer (890 µL)
was added and the mixture was vortexed and sonicated
in a sonicator bath for 30 s.
4.5. Turbidimetry
The absorbance at 600 nm of a 1-mL suspension of
oil-bodies or reconstituted oil-bodies (both having a
lipid content of 100 mg) was read at time intervals in
a spectrophotometer cuvette as described by Tzen and
Huang [26].
4.6. Lipase assay
The release of fatty acids was continuously assayed
potentiometrically with a pH-stat apparatus at 37 °C,
by adding 0.1 M NaOH under mechanical stirring in a
reaction vessel. The 5-mL reaction medium contained
1 mL emulsion (oil-bodies or reconstituted oil-bodies
both containing 100 mg lipids) suspended in 0.3 mM
Tris-HCl, 6 mM CaCl2, 150 mM NaCl (final concen-
trations). The pH was adjusted to 8, and 5 µg HPL
(with or without 5 µg colipase) was added at zero time
after recording the background level for 2 min. Activi-
ties were expressed in katals: 1 katal corresponds to
the release of 1 mole of free fatty acid per second.
4.7. Proteinase digestion and peptide analysis
Digestion was performed on native or reconstituted
oil-bodies. A 0.5-mL suspension of oil-bodies contain-
ing 5 mg lipids in a 25-mM Tris-HCl (pH 8)/150 mM
NaCl/1 mM CaCl2 buffer was incubated at room
temperature during 2 h , under shaking, with 10 units
(0.5 mg powder) proteinase K from Tritirachium album
(Boehringer Mannheim). Digestion was stopped by the
addition of PMSF (final concentration: 1 mM). The fat
pad was then rinsed twice with 1 mL digestion buffer
and once with 1 mL water. After the last rinse, water
was added to the fat pad so that the volume of the
sample was about 100 µL. The sample was finally
extracted three times by 1 mL diethylether and it was
subjected to SDS-PAGE analysis.
Acknowledgments. We thank Dr Daniel Campese
(IBSM, Marseille) for protein sequencing and Josiane
 F. Beisson et al. / Plant Physiol. Biochem. 39 (2001) 623–630
De Caro at the Laboratoire de lipolyse enzymatique
(LLE-Marseille) for the generous gift of purified
colipase and HPL. We thank M. Ivanova and T. Ivanova
for performing Wilhemly plate experiments. The assis-
tance of Dr Jessica Blanc for revising the English
manuscript is acknowledged. We wish to express our
appreciation to Dr Marcel Teissère and Dr Robert
Verger (LLE-Marseille) for helpful discussions and to
Prof. Georges Noat for critically reading the manu-
script. Frédéric Beisson gratefully thanks the Labora-
toires de biologie végétale Yves Rocher (Issy-les-
Moulineaux, France) and the ANRT for his CIFRE
Ph.D. studentship. Part of this work was supported by
the Laboratoires de biologie végétale Yves Rocher.
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