Characterisation of lipoxygenase isoforms from olive callus cultures

Article abstract of DOI:10.1016/j.phytochem.2008.08.001

Two lipoxygenase isoforms from olive callus cultures were separated from each other. Acetone powders were made to stabilise activity and remove lipids. Separation was then achieved by salt precipitation and ion-exchange chromatography. Both isoforms had comparable activity with linoleic and α-linolenic acid substrates, a basic pH optimum and had molecular masses of around 95 kDa. The callus extracts preferentially formed the 13-hydroperoxy products, in keeping with the pattern of volatile derivatives found in olive tissues and oils derived therefrom.

Full text of DOI:10.1016/j.phytochem.2008.08.001

Phytochemistry 69 (2008) 2532–2538
Contents lists available at ScienceDirect
Phytochemistry
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Characterisation of lipoxygenase isoforms from olive callus cultures
1
*
Mark Williams , John L. Harwood
School of Biosciences, Cardiff University, Cardiff CF10 3AX, Wales, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 April 2008
Received in revised form 31 July 2008
Available online 13 September 2008
Two lipoxygenase isoforms from olive callus cultures were separated from each other. Acetone powders
were made to stabilise activity and remove lipids. Separation was then achieved by salt precipitation and
ion-exchange chromatography. Both isoforms had comparable activity with linoleic and a-linolenic acid
substrates, a basic pH optimum and had molecular masses of around 95 kDa. The callus extracts prefer-
entially formed the 13-hydroperoxy products, in keeping with the pattern of volatile derivatives found in
olive tissues and oils derived therefrom.
Keywords:
Olea europaea
Olive callus
Ó 2008 Elsevier Ltd. All rights reserved.
Lipoxygenase isoforms
Purification
Properties
1. Introduction
of these enzymes in higher plants is more complex. LOXs are in-
volved in a number of functions, including defence (Porta et al.,
Lipoxygenases (LOXs) (EC 1.13.11.12) are a class of non-haem,
iron-containing dioxygenases that use molecular oxygen for the
oxygenation of polyunsaturated fatty acids with a 1,4-cis, cis penta-
diene moiety. LOXs are ubiquitous in the plant and animal king-
doms. In plants, the products of the reaction are conjugated cis,
trans hydroperoxy derivatives, which are highly reactive and toxic
to cells. They are rapidly metabolised to non-toxic but physiologi-
cally-active components (see Hildebrand, 1989; Gardner, 1991;
Siedow, 1991; Porta and Rocha-Sosa, 2002; Wasternack, 2007).
There are two distinct pathways in higher plants which lead to
either traumatin or jasmonic acid production, both of which are
known to have regulatory roles. By-products of traumatin forma-
tion include short chain aldehydes which can be further metabo-
lised to form numerous volatile oxylipins (Blée, 1998; Feussner
and Wasternack, 1998). In addition to the above mentioned path-
ways for fatty acid hydroperoxide metabolism (catalysed by allene
oxide synthase and hydroperoxide lyase) there are several other
potential catabolic routes for utilisation (Hildebrand et al., 2000;
Stumpe et al., 2008).
1999; González-Aquilar et al., 2004; Prost et al., 2005; Nemchenko
et al., 2006; Mita et al., 2007), signal transduction (Blokhina et al.,
2003; Kourtchenko et al., 2007), plant growth and development
(Hildebrand et al., 2000: Kolomiets et al., 2001; Santino et al.,
2003; Vellosillo et al., 2007) and fruit ripening (Zhang et al.,
2006). Major roles for products of the lipoxygenase pathways are
in defence against pathogen attack and wounding (Croft et al.,
1993; Fournier et al., 1993; Vick, 1993; Creelman and Mullet,
1995; Blée, 1998; Weber et al., 1999) and senescence (Siedow,
1991; Paliyath and Droillard, 1992; Page et al., 2001; Leverentz
et al., 2002). LOXs may also function as vegetable storage proteins
in soybean leaves and developing organs (Tranbarger et al., 1991;
Fischer et al., 1999). In addition, they have been reported to be in-
volved in lipid reserve mobilisation from lipid bodies (Feussner
and Kindl, 1992; Feussner et al., 1996, 2001).
An understanding of the cellular and subcellular localisation of
LOXs can often help to determine their physiological functions. The
majority of LOXs which have been characterised are, in fact, soluble
(Siedow, 1991) but there is increasing evidence to suggest that
LOXs are diversely distributed in subcellular organelles as well as
being membrane-bound (Todd et al., 1990; Bowsher et al., 1992;
Feussner and Kindl, 1992; Nellen et al., 1995; Stephenson et al.,
1998). Further confusion occurs because LOXs often exist as multi-
ple isoforms (Domoney et al., 1990; Todd et al., 1990; Siedow,
1991; Chen et al., 2004). The compartmentalisation of the isoforms
may well be associated with distinct physiological functions.
There is considerable interest in the food industry concerning
plant LOXs because the oxylipin by-products have characteristic
tastes and smells (desirable and undesirable). LOX activity in
In animals, the LOX pathway is responsible for the production of
the physiologically-active leukotrienes and lipoxins. Whereas the
physiological role of mammalian LOXs have been clearly delin-
eated (Funk, 1996; Yamamoto et al., 1997; Brash, 1999), the role
Abbreviations: LOX, lipoxygenase; HPO, hydroperoxy.
* Corresponding author. Tel.: +44 (0) 29 2087 4108; fax: +44 (0) 29 2087 4116.
E-mail address: Harwood@Cardiff.ac.uk (J.L. Harwood).
1
Present address: St. George’s University School of Medicine, P.O. Box 7, Grenada,
West Indies.
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.08.001

M. Williams, J.L. Harwood / Phytochemistry 69 (2008) 2532–2538
2533
tomatoes (Smith et al., 1997), barley (Doderer et al., 1992; Holtman
et al., 2000), leek (Nielsen et al., 2004), potato leaves (Salas et al.,
2005), oilseed rape (Terp and Brandt, 2000) and soybean (Saravitz
and Siedow, 1995) have been investigated, amongst other impor-
tant food crops. Virgin olive oil is recognised as one of the finest
vegetable oils because of its delicious taste (Aparicio, 2000). It is
also particularly appreciated for its organoleptic properties which
are derived from the by-products of the LOX pathway. Moreover,
sensory analysis of the volatiles in olive oil has led to the identifi-
cation of many of the constituent components including (Z)-2-hex-
enal, 3-hexenal and 3-methylbutanal which each contribute to
virgin olive oil’s sweet and fruity aroma (Morales et al., 1994)
and are derived directly from activity of the LOX pathway. LOX
activity has been found in virgin olive oil (Georgalaki et al.,
1998a, b) as well as in olive fruit (Olea europaea) (Salas and
Sanchez, 1998a; Georgalaki et al., 1998b; Ridofi et al., 2002). LOX
activity in olive fruits (Salas et al., 1999) and callus has been previ-
ously reported by the authors with a number of different isoforms
being identified, both soluble and membrane-bound (Williams and
Harwood, 1998; Williams et al., 2000). Such LOXs are thought to
pay a pivotal role in the generation of important sensory compo-
nents of olive oil during extraction and processing (Salas and
Sanchez, 2000). Thus, a better knowledge of the properties of olive
LOXs has clear relevance to industry.
LOXs bound to a column of DEAE Sephadex A50 were eluted
with a gradient of 0–0.5 M sodium chloride (Fig. 1). Two distinct
peaks of LOX activity were detected (designated Peaks 1 and 2)
which were purified 48- and 55-fold from the olive acetone prep-
aration when linoleic acid (pH 8.8) was used as substrate (Table 1).
Similar purification values were found when
a-linolenic acid was
used at pH 8.0 (data not shown). Recovered activity was also dis-
tributed evenly between the two peaks and the pH optima were
unchanged for the two substrates, remaining at 8.8 and 8.0 for lin-
oleic and
a-linolenic acids, respectively (Fig. 2) which were in
agreement with previously published data for crude acetone pow-
ders of olive tissue cultures (Williams et al., 2000). At pH 8.0 there
was a minor peak of activity with linoleic acid which we suggest
may be as a result of some activity when the enzyme is in a confor-
mation that favours activity with a-linolenate. Indeed, because the
pK values for the two substrates are very similar, one presumes
that the subtle differences in pH optima for the two substrates is
due to alteration in the 3-dimensional architecture of the LOX pro-
tein(s). The total recovery of activity of the partially purified LOX
fractions was 10–19% in different preparations which was ex-
pected, due to the unstable nature of many plant LOXs. These val-
ues are similar to recoveries reported for other plant tissues such
as tomato fruit (Smith et al., 1997) and barley (Doderer et al.,
1992).
In order to gain more information about such enzymes, we have
used tissue culture systems which not only provide reliable plant
material to use in enzymological and metabolic studies (Williams
and Harwood, 2000) but also appear to have low levels of proteases
and enzyme inhibitors (Rutter et al., 1997) thus making enzyme
purification easier. Previous work (Williams et al., 2000) had
shown that the highest olive callus LOX activity towards both lin-
oleic and
a-linolenic acids was soluble and so we concentrated on
this fraction for further characterisation. Here we report informa-
tion on two isoforms which both have comparable activities
toward the two main endogenous substrates, linoleic and
a-linolenic acids.
2. Results and discussion
2.1. LOX purification
Acetone powders were found to be necessary for optimisation
of LOX extraction from olive callus and preservation of activity. En-
zyme activities in olive fruit (Salas and Sanchez, 1998b) and olive
callus (Williams et al., 2000) have been shown previously to be
maintained by this extraction method. Solubilised acetone pow-
ders were subject to ammonium sulphate precipitation as a first
step of LOX purification. A 30-60% cut contained the majority of
the activity and this was used for ion-exchange chromatography
(Table 1). After testing a number of methods to preserve activity,
it was found eventually that, following ammonium sulphate pre-
cipitation and ion-exchange purification, introducing 30% (v/v)
glycerol into the final storage solution provided the best conditions
for stabilisation of LOX activity.
Fig. 1. Elution profile of soluble lipoxygenase isoforms from olive callus on a DEAE
Sephadex A50 anion-exchange column. The column was loaded with a 30–60%
ammonium sulphate cut as detailed under Section 4.3 and assayed with linoleic
acid at pH 8.8. The profile is typical of three separate experiments.
Table 1
Purification of LOX isoforms from acetone powders of olive callus
Fraction
Protein (mg)
Total activity (
l
moles/s)
Specific activity (
l
moles/s/mg protein)
Recovery (%)
Purification (-fold)
Km values (lM)
Crude extract
883
217
23
1.2
0.9
151 (177)
75 (89)
47 (53)
9.8 (10.7)
8.5 (7.1)
0.17 (0.20)
0.34 (0.40)
2.0 (2.3)
8.2 (8.9)
9.4 (7.9)
100 (100)
50 (50)
31 (30)
6.5 (6)
1 (1)
2 (2)
12 (11)
48 (46)
55 (39)
n.m
n.m.
n.m
80
30% (NH4)2SO4 supernatant
30-60% (NH4)2 SO4 cut
ANION DEAE A50 (Peak 1)
ANION DEAE A50 (Peak 2)
5.6 (4)
43
Data represent means (n = 3). Fractions were assayed at pH 8.8, the optimal value for linoleic acid. Numbers in parenthesis represent incubations for a-linolenic acid at pH 8.0.

2534
M. Williams, J.L. Harwood / Phytochemistry 69 (2008) 2532–2538
characterisation of some of the basic properties. Accordingly, the
two peaks were examined and both were found to contain LOXs
of around 95 kDa, as judged by Western blotting (Fig. 4) and gel
filtration (Fig. 5). These results were in good agreement with
previously published data for other plant LOXs such as soybean
(Shibata et al., 1988), pea (Ealing and Casey, 1989), tomato
(Bowsher et al., 1992), cucumber (Feussner and Kindl, 1994),
parsley (Noehringer et al., 2000) and oilseed rape (Terp and Brandt,
2000).
However, Western blots using polyclonal antisera raised against
pea LOX showed cross-reactivity with a 50 kDa peptide (Fig. 4)
even though the crude acetone powder showed little cross-reactiv-
ity except at 95 kDa. Peptides smaller than 90–95 kDa plant LOXs
have been reported before and, because they were inactive, were
assumed to be degradation products. Thus, van Aarle et al.
(1991) found two fragments of around 63 kDa in addition to a
90 kDa LOX in barley seeds, Doderer et al. (1992) detected a
65 kDa protein in barley leaves and Terp and Brandt (2000) found
an 80 kDa polypeptide in oilseed rape. However, gel filtration of
olive LOX isoforms (Fig. 5) showed that the band corresponding
to 50 kDa proteins retained some, albeit low, activity. Unfortu-
nately, this activity was highly unstable and attempts to purify
or analyse it further were unsuccessful. It has been shown previ-
ously that soybean LOXs, for instance, contain an active site which
is embedded deep within the molecular structure (Boyington et al.,
1993) which might account for retained but reduced activity in the
50 kDa polypeptide. Therefore, in the absence of further evidence
Fig. 2. pH profile of Peak 2 for LOX activity from a DEAE Sephadex A50 anion
column assayed with linoleic or a-linolenic acid substrates.
When linoleic acid was used as substrate, Peak 1 had an appar-
ent Km value of 80 M at pH 8.8 whereas Peak 2 had a Km value of
43 M (Table 1). Together with their effective separation, the dis-
l
l
tinct Km values suggested that these two peaks might represent
isoforms. In contrast to data with linoleic acid (data not shown),
Michaelis–Menton kinetics were never obeyed when
a-linolenic
acid was used as substrate and sigmoidal curves were repeatedly
observed. The Hill equation was used with Chemstation Micromath
Scientist Software (Fig. 3) and suggested a co-operative effect (Hill
constant = 3.15 0.06). We have no explanation for this phenome-
non and, without detailed knowledge of any olive lipoxygenase, it
would seem premature to speculate further. Nevertheless, the sig-
moidal kinetics, even though interesting, precluded any meaning-
Fig. 4. SDS-PAGE and immunological identification of olive callus lipoxygenases.
Lane 1, molecular mass markers: phosphorylase
B 97.4 kDa, albumin (bovine
serum) 66.2 kDa, ovalbumin 45 kDa; lane 2, soluble LOX fraction (peak 1) eluted
from anion exchange chromatography; lane 3, soluble LOX fraction (50 kDa) eluted
from Sephadex G150 column.; lane 4, immunological identification of LOXs from
solubilised acetone powders.
ful calculation of Km values for a-linolenic acid.
2.2. Further properties of the LOX isoforms
The purpose of this study was not to purify any olive LOX iso-
form to homogeneity but to achieve sufficient purification to allow
Fig. 5. Elution profile of 30–60% ammonium sulphate fraction of olive callus
lipoxygenase activity from
a Sephadex G-150 column. The profile shown is
Fig. 3. Determination of enzyme activity for Peak 2 for LOX activity from a DEAE
Sephadex A50 anion column and assayed with a-linolenic acid at pH 8.0 (n = 6).
representative of three separate purifications. The column was calibrated with
standards of known molecular weight. Assay was made with linoleic acid.

M. Williams, J.L. Harwood / Phytochemistry 69 (2008) 2532–2538
2535
Table 2
Radiolabelled hydroperoxy fatty acid and aldehyde production following in vitro incubations of solubilised LOX fractions from resuspended acetone powders of olive callus with
either [U-14C]linoleate or [U-14C]linolenate at optimal pHs
Radioactive HPOs (%)
Substrate
Radiolabelled hexanal d.p.m. (Â10À3/mg protein)
Radiolabelled HPO d.p.m. (Â10À3/mg protein)
13-HPO
75.1 3.1
72.6 4.2
9-HPO
24.9 2.4
27.4 2.7
[U-14C]Linoleate
[U-14C]Linolenate
1.91 0.21
n.d.
2.62 0.31
3.27 0.37
Analyses of HPOs was by TLC and HPLC as detailed under Experimental 3.6.
Data represent means S.D. (where n = 3) for separate experiments. Abbreviation: HPO = hydroperoxy fatty acid; n.d. = not detected.
to the contrary, we assume that this polypeptide fragment repre-
sents a degradation product of one or more of the 95 kDa LOXs
but which still retains its active site.
9-LOX to 13-LOX activities fits well with the balance of volatile
products which can be detected in virgin olive oil (Morales et al.,
1994) and olive tissue cultures (Williams et al., 1998). Thus, we be-
lieve that the pattern of sensory products in olive oils, which are so
important to the latter’s quality (Salas and Sanchez, 2000) is lar-
gely influenced by the regiospecificity of the olive LOX activity.
2.3. Reaction products of LOX activity
The immediate products of the LOX reaction are hydroperoxy
(HPO) fatty acids (Siedow, 1991). We analysed HPO fatty acid pro-
duction using solubilised acetone powders at short incubation
times. Analysis of the HPO products showed that they consisted
of a mixture of 13-HPO and 9-HPO derivatives, with the former
being favoured (approximately 3:1 ratio) (Table 2). The ratio was
consistent throughout the linear accumulation of products in the
10 min incubation period confirming that further metabolism in
the LOX pathway, which could have influenced the ratio, was not
significant. The results are consistent with those obtained from ol-
ive fruit (Salas and Sanchez, 1998a) and with preliminary experi-
ments with acetone powders of calli (Williams and Harwood,
1998) where the ZE/EE ratios were reported. The production of
9- and 13-HPO fatty acids no doubt contributes to the array of vol-
atile oxylipin by-products that have been detected in virgin olive
oil (Morales et al., 1994) and in olive callus (Williams et al.,
1998). Moreover, the preferential production of 13-HPO fatty acids
is consistent with data from Georgalaki et al. (1998b) and with the
high amounts of short chain C6 compounds which characterise vir-
gin olive oil (Morales et al., 1994) and which would be formed fol-
lowing HPO lyase activity in the LOX pathway.
3. Conclusions
We have succeeded in separating two soluble LOX fractions
from olive callus cultures. Both of the partially purified LOXs had
alkaline, but distinct pH optima for either linoleic acid or
a-linole-
nic acid as substrates and had molecular masses of about 95 kDa.
They were clearly different from other olive LOX isoforms which
have acidic pH optima (Salas et al., 1999). Regiospecificity studies
showed that acetone powders of cultures yielded preferentially 13-
hydroperoxy products in agreement with experiments for matur-
ing olive fruits and consistent with the main products of the LOX
pathway detected in tissues or in olive oils derived therefrom.
The simple (2-step) method of LOX purification used here and
the demonstrated possibility of using gel permeation chromatogra-
phy (Fig. 5) further, mean that more work on these important olive
enzymes should be easily accomplished in the future. Moreover,
the apparent sigmoidal kinetics observed when
was incubated at pH 8.0 with Peak 2 LOX isoform, was novel and
merits further investigation.
a-linolenic acid
HPO lyases are responsible for the next step in the LOX path-
way, producing short-chain C6 aldehydes and 12-oxo-(Z)-dodece-
noic acid. Changing the pH to that optimal for HPO lyases (Salas
and Sanchez, 1998b) and prolonging the incubations (see Section
4.5) allowed further metabolism with solubilised acetone powders.
When [U-14C]linoleate was used in the incubation mixture,
4. Experimental
4.1. General experimental procedures
LOX activity was determined spectrophotometrically according
to a method described by Axelrod et al. (1981) using a Hewlett
Packard Diode Array Spectrophotometer incorporating a Hewlett
Packard 8452 UV–vis Chemstation Micromath Software pro-
gramme. Protein concentration was determined as described by
Bradford (1976). [1-14C]Acetate was purchased from GE Healthcare
Ltd. Radioactivity was measured using a 1209 Rackbeta Liquid
Scintillation Counter. Radiolabelled hydroperoxy (HPO) fatty acid
products were analysed by reverse-phase HPLC on a Hypersil-
[
14C]hexanal was detected in the products which was indicative
of hydroperoxy lyase activity deriving from the cultures (Table
2). Apart from hexanal, small amounts of radioactivity were also
found in nonanal, isoamyl alcohol, ethyl acetate and hexylacetate.
When [U-14C]linolenate was substrate, radiolabelled nonenal and
hexenal were also detected. These results were qualitatively simi-
lar to those obtained for tomato fruits by Stone et al. (1975).
As a generalisation, 13-hydroperoxy products are favoured by
most plant tissues (Hildebrand et al., 2000). Plant LOXs differ in
their regioselectivity and catalyse the oxidation of unsaturated
18C fatty acids into either 9- or 13-hydroperoxyoctadecadi (tri)
enoic acids, or a mixture of both, depending on the source of en-
zyme. Often the same tissues can yield both 9- and 13-specific
LOXs. For example, parsley cell cultures have been shown to con-
tain a constitutive 9-LOX and to yield a 13-LOX also when chal-
lenged with a fungal elicitor (Noehringer et al., 2000). Likewise,
in barley seedlings, two isoforms have been described which are
specific for the formation of 9S- and 13S-hydroperoxide deriva-
tives, respectively (Holtman et al., 2000). Moreover, mutagenesis
involving single point mutations can be used to convert a soya
bean 13-LOX into a 9-LOX (Hornung et al., 1999).
C18 column (5
l
m, 4.6 Â 100 mm) (Hewlett Packard). Linoleic acid,
a-linolenic acid, hexanal and soybean lipoxygenase were pur-
chased from Sigma–Aldrich (Poole, Dorset, UK).
4.2. Plant materials
Olive callus cultures (cv. Coratina) were established from sterile
olive pericarps as previously described (Williams et al., 1993). The
calli were routinely subcultured at 30-day intervals onto
Murashige and Skoog medium (Murashige and Skoog, 1962) at
25 °C with a 12 h light/dark cycle. The light was provided by cool
white fluorescent tubes (20
was supplemented with an auxin-cytokinin combination consist-
ing of 2,4-dichlorophenoxyacetic acid (12 M) and benzylamino-
purine riboside (0.56 M) (Williams et al., 1998).
l
mol mÀ2 sÀ1). The growth medium
l
For olive tissues, whether they be cultures (Table 2) or maturing
fruits (Georgalaki et al., 1998b; Salas et al., 1999) the balance of
l

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M. Williams, J.L. Harwood / Phytochemistry 69 (2008) 2532–2538
4.3. Lipoxygenase extraction and purification
saturated (0.2 M) sodium borate buffer for 10 min in order to ana-
lyse LOX products. The pH values were optimal for soluble olive
LOX isoforms with each substrate (see Results and Discussion).
Air was gently bubbled through the mixture (flow rate of
5 ml minÀ1). Autoxidation of the radiolabelled substrate was mon-
itored by using controls containing heat-denatured LOX prepara-
tions. The reaction was stopped with 2 M HCl. The radiolabelled
HPO fatty acid products of the reaction were extracted from the
incubation mixture using diethyl ether and then separated by
TLC on silica gel Merck 60 F254 plates at 4 °C using a solvent system
consisting of hexane/diethyl ether/formic acid (60:40:1, by vol.).
The bands were visualised by UV and were further purified and
identified by HPLC as described by Doderer et al. (1992). The reac-
tion products were then analysed by reverse-phase HPLC on a
Olive callus (50 g) was ground and extracted three times in
500 ml acetone (total volume) at À20 °C using a polytron blender.
The acetone powders were filtered under vacuum, rinsed with
diethyl ether, air-dried and stored desiccated at À70 °C until re-
quired, essentially according to a method of Salas and Sanchez
(1998b). LOX activity has been previously shown to be preserved
completely under these conditions (Williams et al., 2000). Acetone
powders were resuspended using a glass homogenizer in 50 mM
Hepes buffer (pH 7.2) containing dithioerythritol (4 mM) and mag-
nesium chloride (10 mM). Protein was extracted from the homog-
enate by gentle stirring of the suspension for 1 h at 4 °C, followed
by centrifugation at 10,000g for 10 min in order to remove solid
debris. The supernatant was taken to 30% saturation with solid
ammonium sulphate and stirred for 60 min. The suspension was
centrifuged (10000g for 10 min) and the pellet discarded. The
supernatant was then taken to 60% (NH4)2SO4 saturation. After
stirring for another 30 min the suspension was centrifuged as
above. During addition of ammonium sulphate, the pH was care-
fully monitored and adjusted to ensure that it remained at 7.2.
The protein pellet was resuspended in a minimum volume of
20 mM potassium phosphate buffer (pH 7.2).
Any traces of ammonium sulphate were removed from samples
by eluting through a column of Sephadex G-25 previously equili-
brated with 20 mM potassium phosphate buffer (pH 7.2). The sam-
ple was then concentrated by ultra filtration in a Centricon C-30
concentrator and loaded on to a 5 Â 60 cm column of DEAE Sepha-
dex A50 anion column previously equilibrated with 20 mM phos-
phate buffer (pH 7.2). LOXs were eluted with a linear gradient of
0–0.5 M sodium chloride in phosphate buffer with a flow rate of
0.5 ml minÀ1. Gel permeation chromatography (gel filtration) used
a Sephadex G-150 column (5 Â 60 cm) equilibrated with 20 mM
phosphate buffer (pH 7.2). The active fractions were pooled, con-
centrated as described above, then glycerol was added to 30%
(v/v) and stored at À70 °C.
Hypersil-C18 column (5
lm, 4.6 Â 100 mm) (Hewlett Packard)
using isocratic conditions. Column elution was with tetrahydrofu-
ran/methanol/water/acetic acid (25:30:44.9:0.1, by vol.) at a flow
rate of 0.9 ml minÀ1. The HPO fatty acid products were detected
by a UV detector set at 237 nm. The identity of the HPO fatty acids
was confirmed by co-chromatography with standards as follows.
9-HPO standards were prepared by incubating sodium linoleate
(1 ml, 10 mM) with a potato tuber LOX preparation (1 ml) in potas-
sium phosphate buffer (5 ml, 50 mM, pH 7.0) (Schmizu et al.,
1984). The potato tuber LOX was prepared using the same method
as that for the olive LOX extract (see Section 4.2). 13-HPO fatty
acids were prepared enzymatically by incubating sodium linoleate
(500 ll, 10 mM) with soybean LOX (Sigma, lipoxidase type 1-S)
(Gardner, 1997). LOX activity was monitored spectrophotometri-
cally at 234 nm, as described in Section 4.3.
In order to examine further products of the LOX pathway, solu-
bilised acetone powders (500 l
l) were incubated with [U-14C]lino-
leate (pH 8.8) in oxygen-saturated (0.2 M) sodium borate buffer for
10 min as before. Thereafter, the pH was reduced to 7.0 which is
regarded as the optimum pH for hydroperoxide lyase in olive fruit
(Salas and Sanchez, 1998b) and the incubation was allowed to con-
tinue for a further 60 min. The reaction was stopped by lowering
the pH to 4.0 with 2 M HCl. Hydrazone aldehyde derivatives were
prepared by adding 2.5 ml of 0.1% 2,4-dinitrophenylhydrazine to
the mixture. The hydrazone derivatives were then extracted three
times with hexane. These were then separated by TLC using silica
gel Merck G60 plates impregnated with PEG 400 using a solvent
system consisting of hexane/diethyl ether (3:2, by vol.). The deriv-
atives were then analysed by reverse-phase HPLC using a Hypersil-
C18 column with an elution solvent consisting of acetonitrile:
4.4. LOX assays
LOX activity was determined spectrophotometrically at 234 nm
by measuring conjugated diene formation using either sodium lino-
leate or linoleic acid (at pH 8.8) or
a-linolenic acid (at pH 8.0) as
substrates at 25 °C as described by Axelrod et al. (1981). The reac-
tion mixture, which had a total volume of 1 ml, consisted of either
20 mM borate buffer pH 8.8 or pH 8.0 (800
l
l), 10 mM linoleic acid/
water: tetrahydrofuran (45:24:1, by vol.) at a flow rate of
linolenic acid (50 l) and 150 l of enzyme preparation. A sodium
l
l
1 ml minÀ1 (Blée and Joyard, 1996). The compounds were detected
using a UV detector set at 350 nm. The identity of the aldehydes
was confirmed by co-chromatography with authentic standards
(Sigma–Aldrich). Radiolabelled aldehydes and HPO fatty acids
were quantified by means of scintillation counting.
borate buffer system (pH 8.0–9.5) and a sodium phosphate buffer
system were used for establishing pH profiles of olive callus.
4.5. Electrophoresis and Western blotting
Western blotting was as reported previously (Towbin et al.,
1979). SDS-PAGE of the protein preparation was on 7.5% acrylam-
ide gels (Laemmli, 1970). The gels were stained with Coomassie
Blue (0.1%, w/v in 40% methanol, 10% acetic acid) and destained
in 10% methanol, 7.5% acetic acid (v/v). Polyclonal antibodies were
raised against purified pea LOX as a primary antibody and alkaline
phosphatase conjugated anti-rabbit IgG was used as a secondary
antibody.
Acknowledgements
We are grateful to Dr. Franca Angerosa (Instituto Sperimentale
per la Elaiotechnica, Pescara, Italy) for kindly providing olive fruits,
Dr Gareth Griffiths (Aston University, Birmingham, UK) for the gen-
erous donation of the pea LOX antisera and to the EC (AIR 3-93-67)
for financial support.
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