A protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H2O extraction and gel filtration of the extract, followed by two steps of high-performance liquid chromatography. The N-terminal amino acid sequence has been determined up to the 28th residue and found to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP does bind fatty acids, but in contrast, it is not significantly homologous to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH  9.6), and it does not show antibiotic properties

Aalen

Atilio Castagnaro

D&#x00E9

Dieryck

Francisco García-Olmedo

Garc&#x00ED

Jakobsen

Linnestad

Molina

Olsen

Ostergaard

Rickers

Segura

Skriver

Tchang

AbstractA protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H2O extraction and gel filtration of the extract, followed by two steps of high-performance liquid chromatography. The N-terminal amino acid sequence has been determined up to the 28th residue and found to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP does bind fatty acids, but in contrast, it is not significantly homologous to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH < 6.8 vs. pH > 9.6), and it does not show antibiotic properties

Castagnaro, Atilio

García-Olmedo, Francisco

Elsevier - Publisher Connector 

FEBS Letters 349 (1994) 117-l 19 
FEBS 14274 
A fatty-acid-binding protein from wheat kernels 
Atilio Castagnaro **, Francisco Garcia-Olmedo* 
Laboratorio de Bioquimica y Biologia Molecular, ETS Ingenieros Agrdnomos - UPh4, E-28040 Madrid, Spain 
Received 18 April 1994; revised version received 15 June 1994 
Abstract 
A protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H,O extraction and gel filtration of the extract, followed 
by two steps of high-performance liquid chromatography. The N-terminal amino acid sequence has been determined up to the 28th residue and found 
to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a 
non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP does bind fatty acids, but in contrast, it is not significantly homologous 
to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH < 6.8 vs. pH > 9.6), and it does not show antibiotic 
properties. 
Key words: Cereal; Fatty acid binding protein; Plant, Wheat 
1. Introduction 
Transcripts whose pattern of expression coincides with 
the accumulation of lipids in developing aleurone cells 
from barley have been previously investigated [l-4]. A 
cDNA corresponding to one of these transcripts, Bl 1E 
(EMBL X15257), has been improperly classified as a 
non-specific lipid transfer protein, LTP2 [3,4]. Trans- 
genie expression of the coding region of this cDNA in 
tobacco, under a constitutive promoter, resulted in the 
premature loss of flower buds [3]. In the course of a 
systematic purification of low-molecular-mass proteins 
from wheat kernels, we have isolated a polypeptide of 
approximatly 7 kDa, designated W-FABP, whose 
N-terminal amino acid sequence is essentially identical 
to that deduced for the barley transcript. Although 
W-FABP does bind fatty acids, as has been reported for 
LTPs [5,6], it does not share with them their antipatho- 
genie properties and other important features. 
2. Materials and methods 
2. I. Materials 
Kernels from the wheat cv Chinese Spring (Triticum aestivum) were 
ground to a powder on a Culatti mill and used to purify the protein. 
The bacterial pathogen Clavibacter michiganensis subsp. sepedonicus, 
strain C5 was from the ETSIA collection (Madrid). Wheat protein 
CM3 was the gift from Dr. R. Sanchez-Monge (Madrid) and thionin 
*Corresponding author. Fax: (34) (1) 336 5757. 
**Present address.. INSIBIO-Universidad National de Tucumhn, 
Chacabuco 461, Tucuman (4000). Argentina. 
Abbreviations: LTP, lipid transfer protein; RP-HPLC, reverse-phase 
high-performance liquid chromatography; SDS, sodium dodecyl sul- 
fate; PAGE, polyacrylamide gel electrophoresis; NAS, normalized 
alignment score. 
and LTP4 were given by Dr. A. Molina (Madrid). Bovine serum albu- 
min and chymotrypsinogen were from Sigma. Radioactive [1 -‘4C]oleic 
acid was from Amersham. 
2.2. Purification procedure 
Ground wheat kernels (40 g) were extracted once with 4 volumes of 
H,O and the extract was freeze-dried, dissolved in 5 ml of 3 M urea, 
and loaded into a Sephadex G-100 column (2.5 cm x 80 cm; 25 ml/h). 
The selected eluate fraction was dialyzed against 5 1 of H,O, using a 
Spectra/Par 7 (MWCO:2000) membrane (Spectrum Medical Ind. Inc.), 
and freeze-dried. The fraction was then dissolved in H,O/O.l% TFA 
and subjected to RP-HPLC on an ultrapore C3 column (1 x 25 cm; 5pm 
particle; 30 nm pore), using a O-50% (180 min) H,O/acetonitrile gradi- 
ent, 0.1% TFA, at 0.5 ml/min. The fraction indicated in Fig. 1B was 
rechromatographed under the same conditions, except hat 2-propanol 
was used instead of acetonitrile. SDS-PAGE in preformed gradient gels 
(620% acrylamide; Bio-Rad) was carried out according to the manu- 
facturers’ instructions. 
2.3. Fatty acid binding assay 
The protein (10 pug) was incubated with [I-‘4C]oIeic acid essentially 
as described [5,6]. Electrofocusing was carried out on a refrigerated 
horizontal apparatus from Pharmacia, using pH 3-10 Pharmalyte. 
Electrode buffers were I M NaOH and 0.04 M aspartic acid. Electrofo- 
cusing was at 600 V for 45 min and at 2000 V for 11 min. The gel was 
stained for protein with Coomassie blue R, air-dried, and exposed with 
X-ray film. Colored isoelectrofocusing standard from Bio-Rad were 
used. 
2.4. Inhibition tests 
Proteins were dissolved in water and added to microtiter wells prior 
to innoculation. Serial dilutions from 0.1 PM to 10 PM were used. 
Bacteria were added at a final concentration of lo4 colony forming 
units/ml at a final volume of 150 fll (100 PI protein + 50 fl1 nutrient 
broth, Oxoid), incubated for 24 h at 28”C, and growth recorded in an 
ELISA plate reader. 
3. Results and discussion 
Ground wheat kernels were extracted with H,O and 
the freeze-dried extract was subjected to gel filtration, as 
shown in Fig. 1A. Fraction I, eluting with an apparent 
molecular weight of about 7 kDa, was further fraction- 
ated by RP-HPLC (Fig. 1B). Fraction II from this col- 
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved 
SSDI 0014-5793(94)00660-N 
118 
6b 240 364 450 
ELUTION VOLUME t-1) 
-1L 
128 135 22 
TIME bid 
TIME (Din) 
0 
Fig. 1. Purification of W-FABP from wheat kernels. (A) Gel filtration 
of H,O extract. Markers of molecular mass were (a) chymotrypsinogen, 
25 kDa, and (b) thionin, 5 kDa. (B) RP-HPLC fractionation of fraction 
I from above, using acetonitrile in the gradient. (C) RP-HPLC fraction- 
ation of fraction II from B, using 2-propanol in the gradient. 
(D) SDS-PAGE of the indicated fractions. Fraction III from C consists 
of homogeneous W-FABP. 
umn, which contained a component that migrated with 
an apparent molecular mass of 5 kDa in SDS-PAGE 
(Fig. lD), was subjected again to RP-HPLC, but using 
2-propanol in the gradient, instead of acetonitrile 
A. Castagnaro, E Garcia-Olmedol FEBS Letters 349 (1994) 117-l I9 
(Fig. 1C). The fraction designated as III contained the 
apparently homogeneous, fast-moving component, des- 
ignated W-FABP (Fig. 1D). 
The N-terminal amino acid sequence of this compo- 
nent was determined up to the 28th residue and no se- 
quence heterogeneity was observed. Except for residues 
4 and 5, this amino acid sequence from wheat was iden- 
tical to that deduced from a previously reported barley 
cDNA, Bl lE, as shown in Fig. 2. The barley sequence 
has been improperly designated LTP2 (EMBL X69793) 
in a recent publication [4], but we have designated it as 
B-FABP for reasons that will be discussed below. The 
two FABPs have been aligned with LTPl from barley 
[7,8], to show that although they have some features in 
common with LTPs, the probability of homology be- 
tween B-FABP and LTPl is extremely low according to 
the criteria of Doolitle [9]. The normalized alignment 
score (NAS = 120) for the LTPl/B-FABP comparison is 
of the same order as those obtained for comparisons 
between random sequences of the same length, and much 
below the minimum value of NAS = 300 that would be 
required for significance [9]. The position of the N-termi- 
nus of W-FABP suggests that the N-terminus of the 
protein deduced from the Bl 1E cDNA (B-FABP) is 
6 amino acid residues downstream from where it was 
originally proposed [l]. The molecular weight of B- 
FABP protein is 6,989 Da, which is compatible with the 
elution volume of W-FABP in gel filtration, but higher 
than the 5 kDa apparent molecular mass estimated for 
this protein by SDS-PAGE. This is a typical behaviour 
of lipid- binding proteins: ie, LTP4, whose size is 8.8 
kDa, appeared as a 6.5 kDa protein in SDS-PAGE [lo]. 
The ability of the FABP to bind fatty acids was inves- 
tigated by a previously described electrofocusing tech- 
nique [6], using [l-‘4C]oleic acid. A wheat protein (CM3), 
which has hydrophobic domains and is soluble in chloro- 
form/methanol mixtures [ 111, was used as a negative con- 
trol, and bovine serum albumin (BSA) as a positive one. 
As shown in Fig. 3, W-FABP did bind oleic acid in a 
similar manner as LTP4 and its isoelectric point was 
more acidic than that of LTP4 (< 6.8 versus >9.6). A 
highly charged motif in the central part of the sequence 
and a hydrophobic stretch downstream from it have been 
proposed as the phospholipid-binding sites of LTPs [ 121. 
These sequences, which are conserved in LTPs, are not 
obviously present in FABPs. Antibodies raised against 
a mixture of barley LTPs [14], which recognized LTPs 
. . 
W-FABP 
~~~“*~~~~~~~~~~~~~ 
B-FABP 
s~~p~~~~~~~~~~~~~~*~~~~~~~“~~~~~~~”~~~~~~~~~~~~~IPtfPHC 
....... ., ., .,...,._._., .,.,. . B_LTP, *LN~~v~~~~~~~~~~~~~~~~~v~~~~~~~~~~~**~~I_#NLHLNNMS*PSKC)(”NYPDCSRIY 
KPt”Yt LHNQAQSSGDRf 1 YTISPDI 
Fig. 2. N-terminal amino acid sequence of W-FABP aligned with those corresponding to B-FABP, deduced from Bl 1E cDNA [I], and LTPl from 
barley [7,8]. Identical residues are shadowed, gaps (-) and insertions (t) are indicated. A highly similar LTPI sequence from wheat has been reported 
Il71. 
,4. Castagnaro, E Garcla-Olmedo/FEBS Letters 349 (1994) 117-119 119 
pn 
i 
Clq3 BSA LTP4 N-FKBP 
n i l  i i  
S R S R S R S R 
t 
Fig. 3. Binding of radioactive oleic acid to W-FABP. Bovine serum 
albumin (BSA) and barley LTP4 [10] were used as positive controls, 
wheat CM3 [l l] as a negative one. The gel was stained with Coomassie 
blue (S) and then exposed to X-ray film (R). The insertion point is 
indicated (i). 
f rom other species, including spinach and Arabidopsis, 
do not recognize W-FABP (not shown). 
We have recently reported that all LTPs tested are 
potent inhibitors of  plant pathogens [10,13]. W-FABP 
was not active against the test microorganism at up to 
l0 /2M concentration, tenfold higher than those giving 
complete inhibition with LTPs or with thionins [10,13], 
which were used as positive controls in the experiment 
(not shown). 
Based on the above dissimilarities of  W-FABP with 
respect o the LTPs, both in their amino acid sequences 
and in their in vitro properties, it would be advisable that 
the amino acid sequence deduced from the barley B11E 
cDNA should not be identified as a member of  the LTP 
family, as has been proposed (EMBL X69793 and [4]). 
The designation LTP2 has been recently given to a pro- 
tein from barley leaves which is significantly homolo- 
gous to LTP1 [10,14]. 
Barley B 11E mRNA was first detected at 10 days after 
pollination (DAP), reached a maximum at about 25 
DAP, and then declined rapidly to an undetectable vel 
in mature aleurone tissue [1,2], whereas W-FABP is pres- 
ent in and can be readily purified from the mature kernel, 
which suggests that it is stable well beyond the moment 
in which its gene is switched off. This persistence points 
more to a structural role in association with lipids than 
to a catalytic function in lipid metabolism. Further re- 
search will be needed to elucidate the function(s) of  both 
FABPs and LTPs, as their in vitro properties, such as 
binding of  fatty acids, coenzyme-A esters, and phosphol- 
ipids, or lipid transfer between artificial l iposomes and 
mitochondria [5,6,15,16], cannot be extrapolated into the 
in vivo situation. The subcellular location of  LTPs seems 
to be incompatible with some of the metabolic roles pro- 
posed for them (see [14]). The subcellular location(s) of  
FABPs need to be investigated. From the above results 
it can be concluded that W-FABP and B-FABP repre- 
sent a distinct protein family with respect to the LTPs, 
although both families might be involved in lipid metab- 
olism. A definite designation of  this family should wait 
until more is known about their roles in vivo. 
,4cknowledgements. Amino acid sequences have been determined by 
Drs. C. Buesa (Servei Sequenciaci6, Universitat Barcelona) and C. 
Lopez-Ofin (Fac. of Medicine, Universidad de Oviedo). Reading of the 
manuscript by Drs. P. Carbonero and G. Salcedo, and technical ssis- 
tance from D. Lamoneda nd J. Garcia are also acknowledged. Finan- 
cial support was from the Comisi6n Interministerial de Ciencia y 
Tecnologia (Grant PB92~0325). 
References  
[1] Jakobsen, K., Klemsdal, S.S., Aalen, R., Bosnes, M., Alexander, 
D. and Olsen, O.-A. (1989) Plant Mol. Biol. 12, 285-293. 
[2] Olsen, O.-A., Jakobsen, K. and Schmelzer, E. (1990) Planta 181, 
462~,66. 
[3] Olsen, O.-A., Klemsdal, S.S., Aalen, R., Linnestad, C., Lenne- 
borg, A. and Kalla, S.R. Proc. EUCARPIA Symp. on Genetic 
Manipulation in Plant Breeding, Reus/Salou, Spain, 26-30 May 
1991. 
[4] Aalen, R., Opsahl-Ferstad, H.-G. and Olsen, O.-A. (1994) Plant 
J. 5, 385 396. 
[5] Rickets, J., Tober, I. and Spener, F. (1984) Biochim. Biophys. Acta 
794, 313 319. 
[6] Rickets, J., Spener, F. and Kader, J.-C. (1985) FEBS Lett. 180, 
29-32. 
[7] Linnestad, C., L6nneborg, A., Kalla, R. and Olsen, O.-A. (1991) 
Plant Physiol. 97, 841-843. 
[8] Skriver, K., Leah, R., Miiller-Uri, F., Olsen, O.-A. and Mundy, 
J. (1992) Plant Mol. Biol. 18, 585-589. 
[9] Doolittle, R.F. (1986) in: Of Urfs and Orfs, pp. 1-103, University 
Science Books, USA. 
[10] Molina, A., Segura, A. and Garcia-Olmedo, F. (1993) FEBS Lett. 
316, 119 122. 
[11] Garcia-Maroto, F., Marafia, C., Mena, M., Garcia-Olmedo, F.
and Carbonero, P. (1990) Plant Mol. Biol. 14, 845-853. 
[12] Tchang, F., This, P., Stiefel, V., Arondel, V., Morch, M.-D., Pages, 
M., Puigdomenech, P., Grellet, F., Delseny, M., Bouillon, P., 
Huet, J.-C., Guerbette, F., Beauvais-Cante, F. Duranton, J., 
Pernollet, J.-C. and Kader, J.-C. (1988) J. Biol. Chem. 263, 16849 
16855. 
[13] Segura, A., Moreno, M. and Garcla-Olmedo, F. (1993) FEBS Lett. 
332, 243-246. 
[14] Molina, A. and Garcia-Olmedo, F. (1993) The Plant J. 4, 983 991. 
[15] D6sormeaux, A., Blochet, J.-E., P6zolet, M. and Marion, D. (1992) 
Biochim. Biophys. Acta 1121, 137-152. 
[16] Ostergaard, J., Vergnolle, C., Schoentgen, F. and Kader, J.-C. 
(1993) Biochim. Biophys. Acta 1170, 109-117. 
[17] Dieryck, W., Gautier, M.-F., Lullien, V. and Joudrier, P. (1992) 
Plant Mol. Biol. 19, 707-709. 


A fatty-acid-binding protein from wheat kernels 

García Olmedo, Francisco

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

A fatty-acid-binding protein from wheat kernels 
Atilio Castagnaro , Francisco García-Olmedo 
Laboratorio de Bioquímica y Biología Molecular, ETS Ingenieros Agrónomos - UPM, E-28040 Madrid, Spain 
Abstract 
A protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H20 extraction and gel filtration of the extract, followed 
by two steps of high-performance liquid chromatography. The N-terminal amino acid sequence has been determined up to the 28th residue and found 
to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a 
non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP does bind fatty acids, but in contrast, it is not significantly homologous 
to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH < 6.8 vs. pH > 9.6), and it does not show antibiotic 
properties. 
Key words: Cereal; Fatty acid binding protein; Plant, Wheat 
1. Introduction 
Transcripts whose pattern of expression coincides with 
the accumulation of lipids in developing aleurone cells 
from barley have been previously investigated [1-4]. A 
cDNA corresponding to one of these transcripts, B11E 
(EMBL XI5257), has been improperly classified as a 
non-specific lipid transfer protein, LTP2 [3,4]. Trans-
genic expression of the coding región of this cDNA in 
tobáceo, under a constitutive promoter, resulted in the 
premature loss of flower buds [3]. In the course of a 
systematic purification of low-molecular-mass proteins 
from wheat kernels, we have isolated a polypeptide of 
approximatly 7 kDa, designated W-FABP, whose 
N-terminal amino acid sequence is essentially identical 
to that deduced for the barley transcript. Although 
W-FABP does bind fatty acids, as has been reported for 
LTPs [5,6], it does not share with them their antipatho-
genic properties and other important features. 
2. Materials and methods 
2.1. Materials 
Kernels from the wheat cv Chínese Spring (Triticum aestivum) were 
ground to a powder on a Culatti mili and used to purify the protein. 
The bacterial pathogen Clavibacter michiganensis subsp. sepedonicus, 
strain C5 was from the ETSIA collection (Madrid). Wheat protein 
CM3 was the gift from Dr. R. Sanchez-Monge (Madrid) and thionin 
and LTP4 were given by Dr. A. Molina (Madrid). Bovine serum albu-
min and chymotrypsinogen were from Sigma. Radioactive [l-l4C]oleic 
acid was from Amersham. 
2.2. Purification procedure 
Ground wheat kernels (40 g) were extracted once with 4 voluntes of 
H20 and the extract was freeze-dried, dissolved in 5 mi of 3 M urea, 
and loaded into a Sephadex G-100 column (2.5 cm x 80 cm; 25 ml/h). 
The selected eluate fraction was dialyzed against 5 1 of H20, using a 
Spectra/Por 7 (MWCO:2000) membrane (Spectrum Medical Ind. Inc.), 
and freeze-dried. The fraction was then dissolved in H2O/0.1% TFA 
and subjected to RP-HPLC on an ultrapore C3 column (1 x 25 cm; 5//m 
particle; 30 nm pore), using a 0-50% (180 min) H20/acetonitrile gradi-
ent, 0.1% TFA, at 0.5 ml/min. The fraction indicated in Fig. IB was 
rechromatographed under the same conditions, except that 2-propanol 
was used instead of acetonitrile. SDS-PAGE in preformed gradient gels 
(4-20% acrylamide; Bio-Rad) was carried out according to the manu-
facturen' instructions. 
2.3. Fatty acid binding assay 
The protein (10 fig) was incubated with [l-l4C]oleic acid essentially 
as described [5,6]. Electrofocusing was carried out on a refrigerated 
horizontal apparatus from Pharmacia, using pH 3-10 Pharmalyte. 
Electrode buffers were 1 M NaOH and 0.04 M aspartic acid. Electrofo-
cusing was at 600 V for 45 min and at 2000 V for 11 min. The gel was 
stained for protein with Coomassie blue R, air-dried, and exposed with 
X-ray film. Colored isoelectrofocusing standard from Bio-Rad were 
used. 
2.4. Inhibition tests 
Proteins were dissolved in water and added to microtiter wells prior 
to innoculation. Serial dilutions from 0.1 fiM to 10 /¿M were used. 
Bacteria were added at a final concentration of 10" colony forming 
units/ml at a final volume of 150 fú (100 fi\ protein + 50 ¡A nutrient 
broth, Oxoid), incubated for 24 h at 28°C, and growth recorded in an 
ELISA píate reader. 
3. Results and discussion 
Ground wheat kernels were extracted with H20 and 
the freeze-dried extract was subjected to gel filtration, as 
shown in Fig. 1A. Fraction I, eluting with an apparent 
molecular weight of about 7 kDa, was further fraction-
ated by RP-HPLC (Fig. IB). Fraction II from this col-
% 
^ ^ ^ K g M M | 
Fig. 1. Purification of W-FABP from wheat kernels. (A) Gel filtration 
of H20 extract. Markers of molecular mass were (a) chymotrypsinogen, 
25 kDa, and (b) thionin, 5 kDa. (B) RP-HPLC fractionation of fraction 
I from above, using acetonitrile in the gradient. (C) RP-HPLC fraction-
ation of fraction II from B, using 2-propanol in the gradient. 
(D) SDS-PAGE of the indicated fractions. Fraction III from C consists 
of homogeneous W-FABP. 
umn, which contained a component that migrated with 
an apparent molecular mass of 5 kDa in SDS-PAGE 
(Fig. ID), was subjected again to RP-HPLC, but using 
2-propanol in the gradient, instead of acetonitrile 
(Fig. 1C). The fraction designated as III contained the 
apparently homogeneous, fast-moving component, des-
ignated W-FABP (Fig. ID). 
The N-terminal amino acid sequence of this compo-
nent was determined up to the 28th residue and no se-
quence heterogeneity was observed. Except for residues 
4 and 5, this amino acid sequence from wheat was iden-
tical to that deduced from a previously reported barley 
cDNA, BllE, as shown in Fig. 2. The barley sequence 
has been improperly designated LTP2 (EMBL X69793) 
in a recent publication [4], but we ha ve designated it as 
B-FABP for reasons that will be discussed below. The 
two FABPs have been aligned with LTPl from barley 
[7,8], to show that although they have some features in 
common with LTPs, the probability of homology be-
tween B-FABP and LTPl is extremely low according to 
the criteria of Doolitle [9]. The normalized alignment 
score (ÑAS = 120) for the LTPl /B-FABP comparison is 
of the same order as those obtained for comparisons 
between random sequences of the same length, and much 
below the minimum valué of ÑAS = 300 that would be 
required for significance [9]. The position of the N-termi-
nus of W-FABP suggests that the N-terminus of the 
protein deduced from the BllE cDNA (B-FABP) is 
6 amino acid residues downstream from where it was 
originally proposed [1]. The molecular weight of B-
FABP protein is 6,989 Da, which is compatible with the 
elution volume of W-FABP in gel filtration, but higher 
than the 5 kDa apparent molecular mass estimated for 
this protein by SDS-PAGE. This is a typical behaviour 
of lipid- binding proteins: ie, LTP4, whose size is 8.8 
kDa, appeared as a 6.5 kDa protein in SDS-PAGE [10]. 
The ability of the FABP to bind fatty acids was inves-
tigated by a previously described electrofocusing tech-
nique [6], using [l-14C]oleic acid. A wheat protein (CM3), 
which has hydrophobic domains and is soluble in chloro-
form/methanol mixtures [11], was used as a negative con-
trol, and bovine serum albumin (BSA) as a positive one. 
As shown in Fig. 3, W-FABP did bind oleic acid in a 
similar manner as LTP4 and its isoelectric point was 
more acidic than that of LTP4 (< 6.8 versus >9.6). A 
highly charged motif in the central part of the sequence 
and a hydrophobic stretch downstream from it have been 
proposed as the phospholipid-binding sites of LTPs [12]. 
These sequences, which are conserved in LTPs, are not 
obviously present in FABPs. Antibodies raised against 
a mixture of barley LTPs [14], which recognized LTPs 
W-FABP ACEVTat*VCWAtl,6ftT«P5«ECC««U 
B-FABP ACEPAQU'Wa5*íl>eG^^SaCC»)WAQQGaCQYVK0PNYG8YVSSPHARDTLNLCGIPVPHC 
B-LTP1 ALNCGQVDSKMCLTVQG-8PGPS«6CCNGVBDaTVCLCLKGIARGI-HNLNLNMAAS!PSKCNVNVPDCSRlY 
KPTVt LHNQAQSSGDRt YTISPDl t 
Fig. 2. N-terminal amino acid sequence of W-FABP aligned with those corresponding to B-FABP, deduced from BllE cDNA [l], and LTPl from 
barley [7,8]. Identical residues are shadowed, gaps (-) and insenions (T) are indicated. A highly similar LTPl sequence from wheat has been reported 
[17]. 
CM3 BSA IITP4 W-FABP 
I ' — ir i i II —i 
pH S R S R S R S R 
Fig. 3. Binding of radioaetive oleic acid to W-FABP. Bovine serum 
albumin (BSA) and barley LTP4 [10] were used as positive controls, 
wheat CM3 [11] as a negative one. The gel was staíned with Coomassie 
blue (S) and then exposed to X-ray film (R). The insertion point is 
indicated (í). 
in vivo situation. The subcellular location of LTPs seems 
to be incompatible with some of the metabolic roles pro-
posed for them (see [14]). The subcellular location(s) of 
FABPs need to be investigated. From the above results 
it can be concluded that W-FABP and B-FABP repre-
sent a distinct protein family with respect to the LTPs, 
although both families might be involved in lipid metab-
olism. A definite designation of this family should wait 
until more is known about their roles in vivo. 
Acknowledgements: Amino acid sequences have been determíned by 
Drs. C. Buesa (Servei Sequenciació, Universilat Barcelona) and C. 
Lopez-Otín (Fac. of Medicine, Universidad de Oviedo). Reading of the 
manuscript by Drs. P. Carbonero and G. Salcedo, and technical assis-
tance from D. Lamoneda and J. García are also acknowledged. Finan-
cial support was from the Comisión Interministerial de Ciencia y 
Tecnología (Grant PB92-0325). 
References 
from other species, including spinach and Arabidopsis, 
do not recognize W-FABP (not shown). 
We have recently reported that all LTPs tested are 
potent inhibitors of plant pathogens [10,13]. W-FABP 
was not active against the test microorganism at up to 
10 fiM concentration, tenfold higher than those giving 
complete inhibition with LTPs or with thionins [10,13], 
which were used as positive controls in the experiment 
(not shown). 
Based on the above dissimilarities of W-FABP with 
respect to the LTPs, both in their amino acid sequences 
and in their in vitro properties, it would be advisable that 
the amino acid sequence deduced from the barley BUE 
cDNA should not be identified as a member of the LTP 
family, as has been proposed (EMBL X69793 and [4]). 
The designation LTP2 has been recently given to a pro-
tein from barley leaves which is significantly homolo-
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binding of fatty acids, coenzyme-A esters, and phosphol-
ipids, or lipid transfer between artificial liposomes and 
mitochondria [5,6,15,16], cannot be extrapolated into the 
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A fatty-acid-binding protein from wheat kernels

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A fatty-acid-binding protein from wheat kernels

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