Proteins extracted with CHCl3-MeOH from wheat endosperm have been fractionated by Sephadex G-100 and the 15 000–20 000 MW range fraction, designated CM protein, has been examined by combined electrofocusing (pH range 5–8) and electrophoresis (pH 3.2) and the heterogeneity of the electrophoretic components has been ascertained. It has been shown by joint mapping and by sequential extraction that CM proteins are extracted by 70% EtOH but not by H2O, although they can be made water-soluble after dialysis against an acid buffer, pH 3.2, 3 M urea, without losing their solubility in CHCl3-MeOH mixtures. It is concluded that CM proteins fit the definition of a Folch—Lees proteolipid. The Triticum aestivum (genomes ABD) map can be reconstructed by mixing T. durum (AB) and Aegilops squarrosa (D). The low intragenomic variability of CM protein is confirmed

Aragoncillo

Bietz

C. Aragoncillo

Charbonnier

Drysdale

F. Garcia-Olmedo

Folch

Folch-Pi

Garcia

Hall

Huebner

Johnson

Jones

Kaltsikes

M.A. Rodriguez-Loperena

Meredith

Pilar Carbonero

Redman

Rohrlich

Tennenbaum

Waines

Wrigley

Zill

English

Rodriguez Loperena, M.A.

Aragoncillo Ballesteros, Cipriano

Carbonero Zalduegui, Pilar

García Olmedo, Francisco

Archivo Digital UPM

Heterogeneity of wheat endosperm proteolipids (CM proteins)

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

HETEROGENEITY OF WHEAT ENDOSPERM 
PROTEOLIPIDS (CM PROTEINS) 
M. A. RODRIGUEZ-LOPERENA, C. ARAGONCILLO, PILAR CARBONERO and F. GARCÍA-OLMEDO 
Departamento de Bioquímica. Universidad Politécnica. E.T.S. Ingenieros Agrónomos. Madrid-3, Spain 
Key Word Index—Triticum aestivum; Triticum durum; Aegilops squarrosa; Gramineae; wheat; proteolipids; CM 
proteins; gliadins; albumins; electrofocusing; electrophoresis. 
Abstract—Proteins extracted with CHCl3-MeOH from wheat endosperm have been fractionated by 
Sephadex G-100 and the 15 000-20 000 MW range fraction, designated CM protein, has been examined 
by combined electrofocusing (pH range 5-8) and electrophoresis (pH 3-2) and the heterogeneity of the 
electrophoretic components has been ascertained. It has been shown by joint mapping and by sequential 
extraction that CM proteins are extracted by 70% EtOH but not by H20, although they can be made 
water-soluble after dialysis against an acid buffer, pH 3-2, 3 M urea, without losing their solubility in 
CHCl3-MeOH mixtures. It isconcluded that CM proteins fit the definition of a Folch-Lees proteolipid. 
The Triticum aestivum (genomes ABD) map can be reconstructed by mixing T. durum (AB) and Aegilops 
squarrosa (D). The low intragenomic variability of CM protein is confirmed. 
INTRODUCTION 
The electrophoretic patterns (pH 3-2) of CHC13-
MeOH (2:1, v/v) extracts of partially delipidated 
endosperm from Triticum aestivum L. consistently 
show three fast main bands, CM1, CM2 and CM3, 
and those of T. durum Desf. only two, CM2 and 
CM3 [1]. García-Olmedo and Carbonero [2] puri-
fied and partially characterized CM1 and CM2, 
showing that they were controlled by chromosomes 
7D and 7B respectively. The third component, 
CM3, has been recently purified by Redman and 
Ewart [3] and by Aragoncillo [4]. The latter has 
also purified a faster variant of CM3, designa ted 
CM3', which is present only in a few T. durum 
varieties. 
There is indirect evidence that more than one 
protein might be present in band CM3 of T. aesti-
vum (genomes ABD): (i) both Aegilops squarrosa 
(D) and T. durum (AB) have a CM3 band [4]; (ii) 
the CM3/CM2 and CM3/CM1 ratios, which are 
rather constant within the species, are significantly 
depressed in both ditelosomics and in the monoso-
mic of chromosome 4A, but no suppression of 
CM3 takes place [4]; (iii) amino acid analysis of 
CM proteins purified by ion exchange [3] and by 
direct elution of trichloroacetic acid precipitated 
bands from an acrylamide slab show a consider-
able lack of agreement [4]. 
The electrophoretic patterns (pH 3-2) of wheat 
endosperm proteins extracted with 70% ethanol 
(EtOH) have a number of components migrating 
ahead of gliadins which are of great taxonomic sig-
nificance, as shown by Johnson and co-workers [5-
7]. Waines [8] has recently implicated homoeolo-
gous chromosome groups 4 and 7 in the control of 
some of these proteins. CM proteins are soluble in 
70% EtOH and the three variant patterns, CM 1-
CM2-CM3, CM2-CM3, and CM2-CM3', can be 
recognized among the more complex patterns of 
EtOH-extracted proteins [3,4]. It has also been 
observed that these proteins are at least partially 
soluble in water [3,4]. 
We report here an investigation of the hetero-
geneity of CM proteins and their relationship with 
the 70% EtOH and H20-extracted proteins. 
RESULTS 
Gelfiltration 
Proteins extracted with chloroform-methanol 
(CHCl3-MeOH) (2:1) from partially delipidated 
Table 1. Distribution of map components in different protein preparations* 
Protein preparation 
Purified CM-protein (CM) 
Chloroform-methanol extract (CME) 
70% ethanol extract (EE) 
Water extract (WE) 
1 
_ 
-
+ 
+ 
2 
+ 
+ 
+ 
-
3 
+ 
+ 
+ 
-
4 
+ 
+ 
+ 
-
5 
_ 
+ 
+ 
+ 
6 
_ 
-
+ 
+ 
7 
_ 
-
+ 
+ 
8 
+ 
+ 
+ 
-
9 
+ 
+ 
+ 
+ 
Component 
10 11 
- + 
- + 
+ + 
+ -
12 
+ 
+ 
+ 
" 
numbert 
13 14 
+ -
+ -
+ + 
- + 
15 
_ 
-
+ 
+ 
16 
+ 
+ 
+ 
+ 
17 
+ 
+ 
+ 
-
18 
_ 
-
+ 
+ 
19 
_ 
-
+ 
+ 
20 
_ 
+ 
+ 
+ 
21 
_ 
+ 
+ 
+ 
Others 
_ 
-
-
+ 
* +, Present; - , absent; ±, detected at the overloading level. 
t See Fig. 2. 
132 242 330 
Elution volume, mi 
Fig. 1. Sephadex G-lOOgel filtration profile of extracts. Crude 
CHCl3-MeOH extract ( ) and the same extract after dialysis, 
centrifugation and freeze drying of the supernatant ( ). Peak 
A are glutenins, peak B gliadins and peak C CM-proteins. 
wheat endosperm can be fractionated on Sephadex 
G-100 (Fig. 1). Three fractions are obtained: peak 
A, which appears with the exclusión volume, is 
precipitated by dialysis against water and is prob-
ably composed of glutenins; peak B elutes in the 
40000-60000MW range and yields an electro-
phoretic pattern identical with that of the classical 
gliadins extracted with 70% EtOH; and peak C, 
which is eluted in 15000-20000MW range, con-
sists of the CM proteins. Proteins extracted with 
70% EtOH or with water also yield a peak in the 
same range as peak C, which includes the proteins 
that move ahead of gliadins in electrophoresis at 
pH 3-2 [9,10]. 
Combined electrofocusing and electrophoresis 
Two-dimensional mapping by combined gel 
electrofocusing and electrophoresis was carried 
out at optimal and at overloading concentrations 
of the following protein preparations: peak C 
(CM), CHCl3-MeOH extract (CME), 70% EtOH 
extract (EE) and water extract (WE). Binary mix-
tures of these extracts were also run to confirm 
homology of spots. A composite map for T. aesti-
vum var. Chínese Spring is presented in Fig. 2 and 
the observed distribution of components in the dif-
ferent preparations is summarized in Table 1. 
Densitometric profiles of the three latter types of 
preparation from T. aestivum var. Candeal are 
superposed in Fig. 3. Maps of CM and CME have 
E . i 
-
-CMI M B 
CM2-M 
CM3HÍ 
OO 
• -
%v¿ 
^ ^ 5 6 7 8 
12' 13' • 99 
II 12 13 
819 
M 
•• 
2021 
— Unfocused gliadins — 
i i 
»IO 
\ 
146 
t" 
i 
pH-gradient 7'4 TO 5'9 4'8 
Electrofocusing 
Fig. 2. Composite map obtained by combined electrofocusing 
and electrophoresis of protein extracts. Spots shown in broken 
line represent components extracted only with water (A com-
ponents). 
Fig. 3. Densitograms of maps obtained with different protein 
preparation of Triticum aestivum var. Candeal. Spot numbers 
are those given in Fig. 2. EE, 70% EtOH extract ( ); WE, 
water extract ( ); CME, CHCl3-MeOH extract (-—). All 
three extracts were obtained from the same amount of endo-
sperm (25 mg). The CM protein densitogram, at the appropriate 
scale, was identical with that of CME but without peaks 5, 
10 and 21. 
the same components in the zone of faster electro-
phoretic migration, with the exception of com-
ponent 5 and the traces of 20 and 21 which are 
missing from CM. 
All CME components are extracted more 
readily with 70% EtOH: spots 2, 3, 4, 8, 9, 11, 12 
and 13 in the CME map ha ve 50-70% of the inten-
sity of those in the EE map while spots 5, 16, 17, 
20 and 21 in the CME map ha ve 5-20% of the in-
tensity; components 1, 6, 7, 10, 14, 15, 18 and 19 
are only present in the EE map. These results are 
consistent with those obtained by sequential 
extraction. Extraction with CHCl3-MeOH after 
70% EtOH yields none of the components of the 
CME map, while extraction with 70% EtOH after 
CHCl3-MeOH gives a complete EE map in which 
spots 2,3,4, 8,9,11,12 and 13 are much weakened. 
The WE map has 9 components in common 
with the EE map (spots 1, 5-7, 10, 14-16 and 21). 
Some of these, 14-16 and 21, are extracted more 
effectively with 70% EtOH than with water. In 
addition WE has a number of components, desig-
nated A in Fig. 2, which are not detected in either 
CME or EE. Extraction with water after 70% 
EtOH yields only the A components plus spots 1 
and 10. The map obtained with the 70% EtOH 
extract of water extracted flour closely resembles 
that obtained with CM. Thus, water does not 
extract CM proteins from flour. WE has two 
minor components overlapping with EE com-
ponents 11-12 but different from them as demon-
strated in T. durum var. Ledesma, whose EE map 
lacks 11 and has a variant of 12 but whose WE 
map still has the two weak components in the 
same position as 11 and 12. 
Proteins extracted with CHCl3-MeOH from EE 
yield the same map as CME. CM proteins are 
readily soluble in CHCl3-MeOH, in water and in 
70% EtOH after gel filtration in acid buffer, pH 
3-2, 3M urea, dialysis against water and freeze-
drying. However, water does not extract CM pro-
teins from CME or EE. 
Analysis oftetraploid and hexaploid wheats 
In Table 2, CME and EE phenotypes of hexap-
loid wheats (genomes ABD) and tetraploid wheats 
(AB), as well as that of Aegilops squarrosa (D), are 
recorded. Equivalence of spots was ascertained by 
joint mapping. All hexaploids have the phenotype 
of variéty Chinese Spring with few exceptions: 
component 2 is absent in six varieties, a variant 
pattern is obtained for components 18-21 in two 
varieties, and some additional components are 
present in the synthetic T. spelta which are contri-
buted by T. carthlicum. 
All tetraploid phenotypes, natural as well as 
obtained by genome extraction, are identical, with 
the exception of variety Ledesma, which has com-
ponents 12-13' of different mobility and isoelectric 
points than 12-13, and the already mentioned ad-
ditional components of T. carthlicum. All com-
ponents of T. durum maps are recognized in T. 
aestivum maps. 
Table 2. Distribution of map components in Aegilops squarrosa and several Triticum genotypes* 
Samples 
Component numbert 
9 10 11 12 13 14 15 16 17 18 
Triticum aesticum var.: 
Chinese Spring 
Candeal 
Prelude 
Rescue 
Thatcher 
Traquejos 
Canalejas 
Chamorro 
Triticum spelta 
Triticum durum var.: 
Senatore Capelli 
Ledesma 
Tetraprelude 
Tetrarescue 
Tetrathatcher 
Triticum carihlkum 
Aegilops squarrosa 
- + + + + 
+ + + - -
* +, Present; - , absent; v, possíble genetic variant present; ?, presence not unequivocally established. 
t See Fig. 2. 
All components of Ae. squarrosa maps are pres-
ent in T. aestivum. Three of them, 12, 13 and 20 are 
also present in T. durum. 
DISCUSSION 
Heterogeneity ofCM proteins 
The abo ve results indica te that not only CM3 is 
heterogeneous, as suspected,but also other electro-
phoretic bands seem to yield more than one com-
ponent. 
The possibility of artifacts cannot be over-
looked. These could arise during electrofocusing 
or in the previous handling of the protein prep-
arations. Our unpublished genetic evidence per-
mits discarding some of the potential cases of arti-
fact formation: the genetic control of component 
2 is independent from that of 3-4; gene(s) for com-
ponent 5 are located in a different chromosome 
than those for 6-7 and 8-9; 11 is equally indepen-
dent of 12-13; component lóiscontrolled by a dif-
ferent chromosome than 17; and the genetic varia-
tion of components 18-21 precludes their being 
artifacts. 
These considerations narrow the possibilities to 
the pairs 3-4, 8-9, 12-13 and 12-13', that could 
represent one protein each, and components 6, 7, 
14 and 15, that are controlled by the the same 
chromosome arm and could be different modifica-
tions of the same protein. 
Different electrofocusing conditions have been 
employed to investígate if these could alter the pat-
terns: no significant changes were observed when 
persulfate was substituted by riboflavin in prep-
aration of the gel or when the sample was applied 
on top of the column instead of incorporated into 
the polymerization mixture. 
It does not seem likely that the pairs are the 
result of incomplete delipidation because the pro-
longed exposure to acid during gel filtration and 
dialysis against water does not alter the pattern. 
However, they could represent in vivo modified 
forms of the same polypeptides. In this connection, 
it is particularly striking that the two components 
of CM3', 12-13', differ to the same extent in their 
isoelectric points and electrophoretic mobility 
from components 12-13 (CM3). In any case, the 
elucidation of this matter will depend on purifica-
tion of the individual components or on further 
genetic studies. 
In conclusión, CM3 and possibly CM1 and 
CM2 are heterogeneous, which would explain the 
already mentioned differences in the amino-acid 
composition data [3,4]. 
Classification oj CM proteins as proteolipids 
Proteolipids were first isolated from brain tissue 
by Folch and Lees [11] and defined as proteins in-
soluble in water and aqueous solvents but soluble 
in CHCl3-MeOH mixtures. Although especially 
abundant in nervous tissue, proteolipids are also 
widely distributed in animal and plant tissues [12]. 
Zill and Harmon [13] have reported on chlorop-
last proteolipids and Rohrlich and Nieder-
auer [14] described a proteolipid from wheat en-
dosperm, a non-photosynthetic plant tissue, which 
was recovered from the interface of the Folch wash 
of endosperm lipids extracted with CHC13-
MeOH. The CM proteins are readily soluble in 
CHCl3-MeOH (2:1) and in water after gel 
filtration in acid buffer, dialysis against water and 
freeze-drying. In a similar manner, the Folch-Lees 
protein is still soluble in CHCl3-MeOH mixtures 
after it has been made water soluble by dialysis 
against solvents of increasing polarity, but is not 
water-soluble in its native state or when obtained 
by vacuum evaporation of the CHCl3-MeOH 
extraction mixture [12, 15]. On this basis, CM 
proteins should be classified as true proteolipids 
and not as intrinsic contaminants of gluten of the 
albumin type, as Meredith [16] tentatively classi-
fied a fraction, designated a, obtained from the 
CHCl3-MeOH extract of gluten and probably 
equivalent to CM protein. 
This proteolipid fraction is also a part of the so-
called [9,10,17-19] albumins plus globulins peak 
of gel filtration profiles of proteins extracted with 
70% EtOH and probably of similar gel filtration 
peaks from other extracts such as those obtained 
with 2M urea, diluted acids and aqueous alcohols. 
Low genetic variability ofCM proteins 
The present results confirm at much greater 
resolution the previous observation by one-dimen-
sional electrophoresis of the low intragenomic var-
iability of the non-gliadin 70% EtOH proteins [5-
7,20] and of the CM proteins [1]. The extreme 
usefulness of these proteins in phylogenetic studies, 
which has been extensively demonstrated by John-
son and coworkers, is based precisely on this pro-
perty, which makes them good genomic markers. 
For the same reasons, these proteins should be 
excellent chromosome markers at the present 
degree of resolution. 
The low genetic variability of these proteins is in 
sharp contrast with the great variability of gliadins 
recently observed by Wrigley and Shepherd [21] 
using combined electrofocusing and electrophor-
esis. 
EXPERIMENTAL 
Biological material. The following wheats were used in this 
study: Triticum aestivum varieties Chínese Spring, Candeal, 
Chamorro, Traquejos, Canaleja, Rescue, Thatcher and Prelude; 
T. durum varieties Senatore Capelli and Ledesma; T. carthlicum 
M-l; Aegilops squarrosa C-l; a synthetic T. spelta (T. carthlicum 
x Ae. squarrosa) and the tetraploid versions of Rescue, 
Thatcher and Prelude obtained by Kaltsikes et al. [22] by D 
genome extraction. 
Extracts. The crude CHCl3-MeOH (2:1) extract was 
obtained from delipidated flour as previously described [1] and 
dispersed in 3 M urea, dialysed against H : 0 for 48 hr, centri-
fuged at 3000 # for 15 min, and the supernatant freeze-dried. 
For the mapping experiments, from 1/2 to 6 kernels were 
crushed between two metal plates with a hammer, defatted with 
light petrol (50-70°, 10 v/w) and the solvent removed in vacuo. 
The samples were extracted 3 x with the appropriate solvent 
(8 v/w each time) and the solvent of the combined extracts eli-
minated in vacuo or by freeze-drying. When sequential extrac-
tions were performed, after the third treatment with the first sol-
vent (100 v/w), the solvent residues in the sample were removed 
in vacuo and the second solvent then added. 
Gelfiltration. Both the crude and the dialysed CHCl3-MeOH 
extracts (100-400 mg of each) were subjected to gel filtration 
through Sephadex G-100 (2-5 x 90 cm column) in aluminium 
lactate buffer 0-1 M, pH 3-2, 3 M urea. Fractions corresponding 
to peak C (Fig.l) were pooled, dialysed against H20 for 48 hr 
and freeze-dried. 
Combined electrofocusing and electrophoresis. The method de-
scribed by Wrigley [23] was used with the following modifica-
tions: polyacrylamide gels were 2 x 140 mm, ampholine pH 
range 5-8, voltage was brought up to 470 V never exceeding 
0-5 mA/tube and then maintained for 6 hr 30 min, electrophor-
esis time was 6hr at 10 V/cm. The unfixed electrofocused gel 
can be stored frozen for several days without affecting the two 
dimensional pattern. Under the above conditions, the gliadins 
are not focused and migrate very little into the starch gel. The 
pH gradient was determined according to Drysdale et al. [24]. 
Densitometry was performed in a Chromoscan (Joyce and 
Loebl) with the 620 nm filter. A wider sample holder was espe-
cially made which allowed scanning of half a map each time. 
Acknowleclgements—We gratefully acknowledge samples given 
by Dr. P. J. Kaltsikes. This work was carried out under contract 
with the Junta de Energía Nuclear, Spain. 
REFERENCES 
1. García Olmedo, F. and García Faure, R. (1969) Lebensm. 
Wiss. U. Technol. 2, 94. 
2. García Olmedo, F. and Carbonero, P. (1970) Phytochemis-
frv 9, 1495. 
3. Redman, D. G. and Ewart, J. A. D. (1973) J. Sci. Fd Agr. 
24, 629. 
4. Aragoncillo, C. (1973) Dr. Ing. Agr. Thesis, Polytechnical 
University of Madrid. 
5. Johnson, B. L. and Hall, O. (1965) Am. J. Botany 52, 506. 
6. Hall, O., Johnson, B. L. and Olered, R. (1966) Proc. 2nd int. 
Wheat Genetics Symp., Lund 1963, Hereditas, Suppl. Vol. 2, 
47. 
7. Johnson, B. L. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 1398. 
8. Waines, J. G. (1973) Proc. Ath Int. Wheat Genet. Symp. Col-
umbio [Sears, E. R. and Sears, L. M. S., Eds] (University of 
Missouri), pp. 873-877. 
9. Huebner, F. R. and Rothfus, J. A. (1971) Cereal Chem. 48, 
469. 
10. Charbonnier, L. (1973) Biochimie 55, 1217. 
11. Folch, J. and Lees, M. (195!) J. Biol. Chem. 191, 807. 
12. Folch-Pi, J. and Stoffyn, P. J. (1972) Ann. N.Y. Acad. Sci. 
195, 86. 
13. Zill, L. P. and Harmon, E. A. (1961) Biochim. Biophys. Acta 
53, 579. 
14. Rohrlich, M. and Niederauer, Th. (1967). Fette Seifen, 
Anstrichmittel 69, 226. 
15. Tennenbaum, D. and Folch-Pi, J. (1966) Biochim. Biophys. 
Acta 115, 141. 
16. Meredith, P. (1965) Cereal Chem. 42, 149. 
17. Jones, R. W., Babcock, G. E., Taylor, N. W. and Dimler, R. 
J. (1963) Cer ra /0^.40 , 409. 
18. Meredith, O. B. and Wren, J. J. (1966) Cereal Chem. 43, 169. 
19. Bietz, J. A. and Wall, J. S. (1972) Cereal Chem. 49, 416. 
20. Waines, J. G. (1969) PhD. Thesis, University of California, 
Riverside, Calif. 
21. Wrigley, C. W. and Shepherd, K. W. (1973) Ann. N.Y. Acad. 
Sci. 209, 154. 
22. Kaltsikes, P. J., Evans, L. E. and Bushuk, W. (1968) Science 
159,211. 
23. Wrigley, C. W. (1970) Biochem. Genet. 4, 509. 
24. Drysdale, J. W., Righetti, P. and Bunn, H. F. (1971) Biochim. 
Biophys. Acta 229, 42. 


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