PURIFICATION AND CHARACTERIZATION OF STORAGE PROTEINS

3. PURIFICATION AND CHARACTERIZATION OF STORAGE PROTEINS

3.1 INTRODUCTION

3.1.1 Characteristic features of storage proteins

The major proteins of seeds display a number of

characteristic features which help distinguish them as

storage proteins.

1. They are synthesized at a particular stage during seed

development to be utilized during germination. These

proteins are therefore called storage proteins.

2. They tend to be rich in certain amino acids .like

aspargine, glutamine, arginine and proline.

3. They accumulate inside the cells in special vacuoles

termed protein bodies.

3.1.2 Classification of storage proteins

on the basis of solubility in different solvents, storage

proteins have been divided into four major classes

(Derbyshire et al., 1976). They are albumins (water

soluble), globulins (salt soluble), prolamins (alcohol

soluble), and glutelins (acid or alkali soluble). Globulins

are the major storage proteins of dicots while prolamins and

glutelins that of monocots. Albumins have been reported to

be present in varying amounts in both monocots and dicots.

68

3.1.2.1 Albumins

Albumins are water soluble proteins which have been

considered as the metabolic proteins. It has now become

evident that the albumins may have storage functions as

well. Furthermore, it has been shown that the albumins of

pea cotyledons are used as a reserve during and after

germination. (Murray, 1979). Two major albumin fractions

with molecular weights of 23,000 and 25,000, have been

reported from pea {Croy et al., 1984; Higgins et al.,1987).

Albumins frequently account for 30% to 40% of

extractable seed protein, as seen in pea and soybean.

Albumin fraction contains a higher percentage of methionine

and cysteine, which makes it nutritionally more important

(Murray, 1984). In addition, some low molecular weight

albumins, which have high methionine content, have also been

purified from various plants. Recently, a gene encoding

methionine rich albumin protein has been used for raising

transgenic Brassica plants (Guerche et al.,1990).

3.1.1.2 Globulins

Globulins are soluble in high salt buffers and they are the

major proteins of most legumes (Higgins, 1984) and Oat

(Brinegar et al., 1982; Higgins, 1984). They are separated

into different classes by cryoprecipitation, differential

69

salt solubility and ultracentrifugation. Most storage

globulins fall into two major groups with sedimentation

coefficients of 11S and 7S. Seeds of many plant species

contain both 11S and 7S storage proteins, although in most

cases, one or the other may predominate (Derbyshire et al.,

1976) .

To a large extent, the molecular structure of these

proteins are conserved among species. Soybean globulins are

the most extensively analysed storage proteins. They are

composed of both 7S and 11S globulins. 7S fraction contains

a group of heterogenous storage proteins called conglycinins

(Ladin et al., 1984) . 11S fraction contains, glycinin, the

major storage protein. It has a molecular weight of about

300 kDa and is composed of 6 to 7 subunits (Shotwell and

Larkins, 1989). Similarly in pea, the globulin fraction is

composed of legumin and vicilin proteins. Legumin has a

sedimentation coefficient of 12S and a molecular weight of

330-400 kDa whereas vicilin sediments at 7S and has a

molecular weight of 186 kDa (Derbyshire et al., 1976). They

are deposited in the seed in an insoluble form in protein

bodies.

3.1.2.3 Prolamins

constitute the major protein fraction in the Prolamins

monocots. They are rich in proline and glutamine but

70

contain less amounts of lysine, methionine and tryptophan.

They are hydrophobic proteins that are soluble in alcoholic

solutions. They exist as families of proteins and are

coordinately synthesized during seed development. They are

secreted into protein bodies.

3.1.2.4 Glutelins

The glutelin protein fraction is soluble in acid and alkali

solutions. Except in rice and sorghum, where glutelins

occur in protein bodies (Larkins, 1981), they are normally

present as an insoluble matrix containing starch grains

and protein bodies. In rice, glutelins occur as major

storage proteins and it constitutes 8 O% of the total

protein. Glutelins are similar to prolamins in their

biochemical properties.

3.1.3

plants

Similarities between storage protiens of different

The storage proteins of evolutionarily diverse groups of

plants show a lot of homology. Globulins occur in both

monocots and dicots. They exist as oligomers of six

subunits. Each subunit is composed of a disulfide bonded

acidic and basic polypeptides (Higgins, 1984). Common

antigenic determinants have been found among the globulins

71

of many members of the leguminosae (Dudman et al., 1975) and

the prolamins of wheat,barley and maize (Dierks-Ventling and

Cozens, 1982).

storage proteins exist as families of proteins rather

than as a single protein species. They are coordinately

synthesized during seed development. They may differ from

each other in their isoelectric points or molecular weight

but they are similar in their primary structure (Messing et

al., 1987).

3.1.4 composition of Amaranth protein

The major cereal crops are all grasses; Amaranth is one of

the few nongrasses to produce significant amounts of grain.

Amaranth grain contains about 16 to 19% protein, of which

albumins and globulins together constitute some 50% of the

total protein. Next major fraction is.the glutelins which is

about 30-33%. Prolamins, the major protein of cereals are

present in very low amounts (less than 3%). (Paredes-Lopez

et al., 1988).

3.2 RESULTS AND DISCUSSION

3.2.1 Sedimentation value of the major protein fractions

Sedimentation value of the major proteins present in

Amaranth seeds was checked on a 5-20% sucrose density

72

gradient. Four major protein fractions corresponding to

sedimentation values of 12S, 7S, 5S, and 2S were obtained.

2S fraction was most abundant (Fig. 4) . Globulins, th.e

major seed proteins of legumes,

7S and 11S ( Derbyshire et al.,

exist in two size classes,

197 6) • In addition some

small molecular weight proteins have also been found in most

of the plants tested (Youle et al., 1978, Murray, 1979, Croy

et al., 1984). When Amaranth albumin and globulin fractions

were separately checked on sucrose-gradient, it was seen

that the major proteins in the 11S and 7S fractions were

globulins, while those of 5S fraction were albumins. Both

the classes of proteins, albumins as well as globulins, were

found in the 2S fraction (data not shown). It may

therefore, represent a heterogeneous population of proteins.

3.2.2 Identification of storage proteins

Amaranth seed extracts contain atleast four polypeptides in

large amounts which may be considered as storage proteins

(Fig. 5). Three of these polypeptides, 54, 34, and smaller

than 12 kDa disappear within a day of germination; however,

the 35 kDa polypeptide disappears only by the third day of

germination (Fig. 5). To further analyze their functions,

two of these proteins, 54 kDa and 35 kDa (indicated by an

arrow in Fig. 5) were purified.

73

2 3 4 5 6 7 8 9 10 11 12 M kDa

-94 -67

-43

-30

-20.1

-14.4

Fig.4 SDS-PAGE of various fractions obtained after sucrose density centrifugation. Crude extract (0.2 ml) containing 500 ug protein vJas layered on 12 ml . linear 5-20% sucrose gradient made in the extraction buffer. 1 ml fractions were collected. 2 0 0 u1 sample of each fraction was precipitated with equal volume of 20% TCA for 30 min. It was centrifuged and the pellet was washed thrice with ethanol:ether (1:1) and dried. Pellet was dissolved in SDS sample buffer and analysed on 12% SDS-PAGE.

Lanes 1 to 12 indicate the fraction numbers. 'M' is standard proteins lane.

Fractions 5, 8, 9, and 11 correspond to sedimentation values of 128, 7S, 5S, and 2S respectively.

1 2 3

54 ... ·

kOa -94 -67

-43

-30

-201

-14.4

Fig. 5 SDS-PAGE analysis of total protein extracted from dry mature seeds -and seeds germinated for different days. 50 mg seeds (wet weight) were homogenized in 0.5ml of lX Laemmli' s sample buffer. 10 ul of the supernatant obtained on centrifugation was analysed on 12% SDS-PAGE.

Lane 1, dry mature seeds; lanes 2 and 3, seeds germinated for 2 and 3 days, respectively. Proteins indicated by arrows were purified for further work.

3.2.3 Purification and Characterization of the 35 kDa

Albumin Protein

3.2.3.1 separation of albumins from globulins

seeds were extracted in high salt buffer and the crude

extract obtained was extensively dialysed against Tris­

acetate buffer pH 8. 5. Globulins (insoluble in low salt

buffers) that precipitated during dialysis, were removed by

centrifugation. Supernatant having the albumin protein was

the source of 35 kDa protein.

3.2.3.2 Chromatofocussing

Chromatofocussing separates ·proteins on the basis of their

isoelectric points. Albumin fraction was bound to a DEAE­

sepharose column previously equilibrated with Tris-acetate

buffer, pH 8.5. The column was washed with the same buffer

and the bound protein was eluted with a multicomponent

buffer system (Table 1), adjusted to a pH of 5. Proteins get

eluted on the basis of their pi values due to a pH gradient

generated in the column. Fractions were analysed on SDS­

PAGE and their pH was determined (Figs. 6A and 6B).

The 35 kDa albumin protein eluted in four

different peaks at pH values of 7.4, 7.1, 6.8, 6.7 and also

in the salt wash (Figs. 6A and 6B). Protein peak, at pH

74

Seed meal (lg)

Two extractions with 20 ml cold acetone

Two extractions with 10 ml 0.5 M NaCl in 25 mM Tris-acetate buffer, pH 8.5

Dialysis against 25 mM Tris-acetate,pH8.5

Supernatant (Source of albumin protein)

Loaded on the Chromatofocussing column

35 kDa albumin protein, referred as AmAl,passed through gel filteration column

AmAl concentrated by centricon

... Lipids

precipitate(source of globulin protein)

Dissolved in Laemmeli buffer and separated on SDS-PAGE.

54 kDa band cut and electroeluted

SDS removed by KCl precipitation

Flow sheet depicting the purification of two proteins from Amaranth seeds.

Fig.6 Fractionation chromatofocussing column.

of soluble proteins on

A, Elution profile of the column eluate. ( - ) 1 Absorbance at 280 nm; ( ....... ) 1 pH of various fractions.

B, SDS-PAGE analysis of different column fractions. L, load; W, wash; M, standarQ.. proteins. Numbers indicate the fractions analysed. ·; Arrow indicates the 35 kDa protein. Equal volume from each fraction was analysed.

A - -

9

8

7

6

J 5 a.

4

I io I I I I I 10 30 40 50 60 70 alo FRACTION NUMBER

B

n

r 0·5 NaCl

91

0 ' 120

~ 0·5

-25

I 0 CXl N <i

130 140

.- 66

-53

-45

-36 - 29

-24

-20.1

-14.2

L W 28 29 30 42 48 67 69 85 88 130 M

7. 4, had the highest yield of 35 kDa protein and so was

taken for further work.

(Amaranth albumin 1).

It is being referred to as AmAl

3.2.3.3 Gel filtration

AmAl, from the chromatofocussing column, was concentrated to

a small volume using centricon-10 (Amicon) and applied to a

gel filtration column. Some small molecular weight

contaminants were retained in the column and the eluted

protein was checked for its purity by one- and two­

dimensional PAGE. About 10 ug protein on one dimensional

and 5 ug on two dimensional-PAGE gave a single band (Fig. 7A

and 7B), so the protein is considered pure.

3.2.3.4 Isoelectric point of AmAl

Isoelectric point of AmA1 calculated from NEPHGE was about

7.8 (Fig. 7B). Proteins are not focussed to equilibrium in

NEPHGE. From the chromatofocussing data, isoelectric

point of AmA1 came to be about 7.4. Proteins are eluted from

the chromatofocussing column on the basis of their

isoelectric points. pi of AmA1 may therefore be around

7.4-7.8.

75

A

1 2 3 '· • ikOa

1--'94 .1 ... 167

! i• 43

........ ! ! .. 30

..... 20·1

8

· + ;kDa: -!94 \

~67

"-.• 43 l

Fig. 7 Purity of AntAl by one-dimensional (A) and two-dimensional (B) gel electrop~resis.

A, 10 ug (lane 1) and 5 ug (lane 2) of the purified protein were analysed on 12% SDS PAGE and stained with Coomassie Blue. Lane 3 shows molecular weight standards.

B, 5 ug protein was analysed on 2-D PAGE (NEPHGE in the first dimension and 10% SDS PAGE in the second dimension) and stained with Coomassie Blue. Dark patch at the bottom of the gel is due to ampholytes.

3.2.3.5 Molecular weight determination

Native molecular weight of AmAl determined by Sephadex G-200

was about 55 kDa (Fig. 8). However, on SDS-PAGE only a 35

kDa protein was observed. To see, if it was a dimer, the

protein was checked on SDS-PAGE without mercaptoethanol

treatment and also after treating the sample with 10 mM

iodoacetate.

the protein.

monomer, with

No difference was observed in the mobility of

It was, therefore, concluded that AmAl is a

a molecular weight of 3 5 kDa. The high

molecular weight observed with sephadex G-200 may be due to

some non specific protein aggregation within the column.

3.2.3.6 Antibody preparation and Immunodiffusion

Antibodies were raised against AmAl by immunization of

rabbits. Titre was checked by Ouchterlony immunodiffusion

technique as described by Garvey et al. ( 1977) . 10 ug of

crude extract and 1 ug of purified protein was titred with

different dilutions of antiserum. Precipitin line could be

observed upto a dilution of 1:16 (data not shown).

3.2.3.7 Specificity of antibodies as checked by Western blot

analysis

Western blot analysis was performed according to the

procedure of Towbin et al. ( 1979) . A single band was

76

20 -------'-:t 0

I . 0 .-->< ..,._____

-+--'

...c Ol

t>l

~ 4

L._

0

::J u

2 t>l

0 2

1 0 0.4 0.5 0.6

K ov

Fig. 8 Estimation of molecular weight of filtration chromatography. Purified chromatographed on Sephadex G-200 column. curve was constructed by plotting Kav values proteins against their molecular wei~hts.

AmAl by gel AmAl vias

Calibration of standard

( o ) indicates standard proteins; ( • ) indicates the size of AmAl.

observed on the blot when checked with crude extract. At

times, some faint high molecular weight bands were observed

when purified protein was used. So antibodies were affinity

purified (Section 2.13) and then used for immunodetection.

Affinity purified antibodies, at a titer of 1:500 could

detect upto 0.1 ng protein (data not shown). High molecular

weight bands were still visible. It may simply be due to

incomplete denaturation of the protein.

3.2.3.8 Isoe1ectric forms of AmA1

35 kDa albumin protein, as reported earlier, eluted from

the chromatofocussing column in four different peaks (Fig.

6) . To see if these proteins were related, the peak

fractions were checked for their reactivity against AmAl

antibodies. They were all seen to react (Fig.9), indicating

that they are the different isoelectric forms of 3 5 kDa

albumin protein. Storage proteins from other systems are

known to exist in families of proteins where the different

proteins may differ on the basis of charge (Higgins, 1984).

'!'he exact mechanism by which different charge variants of a

protein originate is not known, but they may be the products

of a single gene (Beachy et al., 1985).

77

1 2 3 kDa

35----.--

4- 5 6

I .I

Fig. 9 Western blot analysis of different peak fractions of chromatofocussing column probed with AmAl antibodies. Equal volume of su.rnple from different fractions obtained after chromatofocussing vlas resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane. Immunoreactive polypeptide was detected with AmAl antibodies.

Lane 1, load; lanes 2-6, indicate fraction numbers 28, 42, 48, 67 and 130, respectively, of the elution profile shown in Fig.6A.

3.2.3.9 Glycosylation and Lectin activity

Lectins often accumulate in seeds in large amounts and are

known to agglutinate erythrocytes (Elzler, 1985). They are

mostly glycosylated. To see if AmAl has lectin activity, it

was incubated with rabbit erythrocytes as described

(Section 2 .15). No lectin activity was seen to be

associated with it. When stained for glycoproteins by PAS

staining, AmA1 was found not to be glycosylated (data not

shown).

3.2.4 Purification of 54 kDa polypeptides

3.2.4.1 Separation of globulin proteins on SDS-PAGE

Globulin fraction was precipitated from the crude

extract by extensive dialysis of crude extract against 25

mM Tris-acetate, pH 8.5 or water.

in SDS-sample buffer of Laemmli

Precipitate was dissolved

(1970) and separated on a

preparative SDS-PAGE. Protein standards were run

simultaneously.

54 kDa was cut

2. 8) •

Band corresponding to a molecular weight of

after staining the gel with KCl (Section

Electroelution

Proteins were electrophoretically eluted from the gel slice

in 50 mM Ammonium bicarbonate containing 0.1% SDS. SDS

78

confers a negative charge on the proteins and disaggregates

them. As a result quantitative recovery is possible. Eluted

protein was concentrated by acetone precipitation. Some SDS

also precipitated along with the protein.

3.2.4.3 Removal of residual SDS

The residual SDS was removed by precipitation with KCl

(Suzuki and Terada,l988). Potassium salts, at a final

concentration of 20 mM have been found to precipitate SDS

from protein solutions with no protein loss (Suzuki and

Terada, 1988). About 50% of the protein was recovered after

precipitation.

3.2.4.4 Purity of the protein

To see if some contaminating protein is copurified, the

eluted 54 kDa polypeptide was rechecked on SDS-PAGE. It

gave a single band, indicating that it is pure (Fig.lO).

3.2.4.5 Antibody preparation and specificity

Antibodies raised against this polypeptide gave a titer of

1: 16 by Ouchterlony immunodiffusion technique. When

specificity of the antibody was checked by Western blot

analysis, it was found to cross react with many bands in the

crude extract. Since the antibodies were raised against gel

79

1 2 kDa

-94 ,.,....., -67 ............... ·-·· ~,.

~ •• ' •

-43

-30

;_20.1

-14.4

Fig 10 Purity of 54 kDa globulin protein. was analysed for purity on 12% SDS-PAGE.

54 kDa protein

Lane 1, crude extract; Lane 2, purified protein.

purified protein, crossreactivity indicates that it shares

some common antigenic determinants with many other proteins

in the crude extract. It remains to be seen, if these

proteins have some common properties, like glycosylation, or

if they are related in some other way.

3.2.5 Amino acid composition

Purified storage proteins were analysed for their amino acid

composition. As seen in Table 2 , 54 kDa polypeptide is rich

in aspartate, glutamate, arginine and proline as compared to

AmAl which has high content of aspartate only. In general

seed storage proteins are known to contain high level of

these amino acids and they are know·n to be the chief

nitrogen transport compounds in plants (Goodwill and Mercer,

1983). In addition in legume seeds, it has been shown that

the amide nitrogen of asparagine is the chief available

source of nitrogen during development (Goodwill and Mercer,

1983). Both these proteins may therefore have storage

functions. AmAl also has high · lysine content ( 6. 6%) and

fairly good methionine level (1.6%) and so it may be more

important, as far as human nutrition goes. It was therefore

chosen for further work.

80

TABLE 2 : Amino acid composition of the two Amaranthus t . * pro e1ns

Amino acids

Phe

Tyr

Leu

Ile

Val

Met

Cys

Ala

His

Thr

Pro

Gly

Glx

Asx

Ser

Arg

Lys

Trp

* mol ~ Values expressed as 0

ND - not determined

AmA1

5.3

4.4

7.6

5.0

4.7

1.6

0.7

5.3

4.4

6.0

3.6

12.0

7.6

17.6

6.4

5.5

6.6

ND

54 kDa Globulin polypeptide

2.8

ND

7.8

3.9

4.8

ND

0.6

8.5

1.6

3.4

8.2

12.5

17.6

10.0

5.4

10.3

4.1

ND

3.2.6 Protein accumulation during development

seeds were classified into seven different developmental

stages on the basis of their weight (Table 3) . Protein

extracts of all the different stages, when analysed by

immunoblotting indicated that the synthesis of 54 kDa

polypeptide begins later than AmAl (Figs. 11A and 11B). AmAl

synthesis (monitored using anti-AmA1 antibodies) starts

soon after fertilization when the seed weight is only about

0.1 mg and reaches a maximum when the weight is about 0.3 mg

(Fig.11B). Accumulation of the 54 kDa polypeptide (studied

using anti-54 kDa antibodies) begins much later. It was

seen to be present in the initial stages of development as

well, though at a very low level (Fig.11A).

This kind of differential accumulation of different

proteins during seed development has been reported in other

plants, as well (Meinke et al., 1981; Goldberg et al., 1981;

Higgins, 1984). For example, in soybean seeds, 2S proteins

predominate during early stages of development but their

proportion decreases later when the 7S and 11S proteins are

synthesized (Hill and Briedenbach, 1974).

Before the accumulation of 54 kDa polypeptide begins, a

35 kDa polypeptide, which cross reacts with the 54 kDa

antibodies, is seen to appear, but disappears at the stage

81

TABLE 3 : Average wei~ht of seeds at different stages of development .

STAGE SEED WEIGHT(in mg)

I 0.05 ~

II 0.1

III 0.2

IV 0.3

v 0.4

VI o.a

VII 1

* Seeds were collected at different days after fertilization and separated into seven stages on the basis of their weight.

A 8

123456 71234567 kDa kOa

~35

Fig 11 Analysis of stage specific synthesis of the two proteins by Western blot. Crude extracts (20 ug protein) of seeds at different stages of development, as given in Table 2, were reso l ved on 12% SDS-PAGE,transferred to nitrocellulose membrane and probe d with A 54kDa antibodies and B, AmAl a nt ibodies .

when the 54 kDa polypeptide gets accumulated. We do not

know as yet, if these two polypeptides are related, since

the antibody raised against 54 kDa polypeptide was seen to

cross react with many other polypeptides.

3.2.7 Presence of translatable AmAl mRNA in mature seeds

Levels of seed protein mRNA is known to increase during seed

development, and then decrease as the seed matures (Goldberg

et al., 1981} . To see if AmAl mRNA is present in mature

seeds, poly(A)+ mRNA was isolated from seeds at stage V,

Table 3, (when this protein was not getting accumulated any

further) and dry mature seeds. Immunoprecipitation of the

in vitro translated p~oduct (Section 2.19} indicated that,

AmAl mRNA was present in mature seeds and was translatable

(Fig.12}.

3.2.8 seed specificity and function

On the basis of the germination-dependent disappearance of

the 54 kDa polypeptide (Fig.5), and also on its aminoacid

composition (Table 2), one can say that it is a storage

protein. But the role of AmAl as a storage protein is not

very clear, since it does not disappear immed~ately on

germination. Albumins have normally been considered as

metabolic proteins, but there are reports to show that some

82

kDa .. A., B 't

. ~.; .

. ' -~~~:,::· .( ·,.:

....

Fig 12 Imrnunoprecipi ta tion of in vitro translated products. Poly (A)+ RNA isolated from seeds, at stage V (Table 2) , (A} , and dry mature seeds, (B) , was translated in vitro in reticulocyte lysate system. Translated product was precipitated with AmAl antibodies bound to Protein A­Sepharose beads and analysed on 12% SDS-PAGE. 35s labeled and immunoprecipi tated product was then detected by autoradiography.

albumins also have storage functions. ·;,·:·~.Croy et al. (1984)

have reported an albumin protein from pea which does not

disappear immediately on germination but acts as a storage

protein. The delayed breakdown of this protein, according

to Higgins et al. ( 198 7) , is due to its cytoplasmic

localization. According to them, the proteolytic enzymes

are not released into the cytosol until the final stages of

cellular disorganisation. It is not known as yet, whether

AmAl is localized within protein bodies, or not, but a

• similar mechanism is possible for AmAl as well.

w~stern blot analysis of crude extracts from different

regions of the plant e.g. root, stem and leaves, did not

show any reactivity when probed with both the antibodies

(data not shown) . On the basis of above results and ·the

fact that they accumulate in large amounts during seed

development, it can be said that they are storage proteins.

3.2.9 Summary

1. Amaranth grain is rich in certain essential amino

acids like lysine and methionine which are normally

deficient in tradi tiona! crops. As a first step in the

isolation of a gene encoding a protein of high nutritional

value two proteins with molecular weights of 54 kDa and 35

kDa, have been purified from the seeds of Amaranthus

hypochondriacus.

83

2. The 35 kDa protein is soluble in low salt buffer

while the 54 kDa polypeptide requires high salt buffer for

solubilization.

3. On germination 54 kDa polypeptide disappears within a

day while the 35 kDa polypeptide is seen to disappear only

by the third day of germination.

4. 35 kDa protein was purified by chromatofocussing and

gel filtration columns. Purity of the protein was checked by

one dimensional and two dime.nsional PAGE.

5. 54 kDa polypeptide was purified by electroelution from

an 80S-polyacrylamide gel.

6. Antibodies, raised against both the proteins were

found to be of high titre.

7. Aminoacid composition of the two proteins indicated

that the 35 kDa protein has higher content of lysine.

8. For further work, the 35 kDa (AmAl) protein was chosen

because of its high lysine content. It was further

characterized to show that

a. It is a monomer of 35 kDa molecular weight.

b. It exists in four different isoelectric forms

c. It is not glycosylated.

84

d. Its accumulation in the seed starts soon after

fertilization when seed weight is only about 0.1 mg as

compared to 54 kDa polypeptide which is synthesized

much later.

e. mRNA encoding AmAl is present in dry mature seeds

as well.

85