18
Plant Science Letters, 30 (1983) 303--320 303 Elsevier Scientific Publishers Ireland Ltd. GROUP FRACTIONATION OF WHEAT GERM RIBOSOMAL PROTEINS MICHAEL M. SIKORSKI a, DANUTA PRZYBYL a, ANDRZEJ B. LEGOCKI a and KNUD H. NIERHAUS b alnstitute of Biochemistry, Agriculture, University, Wolyhska 35, 60-637 Poznati (Poland) and b Max-Planck-lnstitut ffir Molekulare Genetik, Abt. Wittmann, lhnestrasse 63-73, lOOO Berlin 33 (F.R.G.) (Received September 22nd, 1982) (Revision received November 25th, 1982) (Accepted December 14th, 1982) SUMMARY Proteins from wheat germ ribosomes were isolated by CM-ceUulose chromatography. The 35 proteins of the small subunit (40S) were fraction- ated into 10 main groups by a linear KC1 gradient at pH 7.6, and the 44 ones o.f the large subunit (60S) into six groups by stepwise elution with increasing concentration of potassium acetate at pH 5.5. The proteins of two fractions were further purified using a linear gradient at pH 7.6. Most of the fractions contained only 2--5 proteins. Five proteins from the small subunit and four proteins from the large one could he isolated .to near homogeneity. Key words: Wheat germ ribosomal proteins -- Protein isolation INTRODUCTION About 50% of the mass of eukaryotic ribosomes consists of a large number of different ribosomal proteins. Since most of the 70--80 ribosomal proteins have similar isoelectric points and molar masses, the isolation and purification of ribosomal proteins is a challenging task. Various strategies for the isolation of proteins from eukaryotic ribosomes, such as salt fractiona- tion, ionic exchange chromatography and gel filtration, have been employed [1--6]. In recent years, the wheat germ system has become a reference system for eukaryotic protein biosynthesis in vitro. Therefore, we focused our interest on structure and function of wheat germ ribosomes. A prerequisite of a structural analysis is the isolation and characterisation of the ribosomal constituents. We have recently shown that cytoplasmic ribosomes derived 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

Group fractionation of wheat germ ribosomal proteins

Embed Size (px)

Citation preview

Page 1: Group fractionation of wheat germ ribosomal proteins

Plant Science Letters, 30 (1983) 303--320 303 Elsevier Scientific Publishers Ireland Ltd.

GROUP FRACTIONATION OF WHEAT GERM RIBOSOMAL PROTEINS

MICHAEL M. SIKORSKI a, DANUTA PRZYBYL a, ANDRZEJ B. LEGOCKI a and KNUD H. NIERHAUS b

alnstitute of Biochemistry, Agriculture, University, Wolyhska 35, 60-637 Poznati (Poland) and b Max-Planck-lnstitut ffir Molekulare Genetik, Abt. Wittmann, lhnestrasse 63-73, lOOO Berlin 33 (F.R.G.)

(Received September 22nd, 1982) (Revision received November 25th, 1982) (Accepted December 14th, 1982)

SUMMARY

Proteins from wheat germ ribosomes were isolated by CM-ceUulose chromatography. The 35 proteins of the small subunit (40S) were fraction- ated into 10 main groups by a linear KC1 gradient at pH 7.6, and the 44 ones o.f the large subunit (60S) into six groups by stepwise elution with increasing concentration of potassium acetate at pH 5.5. The proteins of two fractions were further purified using a linear gradient at pH 7.6. Most of the fractions contained only 2--5 proteins. Five proteins from the small subunit and four proteins from the large one could he isolated .to near homogeneity.

Key words: Wheat germ ribosomal proteins -- Protein isolation

INTRODUCTION

Abou t 50% of the mass of eukaryotic ribosomes consists of a large number of different r ibosomal proteins. Since most of the 70--80 ribosomal proteins have similar isoelectric points and molar masses, the isolation and purification of r ibosomal proteins is a challenging task. Various strategies for the isolation of proteins from eukaryotic ribosomes, such as salt fractiona- tion, ionic exchange chromatography and gel filtration, have been employed [1--6].

In recent years, the wheat germ system has become a reference system for eukaryotic protein biosynthesis in vitro. Therefore, we focused our interest on structure and funct ion of wheat germ ribosomes. A prerequisite of a structural analysis is the isolation and characterisation of the r ibosomal constituents. We have recently shown that cytoplasmic ribosomes derived

0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

Page 2: Group fractionation of wheat germ ribosomal proteins

304

from wheat germ contain at least 79 ribosomal proteins [7]. Here we report a simple method for a group fractionation of these ribosomal proteins from both subunits, including the purification of 9 proteins to near homogeneity.

MATERIALS AND METHODS

Preparation of ribosomes, ribosomal subunits and ribosomal proteins Ribosomes were isolated from wheat germ (General Mills, Vallejo, CA)

as described previously [7] with minor modifications. The ribosomal pellet was dissolved in a dissociation buffer: 20 mM Tris--HC1 (pH 7.6), 400 mM KCI, 3 mM MgCI2, 5 mM 2-mercaptoethanol and 5% sucrose (w/v) containing 1 mM puromycin and 1 mM GTP. Ribosomes were then incubated for 30 min at 37°C and the subunits were separated by zonal centrifugation in a Ti 15 Beckman rotor using a 10--38% (w/v) linear sucrose gradient in dissociation buffer. Fractions corresponding to each subunit were pooled and recovered by centrifugation at 35 000 rev./min for 12 h at 4°C in a 42.1 Beckman rotor. The pelleted subunits were suspended in 20 mM Tris-- HOAc (pH 7.6), 50 mM KC1, 5 mM Mg(OAc)2, 5 mM 2-mercaptoethanol containing 10% (v/v) glycerol. Ribosomal proteins were extracted from 40S and 60S subunits by 67% acetic acid according to Hardy et al. [8].

Carboxymethylcellulose Chromatography The proteins extracted from each subunit were applied onto a CH-ceUulose

column at room temperature (CM 52 preswollen, Whatman Inc.; 1.2 × 12 cm). Routinely, 60 A280 units of the total 40S subunit proteins suspended in 15 mM Tris--HOAc (pH 7.6) and 6 M urea were separated on the column which was equilibrated with the same buffer.

The column was washed with the buffer to remove unadsorbed proteins and then developed with 1 1 of a 0--400 mM KC1 linear gradient in the above buffer. Fractions (3-ml) were collected at a flow rate of 15--20 ml/h and were measured for protein content by adsorption at 280 nm.

Total proteins (90 A2s0 units) from the 60S subunit (TP60) in 5 mM KOAe (pH 5.5) and 6 M urea were applied to a CM-cellulose column (1.4 × 15 cm) equilibrated with the same buffer. Adsorbed proteins were then eluted from the column by increasing stepwise the concentration of KOAc (pH 5.5) (5 raM, 250 raM, 400 mM and 600 mM KOAc) in the presence of 6 M urea. Two groups of 60S subunit proteins obtained by this stepwise fractionation were resolved further by chromatography on CM- cellulose at pH 7.6 using a linear gradient of 0--400 mM KC1 in 15 mM Tris--HOAc (pH 7.6) with 6 M urea.

Polyacrylamide gel electrophoresis and identification of proteins The ribosomal proteins were identified by two-dimensional gel electro-

phoresis according to Kaltschmidt and Wittmann [9], except that the second dimension gel contained 16% (w/v) acry!Amide as described earlier [7]. The

Page 3: Group fractionation of wheat germ ribosomal proteins

305

A

O

- r (3.

e

B

O

"1" a.

e

$7

L8 * ..!:" Lm

Li:l'" ~-':

S26

L2 L&

S13

L6

L18

L39 L40 1 L42 ~ ~ L 4 1

I

pH 8.2 -~ o

$1 E" S2

:.I"~.,$3 Sl. S8 "LI: SS:~lm~.~6

S 1 6 ~ I "" SI m

$25 ~ i ~ C s29

$32 :~i~: $ 3 3

r :

C; S 34

L•43 ~ 4

pH 8.2 o

L1 L3

Q

L7 L9 LIO L12 L13 "L11,

2 ~ ~ L 1 5 L19 L22 L 2 0 ~ l l ~ ' ~1~'

s L z ? ~ ~ z e .,L3z

L35 L36

L39

L42 C:.

z, OS

60S

L37

L38

L40 q~.41

$35

e

Fig. 1. Two4iimensional polyacrylamide gel electrophoresis of proteins from the 40S (A) and 60S (B) ribosomal subunits from wheat germ. Dotted circles indicate the proteins weakly stained. The inset in B shows the fastest migrating proteins L43 and LA4 which run out of the gel under standard conditions (12 h in second dimension), whereas here the run lasted 8 h in the second dimension.

Page 4: Group fractionation of wheat germ ribosomal proteins

306

purity of the separated proteins was assessed by slab SDS-gel electrophoresis according to Laemmli [10]. The proteins isolated by CM-cellulose chromato- graphy were extensively dialyzed against 0.5% acetic acid, lyophylized, and dissolved in sample buffer. Identification of ribosomal proteins by two- dimensional gel electrophoresis was facilitated by the comparison of the sample pattern with that of a mixture containing the sample and a small amount of corresponding total proteins.

Determination of protein concentration The concentration of protein was measured by the Bradford method [11]

using bovine serum albumin as a standard.

RESULTS AND DISCUSSION

Figure 1 shows two<iimensional patterns of 40S and 60S ribosomal proteins. The total number of ribosomal proteins revealed from both isolated subunits is 79.

Thirty-five proteins were found to be constituents of the small subunit in reasonable agreement with a recently published number of 32 [6], 44 proteins to be constituents of the large subunit. In order to facilitate separation and further purification of individual proteins a group fractiona- tion procedure was developed for both the 40S and 60S subunits.

A general strategy used for group fractionation of ribosomal proteins from various sources is often based on successive washing of the subunits with buffers of increasing ionic strength, followed by chromatography according to charge or by gel filtration according to molecular weight. However, preliminary fractionation of wheat germ ribosomal proteins by the NH4C1/EtOH or KC1/EtOH wash procedure or LiC1 splitting appeared to be insufficient, since we observed that the majority of proteins appeared in almost all the fractions obtained (unpublished data).

Fractionation of 40S sub unit proteins The proteins of the small ribosomal subunit were separated into 10 main

groups by gradient fractionation on CM-cellulose (Fig. 2). The proteins of each fraction were then identified by both SDS one<limensional and urea two<iimensional gel electrophoresis (Fig. 3).

All three acidic proteins of the 40S subunit: $7, $13 and $26 together with four basic proteins, $8, $15, $17 and $32, were present in the first fraction (flow through; Fig. 3 (la,b)). Several 40S proteins were recovered in a quite pure state. These were $8 in fraction 2, $21 in fraction 3, $30 in fraction 8 and $9 in fraction 10. In two other fractions two proteins were detected with only minor contaminants: $4 and $14 in fraction 6, $18 and $23 in fraction 7. Table I summm~zes the fractionation of the 40S ribosomal proteins by CM~ellulose chromatography.

Page 5: Group fractionation of wheat germ ribosomal proteins

D

!I

0.1

0.05

B

B

O

~S

1

2 3

4 5

M

6 7

8 9

10

t~

E

60

10

30

6

J .L

1

--

5 80

12

0 16

0 20

0 F

RA

CT

ION

S

Fig

. 2.

Cl~

om

ato

gm

pb

y o

n c

arb

ozy

me

t~yl

ee

llu

lose

of

40S

rib

oso

ma

l su

bu

nit

pro

tein

s. T

he

pro

tein

s w

ere

an

aly

zed

by

two

-dim

e]~

ion

al

po

lya

czT

lam

ide

gel

e]e

etro

pbor

esis

in u

rea

(F

ig.

3) a

nd

by

SD

S-g

el e

Ject

ropb

ores

is (s

ee p

ho

to).

(M

--

mo

lecu

lar

we

igh

t m

arke

rs:

phos

- p

bo

ryla

se B

-94

00

0;

bo

vin

e s

erum

alb

um

in -

- 67

00

0;

ova

lbu

min

--

43

00

0;

carb

on

ate

ar~

ydra

se -

- 3

00

0;

soyb

ea

n tr

ypsi

n i

nh

ibit

or

--

20 1

00; ]

yso

zy

m-

13 9

00).

Page 6: Group fractionation of wheat germ ribosomal proteins

4a

~ °

~

II

B

t m

4b

O

lb °

1MI5

e

$22

Q

3

T~

!L

- $23

MI

Page 7: Group fractionation of wheat germ ribosomal proteins

e~

7

~ ~

8 g

st

Qs~7

qB

#

Q

t0

g

$~

Fig

. 3.

Tw

o~ii

men

sion

al p

olya

cryl

amid

e ge

l el

ectr

oph

ores

is o

f 40

S r

ibos

omal

pro

tein

s af

ter

sepa

rati

on i

nto

ten

gro

ups

on a

car

box

y-

met

hyl

-cel

lulo

se c

olu

mn

at

pH 7

.6.

Th

e p

rote

ins

of e

ach

grou

p w

ere

anal

yzed

by

two-

dim

ensi

onal

gel

ele

ctro

ph

ores

is t

o as

sess

th

eir

pu

rity

, and

th

ey w

ere

also

ele

ctro

ph

ores

ed w

ith

a s

mal

l am

oun

t (a

bou

t 50

~g)

of

TP

40S

as

a b

ack

grou

nd

for

th

e id

enti

fica

tion

of

the

prot

eins

.

r~

Page 8: Group fractionation of wheat germ ribosomal proteins

310

TABLE I

GROUP FRACTIONATION OF THE PROTEINS FROM THE 40S RIBOSOMAL SUBUNITS

The symbols used are: +++, present in large amounts; ++, present in small amounts; +, present in traces. P~roteins S1, $2, $3, $11, $20, $31, $33, $34 and $35 were not found in any group.

Protein Group

1 2 3 4 5 6 7 8 9 10

$4 $5 $6 S7 + $8 + +++ S9 S10 S12 S13 ++ S14 S15 + S16 + S17 + + S18 S19 821 $22 ++ S23 $24 $25 $25a $26 +++ $27 $28 $29 $30 S32 ++

÷ ÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷

÷ ÷ ÷

÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷

÷ ÷ ÷

÷ ÷ ÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷ ÷ ÷

• ÷

÷ ÷

÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷ ÷

÷ ÷

÷ ÷ ÷

÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷

÷ ÷ ÷ ÷ ÷

÷ ÷ ÷

Fractionation o f 60S subunit proteins T h e 60S p ro te ins were f r ac t i ona t ed into six g roups (A, B1, B2, C1, C2

and D) b y s tepwise e lu t ion f r o m CM-cellulose wi th increasing concen t ra - t i ons o f p o t a s s i u m a c e t a t e ( p H 5.5) , in t h e p resence o f 6 M urea {Fig. 4). F r a c t i o n A c o n t a i n e d a g roup o f s t rong ly acidic p ro te ins which d o n o t b ind to CM~cellulose u n d e r these condi t ions . T h r e e o f these p ro t e in s were LS, L l l and L16 , wh ich in te res t ing ly can be s t rong ly p h o s p h o r y l a t e d in v i t ro [7] . Peak B~ consis ts p r e d o m i n a n t l y o f t he acidic p ro t e in L18. T h e f rac t ions B: and C1 con ta in a b o u t 20 p ro t e in s (see T a b l e II) . T h e r e is a lmos t no over lap b e t w e e n these t w o f rac t ions . In con t ras t , t he f rac t ions CI and C2 con ta in v i r tua l ly t he s ame set o f p ro t e in s e x c e p t L30 in C2 (Fig. 5). T h e

Page 9: Group fractionation of wheat germ ribosomal proteins

U3

C:)

tO

n U

D

1.5

I --

1.0

• "-

94.0

00

"-67

000

• -,,-

43.

000

-'---

30.

000

! 0.

5~

i ....

..

....

. -

| :

B 20

• "--

20.

100

• '--

13.9

30

I 40

C

r'

C2

I 60

FR

AC

TIO

NS

0.8-

T- i

0.4

Fig.

4.

Gro

up f

ract

iona

tion

of

60S

ribo

som

al s

ubun

it p

rote

ins

by c

hrom

atog

raph

y on

car

boxy

met

hylc

ellu

lose

col

umn

(ste

pwis

e el

utio

n at

pH

5.5

). T

he i

nset

pho

togr

aph

show

s SD

S-ge

l el

ectr

opho

resi

s of

pro

tein

s pr

esen

t in

eac

h gr

oup.

The

sta

ndar

d pn

)tei

ns f

or

the

mol

ecul

ar m

ass

(M)

are

give

n in

the

leg

end

to F

ig.

2. F

or d

etai

ls s

ee M

ater

ials

and

Met

hods

. k

~

t.a

Page 10: Group fractionation of wheat germ ribosomal proteins

| +

""

i I

+L__

t e

° 4

!

r

r--

I

Ii

It.

" 0

Page 11: Group fractionation of wheat germ ribosomal proteins

+ 0

lip Ill

L'b

Cl L3

70

~ 8 L32

L35 L36 ~ L 37

, ~ L 3 ~

" 0

'Hill

0

o q P

LI

L L~

O

C2

0

tL3,r,

-L37

4 P L~a

Fig. 5. Two~timensional polyacrylamide gel electrophoresis of proteins from the 60S ribosomal subunit. The isolation of these proteins is shown in Fig. 4.

Page 12: Group fractionation of wheat germ ribosomal proteins

314

TABLE II

GROUP FRACTIONATION OF THE PROTEINS FROM THE 60S RIBOSOMAL SUBUNITS

Symbols +++, ++ and + are the same as in Table I. Proteins L2, L4, L5, L6, L10, L15, L40, L43 and L44 were not found in any group.

Protein Group

A B~ B2 C, C2 D

L1 L3 L7 L8 L9 L l l L12 L12a L12b L13 L14 L14a L16 L17 L18 L19 L20 L21 L21a L22 L23 L24 L25 L25a L25b L25/26 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L41 L42

4-4-

Jr Jr

4-jr

+ j r

Jrjr 4-4-jr 4-4-

Jr4- 4-4-

4 - j r÷ J r ÷

÷ ÷ j r 4- j r j r

Jr-I-jr

-I-jr

Jr4-

4-jr Jr

Jrjr4-

4-

4-4-4-

4-4-

-I-4-

4-jr4-

+

4-4-

4-4-

4-4-

÷4-4-

Jr4-

Jr4-

4-4-4- ÷ j r ÷

4-4-4- Jr4-jr

4-4- 4-4-4-

4-4-4-

÷ ÷ ÷ 4-4-4-

4-4-

4-4-4- 4-4-4-

4-4-4-

4-

4-÷4- 4-

4- 4-4-

4-4- 4-4-4-

4- 4-

4-4- 4-4-4-

4- 4-4-4-

4-

4-4- 4-

4-4-4- 4-4-4-

4-4-4- 4-4-4-

4-4-4-

4-4-4-

+4-

+4-

Jr-t-

4-4-

Page 13: Group fractionation of wheat germ ribosomal proteins

0,08

60S

B2M

3

4 5

6 7

8 9

10

11

12

13

14

15

16

17

18

0.04

o

---

^ o_

._..~

o ~

_~

_~

--~

88

°

v tn

11

12

13

j"X

....

.""~

.

4,7

,a

2 ~

45 6

7

u ,u

"J

,-

"~

~ "

.~

L .L

t

L J

----

-.-

40

80

120

160

FR

AC

TIO

NS

Fig

. 6

. C

arb

ox

ym

eth

ylc

ellu

lose

ch

rom

ato

gra

ph

y o

f 6

08

rib

oso

mal

pro

tein

s in

gro

up

B 2

(li

near

gra

dien

t at

pH

7.6

). T

he

in

set

ph

oto

- gr

aph

show

s SD

S-ge

l ele

ctro

phor

esls

of

prot

eins

pre

sent

in

each

elu

ted

frac

tion

. M

, sta

ndar

d pr

otei

ns f

or

the

mol

ecul

ar m

n~

(se

e Fi

g. 2

). F

or

furt

her

deta

ils

see

Mat

eria

ls a

nd M

etho

ds.

i t/) E

~5

30

15 r~

t.A

C

J1

Page 14: Group fractionation of wheat germ ribosomal proteins

t

P

411

b

- ql

b

qb

B

Ill

P

0

0

Page 15: Group fractionation of wheat germ ribosomal proteins

L25/)

b

13

L2~/2~

? L

~

11.2

5j26

~t

,2~

.... ¢

~i

0

15

16

D

B

t

~J ~

a t2

7

18

Fig

. 7.

Tw

o~ii

men

sion

al p

olya

cryl

amid

e ge

l el

ectr

oph

ores

is o

f p

rote

ins

in g

rou

p B

2 of

60S

rib

osom

al s

ub

un

it.

Th

e is

olat

ion

of

thes

e p

rote

ins

is s

how

n i

n F

ig.

6. T

he

pro

tein

s of

eac

h f

ract

ion

wer

e an

alyz

ed b

y tw

o-d

imen

sion

al g

el e

lect

rop

hor

esis

to

asse

ss t

hei

r p

uri

ty

and

wit

h 5

0 u

g of

TP

60S

as

bac

kgr

oun

d f

or t

he

iden

tifi

cati

on o

f th

e p

rote

ins.

k-4

--3

Page 16: Group fractionation of wheat germ ribosomal proteins

e.O

i.

I

0.04

0.02

R~k_S

CI

I',~

9 10

I;I

12

13 1

'4 15

16

1'7 1

8 19

~

21 2

2

oo~ j°

.,

A~

~~

~~

/2

.

23

,

I I

65

130

195

FRA

CTI

ON

S

E

60

30

Fig.

8.

Cal

boxy

mat

hylc

ellu

lose

chr

omat

ogra

phy

of

608

ribo

som

al p

rote

ins

in g

roup

C 1

(lin

ear

grad

ient

at

pH 7

.6).

The

ph

oto

gra

ph

sh

ows

8DS-

gel e

lect

roph

ores

is o

f pr

otei

ns i

n ea

ch e

lute

d fr

acti

on. M

, st

anda

rd p

rote

ins

for

the

mol

ecul

ar m

ass

(see

leg

end

to F

ig.

2).

For

fur

ther

det

ails

see

Mat

eria

ls a

nd M

etho

ds.

Page 17: Group fractionation of wheat germ ribosomal proteins

ii! ~ i~ i ~ ~

if?if! ~ ~ i i ~

!!iii ~ i ~I ~ iii ~ i . . . . ~ ~

Fig. 9. Two~timensional polyacrylamide gel electrophoresis of proteins isolated as shown in Fig. 8.

Page 18: Group fractionation of wheat germ ribosomal proteins

320

proteins of fractions B2 and C1 were submitted to a second fractionation step on CM~ellulose, applying a linear KC1 gradient at pH 7.6 (see Figs. 6 and 7). From the B2 group proteins L12 and L25 could be purified to near homogenei ty with increasing ionic strength, and from the C1 fraction protein L22 was obtained in a rather pure state. All the other fractions contained two or more proteins (see Figs. 8 and 9).

In summary, the technique described allows an efficient fractionation of the 79 proteins from wheat germ ribosomes. Most of the fractions contain two to five ribosomal proteins. Five proteins from the small and four from the large subunit could be isolated in a rather pure state.

ACKNOWLEDGEMENTS

We thank Drs. H.-G. Wittmann and R. Brimacombe for advice and discussions.

REFERENCES

1 C. Gualerzi, H.G. Janda and G. StSffler, J. Biol. Chem., 249 (1974) 3347. 2 H. Bielka and J. Stahl, Int. Rev. Biochem., 18 (1978) 79. 3 I.G. Wool, Annu. Rev. Biochem., 48 (1979) 719. 4 K. Tsuguri and K. Ogata, Eur. J. Biochem., 101 (1979) 205. 5 T. Itoh, K. Higo and E. Otaka, Biochemistry, 18 (1979) 5787. 6 C.-Y. Ting Shih, J.E. Toivenen and G.R. Craven, Eur. J. Biochem., 97 (1979) 189. 7 M.M. Sikorski, D. Przybyl, A.B. Legocki, W. Kudlicki, E. Gasior, J. Zajac and T.

Borkowski, Plant Sci Lett., 15 (1979) 387. 8 S.J.S. Hardy, C.G. Kurland, P. Voynow and G. Mora, Biochemistry, 8 (1969) 2897. 9 E. Kaltschmdit and H.G. Wittmann, Anal. Biochem., 36 (1970) 401.

10 V.K. Laemmli, Nature, 227 (1970) 680. 11 H.H. Bradford, Anal. Biochem., 72 (1976) 248.