Specific purification of elongation factor 2 and isolation of its antibody

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 134, No. 2,1986

Pages 1015-1021

January 29,1986

S P E C I F I C P U R I F I C A T I O N OF E L O N G A T I O N F A C T O R AND I S O L A T I O N OF ITS A N T I B O D Y

Kiyoharu

Institute

Takamatsu*,

Tsuyoshi

Uchida

and Y o s h i o

for M o l e c u l a r and C e l l u l a r Biology, I-3, Yamada-oka, Suita, Osaka 565,

Osaka Japan

2

Okada

University

Received December 9, 1985 E l o n g a t i o n factor 2 (EF-2) was p u r i f i e d from rat l i v e r e x t r a c t s by a f f i n i t y c h r o m a t o g r a p h y using f r a g m e n t A of d i p h t h e r i a toxin as the ligand. P u r i f i e d EF-2 has a m o l e c u l a r w e i g h t of 96,000 and i s o l e c t r i c point of 6.6-6.8. The sequence of the n i n e t e e n N - t e r m i n a l amino acid is V a l - A s n - P h e - T h r - V a l - A s p - G l n - I l e Arg-Ala Ile-Met-Asp-Lys-Lys-Ala-Asn and the C - t e r m i n a l amino acid is leucine. P u r i f i e d rat EF-2 m o d i f i e d w i t h A D P - r i b o s e was injected into rabbits to p r e p a r e a n t i b o d i e s a g a i n s t EF-2. The a n t i - E F - 2 a n t i b o d i e s can i m m u n o p r e c i p i t a t e w i t h EF-2 from v a r i o u s e u k a r y o t i c cells. ©1986AcademicPress,lnc.

Eukaryotic

elongation

(Mr:

about

tRNA

from the a m i n o a c y l

eukaryotic

93,000),

the p r e s e n c e diphtheria of EF-2

ribosylation been could

studied

to the p e p t i d y l

The t r a n s l o c a t i o n

(5,6).

localization and GTP b i n d i n g (10).

a l s o bind

*Present address:

chain

of p e p t i d y l -

site

(2,3) on the is c o u p l e d

is A D P - r i b o s y l a t e d

activity

of fragment

decreases

in r e d u c i n g

synthesis

polypeptide

of p e p t i d y l - t R N A

(4). E F - 2

Ribosylation

results

protein

a single

the t r a n s l o c a t i o n

of NAD by the c a t a l y t i c

toxin

Recently,

site

of GTP to GDP

(7) which

inhibiting

EF-2,

(I) c a t a l y s e s

ribosome.

to the h y d r o l y s i s

factor

ribosomal

GTP h y d r o l y s i s

in

A of binding

(8) and

in

(9).

of the sites r e s p o n s i b l e in the e u k a r y o t i c

We a s s u m e d

that

the EF-2 molecule.

fragment In this

for ADP-

EF-2 m o l e c u l e s A of d i p h t h e r i a

paper

Biochemical Research Laboratory Company, Osaka, Japan.

we show

has toxin

that EF-2

of Fuji Oil

0006-291X/86 $1.50 1015

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

VoI. 134, No. 2, 1986

can be p u r i f i e d diphtheria

have

been

EF-2 we h a v e

N-terminal prepared

by a f f i n i t y

toxin as the

purification purified

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reactivity

ligand, already

with EF-2

reports

presented

(11-13).

Using

against

the

rat EF-2

from v a r i o u s

MATERIALS

fragment

previous

and the C - t e r m i n a l

antibody

using

although

now d e t e r m i n e d

amino acids

rabbit

chromatography

cell

AND

sequence amino

A of on EF-2 this

of the n i n e t e e n

acid.

and we h a v e

We h a v e tested

also

its

species.

METHODS

MATERIALS [U14C] N~p (280 mCi/mmole), [ a d e n y l a t e - 3 2 p ] NAD (1,000 Ci/mmole) and L-[J~S] m e t h i o n i n e (1,000 Ci/mmole) were o b t a i n e d from Amersham. P r e p a r a t i o n of f r a g m e n t A - c o n j u g a t e d S e p h a r o s e 4B Fragment A of d i p h t h e r i a toxin was p r e p a r e d from c u l t u r e s u p e r n a t a n t of strain C7(~22) , by a m m o n i u m s u l f a t e p r e c i p i t a t i o n and c o l u m n c h r o m a t o g r a p h y on DE-52(Whatman) and S e p h a d e x G-150(Pharmacia) as d e s c r i b e d p r e v i o u s l y (14). F r a g m e n t A was then c o n j u g a t e d to C N B r - S e p h a r o s e 4B(Pharmacia) (i mg p r o t e i n / m l of wet gel volume). P r e p a r a t i o n of EF-2 from rat l i v e r P r e p a r a t i o n of rat l i v e r extract: Fresh l i v e r s from about 60 rats were h o m o g e n i z e d with 5 v o l u m e s of 0.25 M sucrose, c o n t a i n i n g 3.3 m M p h e n y l m e n t h y l s u l f o n y l f l u o r i d e ( P M S F ) in a W a r i n g b l e n d e r at 0-4~C. The h o m o g e n a t e s were f i l t e r e d t h r o u g h a cheesecloth, then c e n t r i f u g e d at 16,000 X g for 20 min. The s u p e r n a t a n t f r a c t i o n were pooled, and d i t h i o t h r e i t o l ( D T T ) was added to a final c o n c e n t r a t i o n of 1.5 mM. A m m o n i u m s u l f a t e f r a c t i o n a t i o n and DEAE c o l u m n chromatography: The s u p e r n a t a n t f r a c t i o n was f r a c t i o n a t e d by 33-65% s a t u r a t e d a m m o n i u m s u l f a t e at pH 7.5, d i a l y z e d a g a i n s t buffer A( 0.02 M TrisHCl (pH 8.3), 2.5 m M EDTA, 1.5mM DTT, 5% g l y c e r o l ) and then a p p l i e d on a DE-52 c o l u m n (2 X 40 cm) e q u i l i b r a t e d with buffer A. The c o l u m n w a s w a s h e d w i t h the s a m e b u f f e r a n d t h e n e l u t e d w i t h a 800 ml linear g r a d i e n t (0-0.25 M) of NaCI in buffer A. EF-2 a c t i v i t y was e l u t e d at 0.1 M NaCl, and the fractions c o n t a i n i n g the h i g h e s t a c t i v i t i e s were p o o l e d and d i a l y z e d against buffer B (0.02 M sodium p h o s p h a t e , p H 7.3, 2.5 m M EDTA, 1.5 m M DTT, 10% g l y c e r o l ) . F r a g m e n t A - S e p h a r o s e 4B A f f i n i t y c h r o m a t o g r a p h y : The d i a l y z e d s a m p l e was d i v i d e d into two or three (column) p o r t i o n s each containing about 5 mg of EF-2. Each p o r t i o n was a p p l i e d to a fragment AS e p h a r o s e 4B c o l u m n (1.4 X 25 cm) e q u i l i b r a t e d w i t h b u f f e r B. The buffer was c h a n g e d to buffer A c o n t a i n i n g 0.2 M N a C l when the unbound p r o t e i n s started to be eluted. F r a c t i o n s c o n t a i n i n g the h i g h e s t A D P - r i b o s y l a t e d EF-2 a c t i v i t i e s were e l u t e d with 0.2 M N a C l - b u f f e r A and w e r e pooled. In each case, proteins w e r e c o n c e n t r a t e d w i t h 30% p o l y e t h y l e n e g l y c o l in buffer C (0.02 M TrisHCL, pH 8.3, I m M MGC12,1.5 m M DTT, 10% glycerol). The concentrated s a m p l e was d i a l y z e d a g a i n s t buffer C c o n t a i n i n g I M NaCl. S e p h a d e x G-150 gel f i l t r a t i o n : The d i a l y z e d s a m p l e was a p p l i e d to a S e p h a d e x G - 1 5 0 c o l u m n ( 1 X 60 cm) e q u i l i b r a t e d with b u f f e r C c o n t a i n i n g I M NaCI to r e m o v e free f r a g m e n t A o r i g i n a t i n g from the i m m o b i l i z e d ligand. The fractions c o n t a i n i n g EF-2 and free of f r a g m e n t A were c o l l e c t e d and c o n c e n t r a t e d with 30% polyethyleneglycol in b u f f e r C. F i n a l l y , the c o n c e n t r a t e d s a m p l e was d i a l y z e d a g a i n s t b u f f e r C and used in the f o l l o w i n g experiments. 1016

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s and i s o l e c t r i c f o c u s i n g SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was p e r f o r m e d a c c o r d i n g to a m o d i f i c a t i o n of the m e t h o d of L a e m m m l i (16). G e l s w e r e s t a i n e d with C o o m a s s i e b r i l l i a n t b l u e R 250. Molecular weights were estimated u s i n g s t a n d a r d p r o t e i n s (Mouse i m m u n o g l o b u l i n G; 150,000, B o v i n e serum albumin; 68,000., o v a l b u m i n 45,000). I s o e l e c t r i c f o c u s i n g was p e r f o r m e d on A m p h o l i n e PAG p l a t e pH 3.5-9.5 (LKB company) a c c o r d i n g to the m e t h o d d e s c r i b e d in the LKB manual. A n a l y s i s of the N - t e r m i n a l s e q u e n c e and d e t e r m i n a t i o n of the Ct e r m i n a l a m i n o acid The N - t e r m i n a l s e q u e n c e was d e t e r m i n e d by the m e t h o d of Edman(17). The s e p a r a t e d p h e n y l t h i o h y d a n t o i n (PTH) d e r i v a t i v e s were i d e n t i f i e d by high p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y and thin l a y e r chromatography(18,19). The s e q u e n c e was a l s o d e t e r m i n e d u s i n g an A p p l i e d B i o s y s t e m s Gas p h a s e sequencer. The C - t e r m i n a l a m i n o a c i d was i d e n t i f i e d u s i n g an a m i n o a c i d analyzer. I m m u n i z a t i o n and p u r i f i c a t i o n of a n t i b o d y P u r i f i e d rat EF-2 was m o d i f i e d w i t h N A D u n d e r the same c o n d i t i o n s as for the ADPr i b o s y l a t i o n a s s a y s y s t e m (ADPR-EF-2). R a b b i t s (about 2 Kg) were i n j e c t e d s u b c u t a n e o u s l y w i t h 0.2-0.3 mg of A D P R - E F - 2 in F r e u n d c o m p l e t e adjuvant e v e r y two weeks. A f t e r ten injections, serum from the i m m u n i z e d r a b b i t s was f r a c t i o n a t e d w i t h 40% s a t u r a t e d a m m o n i u m sulfate. A n t i b o d y a c t i v i t y was t e s t e d by i m m u n o p r e c i p i t a t i o n of [~=P] ADPribose-EF-2. The a n t i b o d y was p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y on rat EF-2 c o n j u g a t e d S e p h a r o s e 4B (2 m g p r o t e i n / m l w e t gel. RESULTS

Purification

of rat liver

Purification cribed EF-2

for each

and Methods.

step,

Step I. Crude extract 33-65% A.S. ppt

3. DE-52

Specific

step are

elution

Table I.

2.

from rat l i v e r was c a r r i e d

purification

chromatography

DISCUSSION

EF-2

of EF-2

in M a t e r i a l s

AND

activities

show in T a b l e

with buffer

out as des-

I.

and y i e l d of For

A containing

the a f f i n i t y

0.2 M NaC1

was

Purification of rat liver Ef-2

Total Total Specific Protein Activity Activity (mg) (nmole) (nmole/mq) 35150 433.2 0.012

Yield (%) 100

Fold I

10990

275.4

0.025

63.6

2

546

224.7

0.411

51.9

33

4. Frag. A affinity

22.0

157.6

7.17

36.4

582

5. Gel filtration

10.7

102.4

9.57

23.6

778

560 g of rat liver was used as starting material. Protein concentration was determined by the method of Lowry(15) and BSA was used as a standard protein. Activity was measured by ADPribosylation according to the method of Gill et al(6) and calculated with 80% counting efficiency. Determination of specific activity and purification fold were calculated from ADPribosylation activity.

1017

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

=) o v

b)

1 2 3

1-]

b 40

×

E

~i

A i t r

0

<

m

i

t

Z 0

Q

i 0<

0.2M Nac, ~ /~", .... -++,+-LJ + 0

25

Q

I o

50

B~A"~ "~=

9

FRACTION NO.

6 pH

Q

3

Fig. 1. Purification of EF-2. Fragment A-Sepharose affinity chromatography. The main fraction of EF-2 purified by DE-52 was applied to a fragment A-Sepharose column. Fraction volumes were 3ml except when elution was performed with 0.2 M NaCl (1.5mi). The position of buffer changes is indicated by an arrow. 1Q4~I of each sample assayed by ADP-ribosylation in the presence of [J C]-NAD and fragment A and the collected fractions were designated as shown on the figure. Fig. 2. Biochemical analysis of rat EF-2. a) Comparison of EF-2 preparations each purification step by SDS polyacrylamide (9%) gel electrophoresis. Samples were analyzed by SDS polyacrylamide gel electrophoresis. Lane I. 10 ~g of protein from the ADP-ribosylation active fraction of DE-52; lane 2. 5 ~g of protein from the peak fraction of fragment A-Sepharose elute; lane 3. 5 ~g of protein from the Sephadex G-150 M. b) Isoelectric point of EF-2 Isoelectric focusing was performed on Ampholine PAG plate pH3.5-9.5 (LKB company). 10 ~g of purified rat EF-2 and 10 ~g of BSA (pI 4.7-4.9) were loaded on an Ampholine PAG plate.

started when finity weak

the first

to the m a t r i x

binding

continously

after

that EF-2 was step.

fragment

specific

However,

of f r a g m e n t

i).

to i m m o b i l i z e d

chromatography

is v e r y

to EF-2.

(Fig.

the u n r e t a r d e d

fication method using graphy

containing

proteins

Since

fragment

but

purified This

A,

has a

at low p r o t e i n

indicated

out.

Fig.2-a

b a n d by the that

the puri-

for a f f i n i t y

chromato-

in spite of its w e a k b i n d i n g a c t i v i t y

ADP-ribosylation

Therefore,

as a s i n g l e

result

no af-

if the c o l u m n was w a s h e d

was w a s h e d

A as a l i g a n d

for FF-2

A contaminated

chromatography.

protein

showing

the EF-2 m o l e c u l e

w i t h b u f f e r B, EF-2 was e l u t e d g r a d u a l l y

1,2) shows

affinity

appeared

activity

concentration (lanes

fractions

assays

in the EF-2 to r e m o v e 1018

revealed

that a s m a l l

amount

fraction

obtained

by a f f i n i t y

remaining

fragment

A from the

Vol. 134, No. 2, 1986

EF-2

pooled

fractions,

the p r e s e n c e fractions central

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 1 M NaCl.

corresponding

purification

assay

(Table

but

fraction

using

purified

part

The p u r i f i e d

part

EF-2

Rat EF-2

isoelectric

experiments.

of the EF-2

such

has a specific

purified

The

point

of

here

as a s i n g l e

(Fig.

amino

those

(data

not

of part

nineteen N-terminal

bound

is leucine.

2-b).

Analysis

acid was valine.

of p r e v i o u s shown).

the EF-2

body

reaction,

This r e s u l t s

against labeled

EF-2 with

ADP-ribose

blocked

anti-ADPR-EF-2

protein

(Fig.

2-a).

(Table

I),

to one EF-2 m o l e c u l e .

the

electrophoresis

and

of PTH d e r i v a t i v e s These

results

are

reports

(21,22),

as the amino

In addition,

in this

report,

protein

was

carried

out.

The

amino acid are V a l - A s n - P h e - T h r - V a l - A s p - G l n - I l e -

will

and C h a r a c t e r i z a t i o n

or EF-2

compound(s),

It has a m o l e c u l a r gel

Arg-Ala-Ile-Met-Asp-Lys-Lys-Ala-Asn-Ile-Arg

EF-2

by this procedure,

of 9.57 n m o l e / m g

molecule

a

chromatography

as NAD or r e l a t e d

activity

reported

in the p u r i f i e d

Thus,

was characterized.

6.6-6.8

with

analysis

Antibody

has been

In our protocole,

96,000 by SDS p o l y a c r y l a m i d e

composition

and EF-2

peak.

by r i b o s y l a t i o n

toxin

contained

column.

was p u r i f i e d

to one A D P - r i b o s e

identical

Preparation

in

The total yield of

by DEAE c e l l u l o s e

A-Sepharose

EF-2

show that the N - t e r m i n a l

acid

eluted

out in

of rat EF-2

of about

sequence

of the

with dipththeria

substance(s), and

Characterization

acid

was carried

A was not detected

23.6% as e s t i m a t e d

EF-2 f r a c t i o n

corresponds

almost

filtration

by this procedures.

to a f r a g m e n t

be r e m o v e d

weight

was

NAD-agarose

that

inactivation

which

to the c e n t r a l

is i n a c t i v a t e d

applied

could

Thus fragment

procedure

it seems

partially

EF-2

gel

i).

A method

was

G-150

part was used in the f o l l o w i n g

this

(12)

Sephadex

be u s e f u l

of a n t i b o d y

obtained

here

had no c o m p e t i t i v e

(data not

shown).

1019

for c l o n i n g agasinst

of

amino

the EF-2

EF-2

did react with

[35S]-methionine.

immunoprecipitation

antibody

and the C - t e r m i n a l

[32p]-ADPR-

In this a n t i g e n - a n t i activity,

but A D P R - E F - 2

[32p]-ADPR-EF-2

with

gene.

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

..~ L Cell extract Yeast extract m inmmnei~reeilD, lemameelmme~ 12348

l

1234

~lfhamQerm extract Rat E F - 2 w ieNegx~, OoiD ilenu~reoip

6

-~

12345

z

12345

Fig. 3. Crossreactivity oZ anti-rat EF-2 antibody with EF-2 from various eukaryotic cell species. L cells(5 x 105 cells) were lysed with 10 mM Tris buffer (pH 7.5) containing I mM MgCl 2 3 mM PMSF, 5 mM DTT and 0.4% Nonidet p40, and were then centrifuged at 6,000 x g for 5min. The supernatant was used as cell extracts. 2 ml of f r o z e n y e a s t c e l l p e l l e t or I g of w h e a t g e r m was c r u s h e d w i t h I g q u a r t s sand in a c h i l l e d mortar. T h e n 3 ml of H e p e s - K O H buffer (pHT.6) containing 0.1 M KCl, I mM MgCl 2 1 mM DTT, I mM PMSF was added to the mixture, and centrifuged. The supernatants were u ~ d as extracts. Each cell extract was ADP-ribosylated with [J~P]-NAD as describe~ in Materials and Methods. Then, each sample containing 1.2-1.3 10 J cpm of ADP ribosylated EF-2 was immunoprecipitated with anti-EF-2 antibody (1.6 mg/ml) and protein A-Sepharose (10 ~i wet volume). Antibodies: lane I; no addition, lane 2; 1.6 ~g, lane 3; 4.7 ug, lane 4; 16 ~g, lane 5; 47 ug. IgG concentration was adjusted to 47 ug with non immune IgG (1.6 mg/ml).

Finally, EF-2

cross

we e x a m i n e d

reacted

ADP-ribosylated germ

and yeast

with

cell were

EF-2

antibody

against

rat EF-2,

germ

had

a higher

that

the

antibodies and

and t h a t

its

course

the

cell

activity

than

could

EF-2.

has a l s o

Fig.

These

common

rat

[32p]_ wheat 3 shows

react with

L cells

site(s)

are p r o b a b l y

from mammalian

species.

the a n t i b o d y .

antigenic

nature

against

from mouse L cells,

tested

yeast

prepared

cell

but EF-2 from mouse

sites

immunological

with

species

recognized

these

of e v o l u t i o n

obtained

precipitated

three

this antibody

from different

extracts

t h a t E F - 2 of a l l

molecule

whether

on

and wheat

results the

suggest

EF-2

among eukaryotes

been conserved

in the

to yeast.

ACKNOWLEDGMENTS

T h e a u t h o r s w i s h to t h a n k Dr. T s u n a s a w a a n d Prof. S a k i y a m a O s a k a U n i v e r s i t y a n d Dr. I c h i r o u K u b o t a of S u n t r y B i o c h e m i c a l L a b o r a t o r y for c r i t i c a l a d v i c e on a m i n o a c i d a n a l y s i s a n d a m i n o a c i d s e q u e n c e , a n d a l s o t h a n k Dr. H e l e n H a y e s of t h i s I n s t i t u t e h e l p w i t h the E n g l i s h text.

1020

of

for

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

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