Differential purification of methionine-tRNA synthetase and lysine-tRNA synthetase from rabbit liver

Vol.

78,

No.

BIOCHEMICAL

4, 1977

DIFFERENTIAL

AND

BIOPHYSICAL

OF

METHIONINE-tRNA

PURIFICATION

AND

LYSINE-tRNA

SYNTHETASE

Sherman

R.

Dickman

Received

August

RABBIT

David

LIVER*

J . Boll

University City,

Lake

COMMUNICATIONS

SYNTHETASE

FROM

and

Department of Biochemistry, College of Medicine, Salt

RESEARCH

Utah

of Utah 84112

31,L977

SUMMARY: Purification procedures for methionine-tRNA synthetase and lysine-tRNA synthetase from rabbit liver cytosol are presented. The purification factor for both enzymes is higher than that reported previously. The data suggest that methionine-tRNA synthetase and lysine-tRNA synthetase occur as a mixture in rabbit liver cytosol rather than as a complex. The

concept

of a large

responding

tRNA’s

in eukaryotic

cher

Smaller

complexes

(1).

and

Hardesty

ideas the

are

(3), mainly

located proteins satisfied.

of very

the

A procedure enzymes,

Olsen

(4).

types:

1.

and

S20,w

which

in complexes,

mixture

rather

than

research

was

Constancy

results

would

and

molecular

above

of activities

enough

not

to break

Copyright 0 I977 by Academic Press, Inc. Ail righ/s of reproduction in any form reserved.

support Such

by

the

for the

evidence

USPHS

are

the

No.

(2),

Som these

activity)

when

steps.

2.

Sedimen-

activities

are

as a mixture

of large

would

suggestive

but

not

conclusive.

between

two

or more

of bonds

that

is included

Grant

above

Deuts-

criteria

types

idea

occur

cor-

supporting

enzyme

of enzyme

in question

cited ratio

in part

(i .e.,

weights,

and

evidence

peaks

and

Bloemendal

purification the

enzymes

the

provide

and

experimental

that

alter

as a complex.

supported

by Vennegoor The

synthetases Bandyopadhyay

of composition

If the

of the studies

was mild

by

of chromatographic

value.

would

postulated

demonstration

properties

which

present

*This

and

and

of aminoacyl-tRNA

fint

Roberts

to a number

similar

Thus,

was

studied

gradient

at a constant

cells been

is subiect

in a sucrose

composed

have

of two

preparation

tation

and

complex

these in this

AM

expected enzymes

also

be

of these to be

occur

as a

report.

00803.

1191 ISSN

0006-291

.Y

Vol.

78,

No. 4, 1977

BIOCHEMICAL

AND

BIOPHYSICAL

MATERIALS

AND

METHODS

RESEARCH

COMMUNICATIONS

3H-L a b e I e d amino Materials. acids were purchased from New England Nuclear Inc. Specific activities were: L-methionine, 190 mC/mmole; L-lysine, 2.2 Ci/mmole; L-leucine, 5 Ci/mmole; and L-isoleucine, 65 Ci/mmole. The latter three were diluted with the corresponding ‘H-amino acid to furnish approximately the same yield of 3Haminoacyl-tRNA as with 3H-Met in the standard assay. ATP and dithioerythritol were obtained from Sigma. Ultrapure ammonium sulfate was purchased from Schwarz/Mann. Yeast-tRNA was from Calbiochem. DEAE-cellulose and HTP hydroxylapatite were bought from Bio-Rod and Sepharose 4B from Pharmacia Inc. Acrylamide, bis-acrylamide, tetramethylenediamine and mercaptosuccinic acid were purchased from Eastman. All other chemicals were reagent grade. Deionized distilled water was routinely used. Assays. Proteinwasstimated Lowry procedure

Aminoacyl-tRNA synthetases were assayed either by A2~~A260 measurements (7) using beef serum albumin as standard.

as described previously (6) or by a modification

(5). of the

Preparative Procedures: Cytosof from rabbit or dog liver and tRNA activator were prepared as described previously (5). Standard Tris buffers (pH 7.5 at 3’) contained the stated concentration of KCI and 0.01 M MgCl2, 0.’ mM dithioerythritol, and 20% glycerol unless specified otherwise. Standard Pi buffers (pm.0) contaiged 0.1 mM dithioerythritol and 20% glycerol. Ai\ preparations were carried out at 4 unless inicated otherwise. Columns were monitored at A280 ond A260 and the wash solution changed when elution of proteins with a given buffer appeared to be completed. DEAE Cellulose. Pre-washed DEAE-cellulose was suspended in M KCI, and a 4.5 x 34 cm column was prepared. Rabbit liver cytosol Gd the column successively washed with 1 L. each of standard Tris-0.02 0.05 M KCI, and iris-O.1 M KCI. Wash 1 and wash 2 were collected wash 3 was collected in lo-ml fractions. The most active fractions of bined and dialyzed vs standard 0.05fi Pi overnight. This was used to following column.

standard Tris-0.02 (200 mt) was added M KCI, Trisin-6atches while wash 3 were comcharge the

Hydroxylapatite. Biogel HTP was suspended in standard 0.05 M Pi and to hydrate. The sample was added to a 2.5 x 12 cm column and it was washed with standard Pi buffers of increasing concentration. Wash 1 and wash 2 were in batches, washes 3 and 4 in smal I fractions.

was allowed step-wise collected

3 -Aminohexyl Sepharose 4B-Hydrophobic Column. Sepharose 46 was treated with CNBr (8) followed by reaction with hexamethylenediamine (9). It was washed with H20, then with standard 0.05 M Pi buffer. A 1 x 24 cm column was charged with 10 ml of a solution prepared from an Fdroxylapatite column eluate which had been precipitated at 45% saturated ammonium sulphate and dialyzed versus standard 0.05 M Pi. The column was washed successively with 40-ml portions of standard phosphate buKrs. Analytical as descrrbed by free with had

Gel Gabriel

Electrophoresis. Disc (10). Mercaptosuccinic

gels

(5% x 0.25 cross acid was pre-run

link) were prepared through the gels to

them from persulphate. Migration of mercaptosuccinate was monitored by reaction dithiobis-2 nitrobenzoic acid. Current was passed (4 mA/tube) until the dye marker exited. Gels were stained for 2 hr in Comassie Blue. They were destained overnight.

1192

Vol. 78,

4,

No.

BIOCHEMICAL

1977

Standard TrisKCI

AND

BIOPHYSICAL

RESEARCH

Standard PI Buffers O.IM 02M

Wfen 05M

0

COMMUNICATIONS

40

80

I20

2 Figure

1.

(200

ml)

M

KC1

n

KC1

were

Chromatography was

added

buffer.

The

buffer,

Tris-0.05

collected

Figure

in

2.

Details

of to a 4.5 column M

over

cm

DEAE-cellulose.

column

of

was

washed

step-wise

KCI

buffer,

Tris-0.1

Rabbit

DEAE-cellulose with -

M

160 200 Volume m ml

liver

KCI

each

buffer.

280

320

cytosol

suspended 1 L.

240

in

of

standard

The

first

Tris-0.02 Tris-0.02

two

washes

batches,

Purification

are

cytosol

x 34

0.3M

of Met-tRNA

described

in

the

synthetase

by

chromatography

over

hydroxylapatite.

text.

RESULTS Purification

of

a DEAE-cellulose tions

and

cific

activity

and

in

45%

versus

standard and

a 32

saturated

was

buffer.

were in

Synthetase.

The

2 contained

slightly

resulted

tite

KCI

wash

appeared

to

column.

of standard

14%

Methionine-tRNA resin The 30% not

of

of in

ammonium

sulfate,

0.05 successively

-

M

the

Pi buffer. washed

successively

pattern

is shown

original

15%

This with

peak

yield. pH

in

in The

7.4,

the

solution increasing

1193

the

0.1

M -

concentrations

Wash

washes

KCI

of

low

spe-

activity This

a column of standard

and

peak

procedure

combined,

collected on

on

1 contained

were

wash. were

precipitate

layered concentra-

synthetase

solutions

placed

was

increasing 1.

Met-tRNA

peak

was

cytosol

Fig.

These

The

protein

liver with

activity.

investigated. the

x purification

washed

elution

further

advance

was

Rabbit

brought dialyzed

of hydroxylapaPi

buffer.

As

Vol.

78, No.

4, 1977

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Standard PI Buffers

Purification 48 column.

Figure 3. Sepharose

shown

in fig.

resulted again

2,

the enzyme

in a considerable brought

dialyzed

versus

saturated

standard

successively

stronger

eluted

from

column

min/mg.

This

calculates

summary

of the

results

The tase these

ore

ratios

presented

ratios

from

similar

to that

similar

procedures.

specific

found

cytosol

-

by Vennegoor

was

and

had

treatment

fractions

was

used

in Fig.

purification

column

and

tube

were

collected

to charge

and

a Sepharose

It was washed

M P. buffer. I 3,

the

a specific

enzyme

with

activity

activity

in an overall

of Met-tRNA

the

This

of 44 nmoles/

yield

of 5%.

A

I.

activities

through

0.05

u’-aminohexyl

Pi buffer.

precipitate

solution

M P. buffer I

in Table

right-hand

the

As shown

a 300-fold

is included

in the the

of Pi buffers.

to about

of the

This

over

The peak

sulphate,

in standard

0.2

in 0.3M

activity.

Pi buffer.

with

by chromatography text.

peak

in specific

(1 x 24 cm)

solutions

this

as a sharp

ammonium

O.OSr\n

column

synthetase given in the

eluted increase

to 45%

4BCU-aminohexyl

was

of Met-tRNA Details are

in the

most

highly

BLoemendaI

1194

synthetase table.

There

purified (2) and

and was

Lys-tRNA little

fractions. by

Som and

synthe-

change This

Hardesty

in

constancy (3

is with

Vol.

78,

No.

4, 1977

BIOCHEMICAL

Table

I.

AND

Purification Synthetases

BIOPHYSICAL

of Methioninefrom Rabbit

Cytosol DEAE-cellulose chromatography

Yield

-fold

%

0.14

3.

Precipitation saturated

4.

Hydroxyapatite chromatography

5.

Hexylamine arm SepharDse 4B, chromatography

various

When

The

by

preparations

gel

band

was

were

considerably

heavy

band

represents

rather

than

stained

demonstrating

of the

15

-

-

7.2

52

14

-

-

30

211

8

44

307

constant

with

buffer

eluate

tube

in peak

enzyme.

1 .3

5

1.6

in any

centrifuged 2 hr versus

at 95,000 10%

the

Sepharose

two

other

very

For

runs

the

we have

bands

this

the

reason, were

yet

wasana-

were

whereas

gels

not

column

light

columns,

samples.

In other

we

two

light

believe

scanned

been

found.

the

at 280

nm

successful

in

iayered

on a

washed

succes-

band.

0.05

Rabbit

-

x g to remove It was

liver

M P. buffer, I

of Pi buffer

aminoacyl-tRNA

glycerol.

and

from

Synthetase.

concentrations

text.

aminohexyl

band

However,

in standard

contained

the

less purified

of Lysine-tRNA

increasing

from heavy

protein.

activity

in the

fractions

in

the active

of hydroxylapatite

sively

peak

heavier

enzyme

described

one

to locate

Purification

tus for

32

are

electrophoresis,

bands

was

2.5

on

an aliquot

disc

heavy

column

100

~~_~ ~~

‘The

lyzed

1

Activity

Ratio Me t/Lys

4.6

with 45% ammonium sulfate

~~~~~

Lysine-tRNA

Purification

nmol/min/mg 1. 2.

and Liver

COMMUNKATIONS

Specific

Met-tRNA Synthetase Specific Activity

stepa

RESEARCH

pH 7.0.

as shown

synthetase particles adjusted

1195

and

cytosol

in Fig. activity.

dialyzed

to 0.01

- M Tris

was It was 2.

The

The

solution

in a hollow buffer,

0.3

-M Pi (115

fiber

pH 7.0,

ml)

apparaand

Vol.

78,

No.

4,

1977

BIOCHEMICAL

Table

II.

AND

Purification

of

Separation

from

BIOPHYSICAL

RESEARCH

Lysine-tRNA

Synthetase

Methionine-tRNA

COMMUNICATIONS

and

Its

Synthetase

Aminoacyl-tRNA Syn the tase Step’

Specific

Specific

Activity

Activity

Ratio

nmole/min/mg Met Cytosol 1st

H.A.

Column,

0.3

DEAE-Cellulose 2nd

1st

0.25

M

0.3

-~Pi

Peak,

aExperimental

layered

on

creasing was

M -I

with

and

-

M

a lower

ratio

showed

the

in

the

0.25 0.03

included.

than

lowest

specific are

any

2.

0.3

-

M

P. has I

washed

The

similar

ml)

column

which

are

previously. for

over

in

The

Lys-tRNA series

800-fold,

1196

Table

Table

while

to

that

for

those

- M and

Met-tRNA

this

of

the

column

solution

a Met/Lys synthetase

of

a 0.25

of Lys-tRNA

from

the

had

Lys-tRNA

P. I

It was

that

0.25

that

Pi

Pi eluate,

synthetase, the

with

M

II.

0.25%

0.3&I

-

buffer.

except

I except

with

and

0.05

same

Pi buffer,

synthetose in

centrifuged,

the

eluted

in-

activity

to standard

contained

eluted

In this

obtained. increased

material

thawed,

were

with

synthetase

adjusted

of Met-tRNA

obtained activity

was

presented

included

manner

Met-tRNA

frozen,

synthetases

activities

those

a stepwise

of standard

tRNA

to

of

was

(100

activities

in

peak

solution

solution

The

have

text.

was

The

column.

specific we

the

concentrations

relative

the

highest

KCI.

Aminoacyt

Their of

in

buffer.

The

increasing

(Met/Lys)

the

35 88

a hydroxylapatite

with

ratios

-M

glycerol.

hydroxyapatite

0.03,

2.8

which

KC!-Tris 0.1

on

P. buffers. I

synthetase

ity

layered

given

column of

10%

was

The

second

are

standard

as before

P. wash

0.3

5

9

details

versus

washed

14 3

a DEAE-cellulose

eluted

buffer

0.4

Pi

concentrations

dialyzed

0.1

7

Column

Peak,

2nd

Pi

t/Lys

0.2 M

Column

H .A.

Me

LYS

had

which

ratio specific synthetase

of activhas

Vol.

78, No.

4, 1977

increased two

by

the

which

usually

concept

The

result

also

is in the

gradient;

ratio

a significant

tained are

a value considered

weight

in the

wide

step

if the

elution.

steps

to be present range

of the

fairly

this

6 x lo5

the

similar

If the

- 1 x 10

6

to those

and

of these

does exposed

not

to conditions

of Vennegoor

The

I, this steps

are

If the data

that

main

Met-

suggest

they

differ

and that

not

affect

Bloemendal Lys-tRNA

each

in charge

a linear

as shown

and

the

in Table (2) ob-

synthetases

possesses by

Bloemenin tech-

used

does

narrowed,

and

difference

They

difference

Vennegoor

of enzymes. the

not

columns.

in Table

as a mixture,

was

material.

occurred.

group

of purification

solution

hydroxylapatite

wide.

separation

complexes.

as starting

of activities

of 165 for

most

As shown

are

separation

are

liver

of elution

since

COMMUNICATIONS

a significant

in degree

of enzyme

above

RESEARCH

that

divergence

complex

mammalian

BIOPHYSICAL

demonstrate

dissociation

reported

method

AND

data

This

in the

used

we used

Met/Lys

These

of an enzyme

experiments

(2) who

nique

1%fold.

has occurred.

support

II,

only

enzymes

dal

BIOCHEMICAL

OS little

a particle CIS one

unit. ACKNOWLEDGMENT We

thank

Sylvia

Bjorge

Berman

for

excellent

assistance.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Bandyopadhyay, A.K. and Deutscher, M.P. (1971) J. Mol. Biol. 60, 113-122. Vennegoor , C . and Bloemendal, H. (1972) Eur. J. Biochem. 26, 462-473. Som, K. and Hordesty, B. (1975) Arch. Biochem. Biophys. 166, 507-512. Roberts, W.K. and Olsen, M.L. (1976) Biochim. Biophys. Ax 454, 480-492. Dickmon, S.R. and Boll, D.J. (1976) Biochemistry 15, 3925-393r Layne, E. (1957) in Methods in Enzymology, III (CoGick, S.P. and Kaplan, N.O., eds) pp. 447-454, Academic Press, New York. Campbell, P.N. and Sargent, J.R., eds (1967) Techniques in Protein Biosynthesis, 1, pp. 299-300, Academic Press, New York. Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3059-3065. Jakubowski, H. and Pawelkiewicz, J. m3) FEBS Lett. 34, 150-153. Gabriel, 0. (1971) in Methods in Enzymology, XX (JakoG, W.B., ed) pp. 565577, Academic Press, New York.

1197