Extraction and partial purification of two histone-specific transacetylases from rat liver nuclei

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL

EXTRACTION AND PARTIAL HISTONE-SPECIFIC

RESEARCH COMMUNICATIONS

PURIFICATION

OF TWO

TRANSACETYLASES FROM RAT LIVER NUCLEI. Dieter

Gallwitz

Physiological Chemical Institute University of Marburg (Lahn), Germany

Received

June 5, 1970

SUMMARY Two histone-specific transacetylases with an approximate molecular weight between 80,000 and 90,000 were solubilized from rat liver nuclei by sonication in the presence of 1 M ammonium sulfate and 20% glycerol followed by precipitation with 3.5 M ammonium sulfate. A SO-fold purification was achieved by DEAEcellulose and Sephadex G-200 chromatography. The acetate transfered by the enzymes from acetyl coenzyme A to histones is N-linked. The pH-optimum of the enzymatic acetylation is approximately 8.6. The occurence histones

has been

E-N-acetyllysine incubation acetate that

of

isolated

(3).

From the

in the

arginine-rich

acetylated

in

a specific

lysine

and takes Methylation other

of histone the

known

histone

for

place

recent

nuclei

on already

of

occuring

IV and may have

presence it from is

This

--in vitro of labelled

is evident calf

thymus

partially

modification

of

be an intranuclear

to

completed

and phosphorylation

2al)

residue

(Q,5,6). seems

after

studies

IV (f

lysine

the S-N-position residue

in the

sequencing

in

In addition,

in histones

histone

a specific

residues

(1,2).

identified

thymus

modifications

acetylserine

some time

has been

and pea seedlings

are

of NH2-terminal

protein

molecules

specific

in the

amino

highly

some influence

process

acid

basic

on the

(7,8). residues portion

binding

of

to DNA.

To establish

a basis

for

an understanding

236

of the

biological

Vol. 40, No. 1, 1970

meanins

of

involved

BIOCHEMICAL AND BIOPHYSICAL

these

modification

should

prove

acetone

fractionation

proteins

catalyzing;

to histones

more

helpful, of the

of

some properties

It

the

study

has already

of

been

the

enzymes

shown that

pieeon

(9)

and rat

liver

(10)

transfer

of

acetate

from

acetyl

or less

The extraction

reactions

RESEARCH COMMUNICATIONS

yields coenzyme

A

unspecifically,

two histone-specific

of the

partially

transacetylases

purified

enzymes

are

and reported

here. EXPERIMENTAL Liver rats

nuclei

initially

(10,111,

the

X 100

(Tris-ECl,

(12).

5 mll,

viscous

125 !Jatt)

3.5

with

suspended

EDTA, 0.25 glycerol,

90 min in

v/v) the

and

x gav for

medium

B or passed

in

solution

equilibrated

at

with

the

in

ZDTA,

over

0.25

2'

in

the

x S,,.

for

2 hrs. a final

cold

medium

concentration

the

extract

onto

B, Proteins

To remove

pellets

237

MgC12,

the

1 mM,

ammonium

centrifugation for

extracted

DEAR-cellulose were

was

5 m?l, 20%

G-25 equilibrated the

the

Crystalline

The protein

dialyzed

20% of

v/v),

15 mV, pH 7.6,

60 min.

Sephadex

adsorbed

mY, 2-mercapto-

20 kHz,

1 hr

after

A

sonifier,

B (Tris-HCl,

about

medium

(Branson

yield

obtained

detergent

in

to

for

al

was sonicated

added

10 min was either

and were

by use of

"i istar

bath

at

120,000

stirred

procedure

et -2

10 mM, 2-mercaptoethanol,

supernatant

40,000

this

stirred

medium

mM, NH4C1,

an ice

bursts

for

Chauveau

homogenized

1 mll,

g bR II

1 ?!, 20% qlycerol,

in

25 2 set

120-18(!

of

study

cjere

sulfate,

stirring

for

the

NgC12,

was slowly

I?. After

sulfate

of

immersed

sulfate

centrifuged

After

state

ammonium

ma le

procedure

and subsequently

ammonium

from

The nuclei

homoqenate

40 ml aliquots

were

later

75 mM, p:i 7.6,

ethanol,

of

prepared

by a modified

in

Triton

were

eluted

at 40 hrs with

against medium

proteins

remained

previously from

B.

the

column

Vol. 40, No. 1, 1970

with

a linear

enzymatic 3.5

0.01-0.3

activity

medium

sulfate

activity

C-acetate

acetyl

0.35

NH,+Cl,

25 @moles

of

glycerol

(v/v),

0.025

56.6

mC/mM),

total

volume

were

discs

KCl,

of

0.5

37')

1 mg of

the

incubation ice

Sephadex

were

with

G-200

equilibrated

Protein

hot

0.1 ymole of

contained EDTA,

act.

protein

in

incubation

time

was added

as carrier

and

as described

precipitated

ml

filter

The filters

(10).

purified with

was estimated

a

0.2

onto

acid.

rat

0.14

or thymus Extraction

precipitation

according 56.6

act.

liver

M NaCl.

N HCl and subsequent

Nuclear

A (spec.

the

were

of

15%

25 ,ug enzyme

25% trichloroacetic

A (spec.

15 @moles

3.5 ,umoles

coenzyme

end of

from

acid

2-mercaptoethanol,

and 2 to the

of

trichloroacetic

of

l4 C-1-acetyl

washing

coenzyme

New England

incorporation

medium

mixture

prepared

by 0.25

14 C-1-acetyl

A into

albumin

and counted

was achieved

from

At

cold

extensive

acetone.

,uC of

ml.

after

of

having

precipitated

as the

2 flumoles

50 pug substrate

treated

nuclei

proteins

over

coenzyme

flumole of MgC12,

in

Histones

The fractions

The incubation

Tris-HCl,

paper

and passed

material.

of

the

was determined

from

precipitable

aliquots

pooled,

RESEARCH COMMUNICATIONS

B.

Enzyme

(20 min at

AND BIOPHYSICAL

M NH&Cl gradient.

were

M ammonium

with

14

BIOCHEMICAL

to

Lowry

et -2

in

10 vol

al

(13).

mC/mM) was purchased

Corporation.

RESULTS AND DISCUSSION The DEAE-cellulose preparation one major about

0.09

from peak

of activity

G-200

liver

of activity

M NH4C1.

The enzyme Sephadex

rat

elution

could

nuclei

is

with

be further in

may point

Fig. to

of

the

shown

an intimate

The purification

as seen

which

profile

in purified 2. There

the 238

in

soluble Fig.

enzyme 1. There

shoulder

this

step

eluting

existence

two distinct of

at

was 6- to

by chromatography are

is

at

least

8-fold. on

peaks two

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 40, No. 1, 1970

Fig. 1. DEAE-cellulose chromatography of soluble nuclear histone transacetylase. 150 mg protein were adsorbed to a 2 x 9 cm DEAE column and eluted with a linear 350 ml 0.01 - 0.3 M NH4C1 gradient at a flow rate of 40 ml/hr.

Fip. 2. Sephadex G-ZOO chromatography of nuclear histone transacetylase. 25 me protein previously chromatographed on DEAEcellulose (fractions pooled as indicated by arrows in Fig. 1) were chromatocraphed on a 1.8 x 90 cm Sephadex G-200 column at flow rate of 5 ml/hr. different the

histone

molecular

acetylating

weight

of the

enzymes. two

enzymes 239

According is

to

gel

approximately

filtration 80,000

a

Vol. 40, No. 1, 1970

to

90,000.

BIOCHEMICAL

The final

adsorbed

to

coenzyme

not

for

Other

basic (Table

1).

of

the

acetate

from

Under two

the

to

conditions

has yet

of nuclear histone transacetylase. on Senhadex G-200 were incubated protein as described in Methods, wmoles

(rat (rat (bovine)

Cytochrome Lysozyme

C (horse

been

at

acetate

5 ~g of ?,I1 8.6

incorporated

acceptor

liver) thymus)

Hemoglobin

the

proteins

test

enzymes

acetyl

fraction.

protein

histone histone

extract

as nonbasic

50 fig Total Total

the

and predominantly

as well

histone

Table 1. Specificity the enzyme purified with 50 pg acceptor

Acceptor

G-ZOO transfer

specificity

one specific

with

50-fold.

to histones

acetylated

no absolute

found

Sephadex

ones.

were

used

from

compared

was about

A specifically

aroinine-rich tested

purification

DEAE-cellulose

The enzymes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein

22 25

heart)

RNase Albumin

(bovine)

In from

earlier

studies

neutral

by acetyl

to

alkaline

coenzyme

these

acetylating In

test

concentrations

enzymes (14,151 acetylases.

histones

by KC1 at

acid,

of

a final

by raising

acetylated

the

10m3 to

obviously

a chemical

concentration

was optimal

to

10e8

activate

of

for

any effect

240

M adenosine histone on the

pH

spontaneously

the

reaction 50 mM. histone

3).

known

was without

is

pH 8.6

(Fig.

that

were

This

conditions

enzymes

monophosphoric

was observed

A (8,101.

and can be prevented Under

it

3',5'-cyclic phosphorylating

histone

trans-

BIOCHEMICAL

Vol. 40, No. 1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

9.0 PH

Pip,. 3. @I-dependence of nuclear buffer (O--C ) and glycine-XaOII The stability in

hot

incorporated

acid

acetate

shows that

The identification

of

the

the

amino

to

the

acetyl

treatment

groups

acid(s)

are

acetylated

is

progress. This

ally

work

has clearly

acetylate

histone is

the

trichloroacetic

?i-linked. in

of

histone transacetylase. Tris-IiCl buffer (A) were used.

histones

synthesis

probably

likely

not

the

internally nuclear

in

are

takes

initiated

established

N-terminal the

located

place with

histone

that

in

in the

enzymes

the

cell

cytoplasm

N-acetyl-seryl-tRNA amino

acid

molecule

but which

which

nucleus.

Since

(16,17,18)

and

(19) an amino is

specific.

it acid

acetylated

is

very

positioned by the

enzymes.

ACKNOWLEDGEMENTS I wish work

to

and Dr.

thank C.E.

Prof.Dr. Sekeris

P. Karlson for

stimulating 241

for

his

interest

discussions.

in Thanks

this are

Vol. 40, No. 1, 1970

due to

Mrs.

Ch.

BIOCHEMICAL

Pfeiffer

for

AND BIOPHYSICAL

helping

to

RESEARCH COMMUNICATIONS

prepare

the

manuscript.

REFERENCES 1. Phillips, D.M.P., Biochem. J. 87, 258 (1963). 2. Phillips, D.M.P., Biochem. J. 107, 135 (1968). 3. Gershey, E.L., Vidali, G. and Allfrey, V.G., J. Biol. Chem. 5018 (1968). 243, 4. DeLange, R.J., Fambrough, D.M., Smith, E.L. and Bonner, J., J.Biol. Chem. 244, 319 (1969). 5. Ogawa, Y., QuaFarotti, G., Jordan, J., Taylor, C.W., Starbuck, C.W. and Busch,,H., J. Biol. Chem. 244, 4387 (1969) 6. DeLange, R.J., Fambrough, D.M., Smith, E.L. and Bonner, J., J. Biol. Chem. E, 5669 (1969). I. Allfrey, V.G., Faulkner, R. and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S. 5l, 786 (1964). 8. Gallwitz, D. and Sekeris, C.E., 2. Physiol. Chem. 2, 150 (1969). 9. Nohara, H., Takahashi, T. and Ogata, K., Biochim. Biophys. Acta 154, 529 (1968). 10. Gallwx, D., Biochem. Biophys. Res. Commun. 32, 117 (1968). 11. Chauveau, J., Moule, Y. and Rouiller, C., Exptl. Cell Res. 11, 317 (1956). 12. Beato, M., Homoki, J. and Sekeris, C.E., Exptl. Cell Res. 55, 107 (1969). 13. Lowry, O.H., Rosebrough, J.N., Farr, A.L., and Randall, R.J., J. Biol. Chem. 193, 265 (1951). 14. Langan, T.A., Science 162, 579 (1968). B. and Dixon, G.H., J. Biol. Chem. E, 425 (1970). 15. Jergil, C. and Borun, T.W., Proc. Nat. Acad. Sci. U.S. 16. Robbins, 57, 409 (1967). 17. Gallwitz, D. and Mueller, G.C., Science 163, 1351 (1969). 18. Gallwitz, D. and Mueller, G.C., J. Biol. Chem. 244, 5947 (1969). 19. Liew, C.C., Haselett, G.W. and Allfrey, V.G., Nature 225, 414 (1970).

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