Turnover of these acetyl groups in histones is observed in the mammalian cells and the rate varies among various tissues( Pogo et al.,1968 ; Byvoet ,1968 >.
Vol. 36, No. 1, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ENZYMATICDEACETYLATION OF HISTONE
Akira Inoue and Daisaburo Fujimoto Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan
Received May 27, 1969
SUMMARY An.enzyme activity which catalyzed the deacetylation of histone was found in the calf thymus extract. 14C-Acetyl-histone prepared by incubating calf thymus nuclei with sodium14C-acetate served as the substate and treatment of the histone with pronase destroyed most of the susceptibility to the enzymatic deacetylation. 14C-Acetyl-histone prepared by chemical acetylation with 14C-acetic anhydride served as the substrate to a less extent.
Acetyl either
groups are present in many histone fractions
and they are attached
to the amino-terminal nitrogen of the polypeptide
chain( Phillips,
1963 ; 1968 ) or to the s-amino groups of lysine residuesc Gershey et al., 1968 ; Vidali acetyl
et al.,1968
; DeLangeet al.,1969
).
groups in histones is observed in the mammaliancells and the rate
varies amongvarious tissues(
Pogo et al.,1968
; Byvoet ,1968 >.
been proposed that the structural
modifications
and deacetylation
the interactions
would influence
DNA and might play a role in regulation ( Allfrey
et al.,1964
which catalyzes
It has
of histones by acetylation between histones and
of information
transfer
from DNA
).
This paper reports that the calf thymus extract ivity
Turnover of these
deacetylation
contains an enzyme act-
of histone.
METHODS Calf thymus ( 2.1 g ) was minced and homogenized in 0.14M NaCl ( 5.0 ml ) 146
Vol. 36, No. 1, 1969
BIOCHEMICAL
in a Potter-Elvehjem
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
type homogenizer for 3 min.
The homogenatewas centri-
fuged at 1OOOOxgfor 15 min and the supernatant was used as the enzyme preparation. Two kinds of 14C-acetyl histones were prepared.
"Biologically
acetylated"
histone was prepared by incubating calf thymus nuclei with sodium l-14Cacetatet. 20 uC / ca. 0.3 g nuclei,
specific
ing to the method of Gershey et al.(
activity
1968 ).
Whole histone was extracted
from the nuclei with 0.25M HCl( MacGillivray ated" histone was prepared by acetylation Whole histone sulfate( with l-14C-acetic
anhydride{ 20 PC ,l.l After
the treatment,
4.0 mg protein
volume of 0.6 ml in 0.025M Tris-HCl buffer, the reaction mixture was acidified 5u mole of carrier
pH 7.3.
) at 37O in a final After
the incubation,
Acetic acid was extracted
by shaking
Two ml of the organic layer was taken, mixed with
solution and the radioactivity
scintillation
with ethanol.
by the addition of 0.1 ml Of O.lM HCl con-
acetic acid.
with 3 ml of ethylacetate.
efficiency
histone was recovered by pre-
14C-acetyl histone( 2.5 mg ) was
incubated with the enzyme preparationc
liquid
acetyl-
u mole ) in 2.5 ml of 2M sodium acetate
with ethanolc 5 ml ) and washed throughly
the scintillator
Yhemically
of histone with 14C-acetic anhydride.
For the assay of the enzyme activity,
taining
,1968 ).
Murray ,1964 ) from calf thymus( 60 mg ) was treated
pH 8.1 at O" for 4 hours. cipitation
40 mC/ m mole ) accord-
counter,
of the extaction
Model GSL-111. with ethylacetatec
was measured by a Kobe Kogyo Values were corrected for the 80 % ) and for the efficiency
of the countingc ca. 80 % ). RESULTSand DISCUSSION
Fig. 1 shows the time course of the deacetylation ally
acetylated"
After
reaction
when "biologic-
histone was incubated with the calf thymus extract.
60 min incubation,
about 55 % of the 14C-acetyl groups was released.
Without the enzyme, no significant
deacetylation
In order to confirm the liberation
occurred.
of 14C-acetate and to check the possible 147
Vol. 36, No. 1, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. Time course 1
( dpm
1
of the deacetylation
( B 1 70
t500
t
e-Q-------------------o---
0
I
I
I
I
I
IO
20
30
60
1080 (
Time
( Minutes
The "biologically acetylated" dpm ) was incubated with ( e calf thymus extract at 37O .
contamination acetate the
of 14C-acetyl-amino
extract,
acetic
ethylacetate
acid
for
As shown in Table
results
out,
from
or acyl-lysine Deacetylation
the
different
90 % of the
ethylacetate
cooperation
rate
known
that
of proteases
with
of the
radioactivity. with
pronase
deacetylation.
acetyl
groups
aminoacylases.
acetate
from
i.e.14C-acetic
extractable
the
the
E.C.3.5.1.17
distillation
distillatec
enzymatic
attacks
other
in the ethyl-
14C-acetyl-histone
to the
deacetylase from
deacylase(
by azeotropic
of the
)
14C-acetyl-peptides
in the
the possibility the
or
Radioactivity
1, treatment
that
and is
therefore,
resulted
acids
most of the susceptibility indicate
molecule
about
)
histone ( 2.5 mg, 2100 ) or without ( o---o
was seperated
extract.
) accounted
destroyed
acid
18hr
1
The in a large
The results
liberation
ruled
from histone
aminoacylase(
E.C.3.5.1.14
)
).
"biologically 148
acetylated"
histone
and that
of
Vol. 36, No. 1, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Comparison
of the
Substrate
deacetylation
Activity substrate
in
( min
preparations
14C-Acetate
released Degree of deacetylation
Activity )
(%I
( dpm )
2100
0 10 30
97 912 1017
5 44 49
2100
0 10 30
36 133 157
2 6 7
10000
0 10 30
349 524 888
3 5 9
Pronase-digested "biologically acetylated"histone* "Chemically acetylated"histone
of the histone
Incubation time
( dpm 1 "Biologically acetylated"histone
1
14C-acetyl-histone( 2.5 mg ) was incubated with the calf thymus extract at 37O. * --The histone( 2.5 mg ) was digested with pronase( 0.014 mg > in 0.3 ml of 0.05M Tris-HCl buffer ,pH 7.3, at 37O for 60 min. After the digestion, the solution was heated at 100° for 15 min and served for the deacetylation reaction.
the
"chemically
acetylated"
After
10 min incubation,
ated"
histone
of the
"chemically
specific
lysine
"chemically
terminal
The deacetylase
acids
the
only
comparedc
et al.,1968
a high
groups
function
are some indications
groups degree
in the
of acetyl that
acetylation
149
groups
to s-amino
into
acetyl
in the
groups
of the
of lysine
The
).
but
a-amino
case
acetylated"
et al.,1969
characterized,
of specificity
groups
"biologically
"biologically
of a number
histone
1 1.
in the
; DeLange
been
non-specifically
and s-amino
may have
to attach
has not
introduced
groups
In the
are known Vidali
Table
5 % of 14C-acetyl
histone.
" histone
acetyl-amino
Although there
residues(
probably
amino
specific
groups
acetylated
are
while
acetylated"
14C- acetyl
were
44 % of 14C-acetyl
was released
histone,
groups
histone
14C-acetyl
groups
of N-
residues.
and distinguish
some
molecule in histone
is
not yet
and deacetylation
clear are
,
involved
in
Vol. 36, No. 1, 1969
the
activation
1968
).
BIOCHEMICAL
and repression
The deacetylase
Nohara
et a1.,1968;
control
mechanism
of the
demonstrated
Gallwitz,1968 in higher
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gene
( Allfrey
et al.,
as well
as histone
here ) may play
an important
1964;
role
Pogo et al.,
acetylases
(
in genetic
organisms.
ACKNOWLEDGEMENT The authors wish to thank Professor ment and Professor S.Seto for his kind counter .
N.Tamiya permission
for
his interest to use liquid
and encouragescintillation
REFERENCES
Allfrev. _ - V.G.,
Faulkner, R., and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 51, 786 ( 1964 1 B,yvoet, P., BiochiK Biophys. Acta, 160, 217 (1968 ) J. Biol. Chem., DeLange, R.J., Fambrough, D.M., Smith, E.L., and Bonner, J., 244, 319 ( 1969 > Gallwitz, D., BiocG; Biophys. Res. Commun., 32, 117 ( 1968 ) Gershey, E.L., Vidali, G., and Allfrey, V.G., J.xol. Chem., 243, 5018 ( 1968 ) Biochem. J., 2, MacGillivray, A.J., 181 ( 1968 ) Murray, K., Biochemistry, 3, 10 ( 1964 ) Nohara, H., Takahashi, T., and Ogata, K., Biochim. Biophys. Acta, z, 529 ( 1968 ) Biochem. J., 87, 258 ( 1963 ) ; ibid.,%, 135 ( 1968 ) Phillips, D.M.P., Pogo, B.G.T., Pogo, A.O., Allfrey, V.G., 'and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 59, 1337 ( 1968 ) J. Biol. Chem., x, 6361 Vidali, G., Gershey, E.L., and Allfrey, V.G., ( 1968 )
150
