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Mechanism of protein synthesis inhibition by fusidic acid and related antibiotics
Vol. 30, No. 3, 1968
MECHANISM
BIOCHEMICAL
OF PROTEIN
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNTHESIS
INHIBITION
BY FUSIDIC
ACID AND
RELATED ANTIBIOTICS
Nobuo
Tanaka,
Institute
Tadatoshi
of Applied
ReceivedJanuary Fusidic
Microbiology,
and helvolinic skeletone,
synthesis
in
the
Harvey
affect
were
et al.,
from
1966;
The
coli,
Fusidic
acid
ribosome-dependent polypeptide both
synthesis
reactions from
the
fusidic
acid
in the
fusidic
reaction, acid.
with
purified
are presented
activity
as well,
absence
enhanced The results
E. coli
and separated
of
transfer in this
antibiotics
enzymes
grade
to
They of
significantly
by GTP and G factor, that
of E.
communication.
of GTP and G factor;
278
GTPase
were observed
and the
indicate
and Lipmann,
steroidal
of G factor.
was not
acid
ribosomes.
Puromycin-dependent
ribosomes
amino
on the
antibiotics
was parallel.
peptide
mycin
of action
GTPase
inhibit
ribosome-dependent
site
and related
of aminoacyl-
T and G (Nishizuka
exhibited
(Yamaki,
They neither
1967).
they
from
factors,
in a system
systems
formation
to protein
factor
results
but
of protoprotein
bacterial
nor
was purified
The detailed
and the
to inhibit
et al.,
complex;
enzyme
latter
was studied
Tanaka
of aminoacyl-sRNA
two complementary
activity.
Tokyo
antibiotics
demonstrated
aminoacyl-sRNA
The transfer
1966).
of Tokyo,
steroidal
and in vitro
sRNA-messenger-ribosome
into
Masukawa
University
acids,
in vivo
synthesis
transfer
and Hiroshi
8, 1968
lanostane
1965;
Kinoshita,
fusidic
inhibit
inhibited
inhibition
of
release
of
affected
by
but
the
was inhibited acid
and
puroby
Vol. 30, No. 3, 1968
related
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
antibiotics
GTPase
affect
activity
of G factor
aminoacyl-sRNA assumption linked
on the
location
ribosomes
synthesis
and inhibiting
ribosomes.
of Nishizuka
to the
polypeptide
It
translocation
(1966)
and G factor,
is
on the
of
seems to support
and Lipmann
of aminoacyl-sRNA
by inhibiting
that
the
GTPase
essential
reaction,
for
trans-
from
the
ribosomes.
Results -Washed
ribosomes,
T and G factors
were prepared
extracts
of E. coli
B by the
of Nishizuka
(1966).
The sRNA of E. coli
acid,
following
the
Lipmann chloroplasts
antibiotics
Institute
of Applied
Y. Nakayama,
The effects alanine
synthesis
factor
both
results tion
are
curves illustrated
fusidic
acid
The effects directed
for
U-directed
Fusidic
By the method were obtained
polypeptide
for
activity
was observed
employed,
similar
reactions.
of
The
65 % inhibi0.185
and for
antibiotics
and ribosome-dependent 279
of G
acid
both
concentration synthesis
synthesis
and
polyphenyl-
Approximately
1.
steroidal
S. Okuda,
of Tokyo,
GTPase
studied.
at the
of various
polylysine
on poly
in Fig.
was demonstrated
by Prof.
The
Ltd.
and ribosome-dependent
reactions.
dose-inhibition
Co,,
acid
were comparatively
to inhibit
chromatography.
University
Kayaku
fusidic
of Conway and
of GDP by spinach
supplied
Microbiology,
Nihon
of
method
by Dowex-1
were kindly
14C-amino
and Lipmann
photophosphorylation
and purification
steroidal
Dr.
by the
and Lipmann
with
of von Ehrenstein
was prepared using
(19641,
B was labelled
method
32P-r-GTP
(1962).
method
mM of
GTPase on poly
activity. AGTPase
Vol. 30, No. 3, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1. Effects of Fusidic Acid and on Ribosome-dependent
Fig.
Fusidic
(a)
on Polyphenylalanine GTPase Activity
Synthesis of G Factor.
acid
Pol eptide Synthesis 10007 = 1,250 cpm/O.Z ml
(b)
GTPase Activity 100% = 1,820 cpm/O.Z
ml
The assay for polyphenylalanine synthesis was performed in reaction mixture, containing, in a total volume of 0.2 ml, 50 mM Tris-HCl pH 7.4, 160 mM NH4Cl, 10 mM MgCls, 6 mM 2mercaptoethanol, 500 pg/ml ribosomes, 40 pg/ml poly U, 500 pg/ml 14C-phenylalanyl-sRNA (150,000 cpm/mg RNA), 80 pg/ml T factor, 2.5 pg/ml G factor, 100 @I GTP and the antibiotic. The ribosome-dependent GTPase activity of G factor was assayed by measuring liberation of radioactive inorganic phosphate from 32P-y-GTP as described by Conway and Lipmann mixture contained, in a total volume of ( 1964 1. The reaction 0.2 ml, 50 mM Tris-HCl pH 7.4, 160 ml4 NH,Cl, 10 mM MgC12, 6 mM 2-mercaptoethanol, 500 pg/ml ribosomes, 2.5 pg/ml G factor, 100 PM 32P-y-GTP (120,000 cpm/mwole), and the antibiotic. Both reaction mixtures were preincubated in the absence of GTP for 10 min. at 30°, and then incubated with GTP for 10 min. at 30". The radioactivity of 14C was determined by a windowless gas flow counter, and that of 32P by a GM counter. the
activity both
of G factor reactions,
As summarized
although in Table
inhibition
of both
steroidal
antibiotics.
The effects polyphenylalanine in the
were
absence
of
comparatively the
grades
fusidic
with
acid the
and presence
They
of inhibition
1, a parallelism
reactions
from
studied.
the
were diverse.
was observed various
U complex
of GTP and G factor. 280
for
derivatives
on puromycin-dependent
ribosome-poly
inhibited
the of
release were
studied
As illustrat-
of
Vol. 30, No. 3, 1968
Table I. Synthesis
Effects of Various Steroidal and on Ribosome-dependent
Steroidal Control Fusidic
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Antibiotics GTPase Activity GTP hydrolyzed 100 27 9
antibiotics acid
Helvolinic
0.37 m.M
1.85
acid
Helvolic
0.36
1.80
0.37
1.85
0.32
Methylhelvolinate
8
27 13
1.60 0.34 1.70
acid
25
22 20 12 12 8
0.36
7-Propionylhelvolinic acid 24,25-Dibromohelvolic acid 3-Dihydrohelvolic
Polylysine synthesized 100
52
1.80
acid
on Polylysine of G Factor.
;; 101
0.37 3.70
92
was 100 = 1,820 cpm/O.Z ml, and Hydrolysis of 32P-y-GTP incorporation of 14C-lysine was 100 = 723 cpm/0.2 ml. The assay conditions for GTPase activity were the same as indicated in Fig. 1. Polylysine synthesis was assayed by poly A--ii;,;ted incorporation of 14C-lysine into polypeptide. Poly U -phenylalanyl-sRNA were replaced by 100 pg/ml poly A and 14C-lysyl-sRNA (90,000 cpm/mg RNA) in the reaction 500 pg/ml mixture, indicated in Fig. 1. Na2W04, containing TCA, was used instead of TCA.
ed in
2, puromycin-dependent
Fig.
significantly
affected
and G factor. and G factor, results dependent
But the
by fusidic
acid
puromycin
reaction,
was markedly
were obtained release
release
inhibited
in the
of peptide in
from
absence
stimulated
by fusidic
experiments,
of polylysine
the
was not
by GTP
acid.
using
the
of GTP
Similar
puromycin-
ribosomes.
Discussion Fusidic
and helvolinic
aminoacyl-sRNA Fusidic
acid
acids
to polypeptide and related
inhibit in the
antibiotics 281
amino
acid
transfer
protein-synthesizing inhibit
ribosome-dependent
from system.
Vol. 30, No. 3, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Effects of Fusidic Acid on Puromycin-dependent of Peptide from the Ribosomes in the Absence Presence of GTP and G Factor.
Fig.
Release and
CPM 5x105
(b) +PU
(cl
+PM,FA
$ 3
2 1
I:: ;I
Tube nmber
(c) +GTP,G,PM
0.2
0.1
10
5 -
-+-
cpm
cpm
+GTP,G,PM,FA
10 5 Tube number
15 OD 260
(f)
c
5x103
15
in a total volume of 0.5 ml, The reaction mixture contained, 14C-phenylalanyl-sRNA-charged ribosomes of E. coli B 4.0 ma/ml, poly U 50 &ml, NH,Cl 150 mM, Mg acetate 12 mM, 2-mercaptoIt was incubated at 35” ethanol 6 mM, and Tris 5 mM, pH 7.2. for 10 min. and sucrose density gradient, linear 3 to 20 %, centrifugation analysis was performed at 40,000 rpm for 60 min. Each fraction of 25 drops was assayed for ODs60 and for at 0'. (a) Control. (b) With puromycin TCA-insoluble radioactivity. 0.1 mM. (c) With puromycin 0.1 mM and fusidic acid 0.5 mM. (d) With G factor 15 &ml and GTP 0.1 mM. (e) d + puromycin (f) d + puromycin 0.1 mM + fusidic acid 0.5 mM. 0.1 mM. GTPase parallel
activity to that
of G factor. of polypeptide
The grade synthesis. 282
of
inhibition GTP split,
is linked
Vol. 30, No. 3, 1968
to the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ribosomes
and G factor,
location
of peptidyl-sRNA
Lipmann,
1966);
antibiotics,
In the
attached
to the
puromycin, may not
and it
be released
and released
inhibits
acid
site"
site"
the
However, attached
(Traut
1966).
Fusidic
analogous
"peptide
to peptide
in the
"peptide
1964;
site"
site"
inhibit
from the
by the
bond formation;
of GTPase activity
of
acid
a review
on the ribosomes.
inhibition
by
presence
"amino
does not
of peptidyl-sRNA site"
peptidyl-sRNA
"amino acid
to the
acid
and related
may be released
to the
and Monro,
trans-
and
acid
to the
may be translocated
seems to be caused by the ribosomes
of ribosomes
by puromycin.
translocation
to the
by fusidic
attached
for
(Nishizuka
absence of GTP and G factor,
by puromycin
reaction,
ribosomes
inhibited
peptidyl-sRNA
and Heintz,
puromycin it
is
but peptidyl-sRNA
of ribosomes
Schweet
on the
"peptide
GTP and G factor, site"
seems to be essential
but "amino
And it of the
and G factor.
References Conway, T.W. & Lipmann, F.: Proc. Natl. Acad. Sci. 52,1462(1964) von Ehrenstein, G. & Lipmann, F.: ibid. 47, 941 (1962) Harvey, C.L., Knight, S.G. & Sih, C.L.: Biochem. 5, 3320 (1966) Nishizuka, Y. & Lipmann, F.: Proc. Natl. Acad. Sci. 55,212(1966) Schweet, R. & Heintz, R.: Ann. Rev. Biochem. 35, 723 (1966) Tanaka, N., Yamaki, H., Lin, Y. & Umezawa, H.: J. Antibiotics 20, 156 (1967) Traut, R.R. & Monro, R.E.: J. Mol. Biol. 10, 63 (1964) Yamaki, H.: J. Antibiotics 18, 228 (1965)
283
MECHANISM
BIOCHEMICAL
OF PROTEIN
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNTHESIS
INHIBITION
BY FUSIDIC
ACID AND
RELATED ANTIBIOTICS
Nobuo
Tanaka,
Institute
Tadatoshi
of Applied
ReceivedJanuary Fusidic
Microbiology,
and helvolinic skeletone,
synthesis
in
the
Harvey
affect
were
et al.,
from
1966;
The
coli,
Fusidic
acid
ribosome-dependent polypeptide both
synthesis
reactions from
the
fusidic
acid
in the
fusidic
reaction, acid.
with
purified
are presented
activity
as well,
absence
enhanced The results
E. coli
and separated
of
transfer in this
antibiotics
enzymes
grade
to
They of
significantly
by GTP and G factor, that
of E.
communication.
of GTP and G factor;
278
GTPase
were observed
and the
indicate
and Lipmann,
steroidal
of G factor.
was not
acid
ribosomes.
Puromycin-dependent
ribosomes
amino
on the
antibiotics
was parallel.
peptide
mycin
of action
GTPase
inhibit
ribosome-dependent
site
and related
of aminoacyl-
T and G (Nishizuka
exhibited
(Yamaki,
They neither
1967).
they
from
factors,
in a system
systems
formation
to protein
factor
results
but
of protoprotein
bacterial
nor
was purified
The detailed
and the
to inhibit
et al.,
complex;
enzyme
latter
was studied
Tanaka
of aminoacyl-sRNA
two complementary
activity.
Tokyo
antibiotics
demonstrated
aminoacyl-sRNA
The transfer
1966).
of Tokyo,
steroidal
and in vitro
sRNA-messenger-ribosome
into
Masukawa
University
acids,
in vivo
synthesis
transfer
and Hiroshi
8, 1968
lanostane
1965;
Kinoshita,
fusidic
inhibit
inhibited
inhibition
of
release
of
affected
by
but
the
was inhibited acid
and
puroby
Vol. 30, No. 3, 1968
related
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
antibiotics
GTPase
affect
activity
of G factor
aminoacyl-sRNA assumption linked
on the
location
ribosomes
synthesis
and inhibiting
ribosomes.
of Nishizuka
to the
polypeptide
It
translocation
(1966)
and G factor,
is
on the
of
seems to support
and Lipmann
of aminoacyl-sRNA
by inhibiting
that
the
GTPase
essential
reaction,
for
trans-
from
the
ribosomes.
Results -Washed
ribosomes,
T and G factors
were prepared
extracts
of E. coli
B by the
of Nishizuka
(1966).
The sRNA of E. coli
acid,
following
the
Lipmann chloroplasts
antibiotics
Institute
of Applied
Y. Nakayama,
The effects alanine
synthesis
factor
both
results tion
are
curves illustrated
fusidic
acid
The effects directed
for
U-directed
Fusidic
By the method were obtained
polypeptide
for
activity
was observed
employed,
similar
reactions.
of
The
65 % inhibi0.185
and for
antibiotics
and ribosome-dependent 279
of G
acid
both
concentration synthesis
synthesis
and
polyphenyl-
Approximately
1.
steroidal
S. Okuda,
of Tokyo,
GTPase
studied.
at the
of various
polylysine
on poly
in Fig.
was demonstrated
by Prof.
The
Ltd.
and ribosome-dependent
reactions.
dose-inhibition
Co,,
acid
were comparatively
to inhibit
chromatography.
University
Kayaku
fusidic
of Conway and
of GDP by spinach
supplied
Microbiology,
Nihon
of
method
by Dowex-1
were kindly
14C-amino
and Lipmann
photophosphorylation
and purification
steroidal
Dr.
by the
and Lipmann
with
of von Ehrenstein
was prepared using
(19641,
B was labelled
method
32P-r-GTP
(1962).
method
mM of
GTPase on poly
activity. AGTPase
Vol. 30, No. 3, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1. Effects of Fusidic Acid and on Ribosome-dependent
Fig.
Fusidic
(a)
on Polyphenylalanine GTPase Activity
Synthesis of G Factor.
acid
Pol eptide Synthesis 10007 = 1,250 cpm/O.Z ml
(b)
GTPase Activity 100% = 1,820 cpm/O.Z
ml
The assay for polyphenylalanine synthesis was performed in reaction mixture, containing, in a total volume of 0.2 ml, 50 mM Tris-HCl pH 7.4, 160 mM NH4Cl, 10 mM MgCls, 6 mM 2mercaptoethanol, 500 pg/ml ribosomes, 40 pg/ml poly U, 500 pg/ml 14C-phenylalanyl-sRNA (150,000 cpm/mg RNA), 80 pg/ml T factor, 2.5 pg/ml G factor, 100 @I GTP and the antibiotic. The ribosome-dependent GTPase activity of G factor was assayed by measuring liberation of radioactive inorganic phosphate from 32P-y-GTP as described by Conway and Lipmann mixture contained, in a total volume of ( 1964 1. The reaction 0.2 ml, 50 mM Tris-HCl pH 7.4, 160 ml4 NH,Cl, 10 mM MgC12, 6 mM 2-mercaptoethanol, 500 pg/ml ribosomes, 2.5 pg/ml G factor, 100 PM 32P-y-GTP (120,000 cpm/mwole), and the antibiotic. Both reaction mixtures were preincubated in the absence of GTP for 10 min. at 30°, and then incubated with GTP for 10 min. at 30". The radioactivity of 14C was determined by a windowless gas flow counter, and that of 32P by a GM counter. the
activity both
of G factor reactions,
As summarized
although in Table
inhibition
of both
steroidal
antibiotics.
The effects polyphenylalanine in the
were
absence
of
comparatively the
grades
fusidic
with
acid the
and presence
They
of inhibition
1, a parallelism
reactions
from
studied.
the
were diverse.
was observed various
U complex
of GTP and G factor. 280
for
derivatives
on puromycin-dependent
ribosome-poly
inhibited
the of
release were
studied
As illustrat-
of
Vol. 30, No. 3, 1968
Table I. Synthesis
Effects of Various Steroidal and on Ribosome-dependent
Steroidal Control Fusidic
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Antibiotics GTPase Activity GTP hydrolyzed 100 27 9
antibiotics acid
Helvolinic
0.37 m.M
1.85
acid
Helvolic
0.36
1.80
0.37
1.85
0.32
Methylhelvolinate
8
27 13
1.60 0.34 1.70
acid
25
22 20 12 12 8
0.36
7-Propionylhelvolinic acid 24,25-Dibromohelvolic acid 3-Dihydrohelvolic
Polylysine synthesized 100
52
1.80
acid
on Polylysine of G Factor.
;; 101
0.37 3.70
92
was 100 = 1,820 cpm/O.Z ml, and Hydrolysis of 32P-y-GTP incorporation of 14C-lysine was 100 = 723 cpm/0.2 ml. The assay conditions for GTPase activity were the same as indicated in Fig. 1. Polylysine synthesis was assayed by poly A--ii;,;ted incorporation of 14C-lysine into polypeptide. Poly U -phenylalanyl-sRNA were replaced by 100 pg/ml poly A and 14C-lysyl-sRNA (90,000 cpm/mg RNA) in the reaction 500 pg/ml mixture, indicated in Fig. 1. Na2W04, containing TCA, was used instead of TCA.
ed in
2, puromycin-dependent
Fig.
significantly
affected
and G factor. and G factor, results dependent
But the
by fusidic
acid
puromycin
reaction,
was markedly
were obtained release
release
inhibited
in the
of peptide in
from
absence
stimulated
by fusidic
experiments,
of polylysine
the
was not
by GTP
acid.
using
the
of GTP
Similar
puromycin-
ribosomes.
Discussion Fusidic
and helvolinic
aminoacyl-sRNA Fusidic
acid
acids
to polypeptide and related
inhibit in the
antibiotics 281
amino
acid
transfer
protein-synthesizing inhibit
ribosome-dependent
from system.
Vol. 30, No. 3, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2. Effects of Fusidic Acid on Puromycin-dependent of Peptide from the Ribosomes in the Absence Presence of GTP and G Factor.
Fig.
Release and
CPM 5x105
(b) +PU
(cl
+PM,FA
$ 3
2 1
I:: ;I
Tube nmber
(c) +GTP,G,PM
0.2
0.1
10
5 -
-+-
cpm
cpm
+GTP,G,PM,FA
10 5 Tube number
15 OD 260
(f)
c
5x103
15
in a total volume of 0.5 ml, The reaction mixture contained, 14C-phenylalanyl-sRNA-charged ribosomes of E. coli B 4.0 ma/ml, poly U 50 &ml, NH,Cl 150 mM, Mg acetate 12 mM, 2-mercaptoIt was incubated at 35” ethanol 6 mM, and Tris 5 mM, pH 7.2. for 10 min. and sucrose density gradient, linear 3 to 20 %, centrifugation analysis was performed at 40,000 rpm for 60 min. Each fraction of 25 drops was assayed for ODs60 and for at 0'. (a) Control. (b) With puromycin TCA-insoluble radioactivity. 0.1 mM. (c) With puromycin 0.1 mM and fusidic acid 0.5 mM. (d) With G factor 15 &ml and GTP 0.1 mM. (e) d + puromycin (f) d + puromycin 0.1 mM + fusidic acid 0.5 mM. 0.1 mM. GTPase parallel
activity to that
of G factor. of polypeptide
The grade synthesis. 282
of
inhibition GTP split,
is linked
Vol. 30, No. 3, 1968
to the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ribosomes
and G factor,
location
of peptidyl-sRNA
Lipmann,
1966);
antibiotics,
In the
attached
to the
puromycin, may not
and it
be released
and released
inhibits
acid
site"
site"
the
However, attached
(Traut
1966).
Fusidic
analogous
"peptide
to peptide
in the
"peptide
1964;
site"
site"
inhibit
from the
by the
bond formation;
of GTPase activity
of
acid
a review
on the ribosomes.
inhibition
by
presence
"amino
does not
of peptidyl-sRNA site"
peptidyl-sRNA
"amino acid
to the
acid
and related
may be released
to the
and Monro,
trans-
and
acid
to the
may be translocated
seems to be caused by the ribosomes
of ribosomes
by puromycin.
translocation
to the
by fusidic
attached
for
(Nishizuka
absence of GTP and G factor,
by puromycin
reaction,
ribosomes
inhibited
peptidyl-sRNA
and Heintz,
puromycin it
is
but peptidyl-sRNA
of ribosomes
Schweet
on the
"peptide
GTP and G factor, site"
seems to be essential
but "amino
And it of the
and G factor.
References Conway, T.W. & Lipmann, F.: Proc. Natl. Acad. Sci. 52,1462(1964) von Ehrenstein, G. & Lipmann, F.: ibid. 47, 941 (1962) Harvey, C.L., Knight, S.G. & Sih, C.L.: Biochem. 5, 3320 (1966) Nishizuka, Y. & Lipmann, F.: Proc. Natl. Acad. Sci. 55,212(1966) Schweet, R. & Heintz, R.: Ann. Rev. Biochem. 35, 723 (1966) Tanaka, N., Yamaki, H., Lin, Y. & Umezawa, H.: J. Antibiotics 20, 156 (1967) Traut, R.R. & Monro, R.E.: J. Mol. Biol. 10, 63 (1964) Yamaki, H.: J. Antibiotics 18, 228 (1965)
283