BIOCHEMICAL
Vol. 64, No. 3,1975
POLYAMINE
AS A FUNCTION
Igarashil,
Seiyu
RESEARCH COMMUNICATIONS
AND MAGNESIUM CONTENTS AND POLYPEPTIDE
SYNTHESIS Kazuei
AND BIOPHYSICAL
OF CELL GROWTH
Katsuko
Hirosel,
Haral,
and Yoshifumi
Yasuhiro
Watanabel,
Takeda2
Faculty of Pharmaceutic 1 Sciences, Chiba University, Yayoi-cho, Chiba, Japan B and Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamada-Kami, Suita, Osaka, Japan2 Received
April
2,1975
SUMMARY: The polyamine and ribosome contents during the logarithmic phase of growth were much higher than those in the late lo arithmic On the other hand, the Mg 2g content or stationary phase of growth, did not vary markedly throughout the different growth phases0 These data, together with the results of a previous communication (l), suggest that increased polyamines during the logarithmic phase of growth may play significant roles not only in the neutralization of the negative charges of increased ribosomes but also in the increase of the velocity of polypeptide chain elongation by binding to the The polyphenylalanine synthetic activity of ribosomes ribosomes, from the logarithmic phase of growth was higher than that of ribosomes obtained from the stationary phase of growth in the presence of optimal concentrations of spermidine.
It in
the
that
has become growth
a partial
can occur reported
sary
of various
protein
cofactor
It
polyamines (2).
in E. coli
an important
We have proposed
not
(3-T)., only
synthesis
but
has been reported
studied
the
the
that
relationship synthesis
a97
role
previously vivo
usually
we have
optimal
recently
concentration
stimulated
synthesis
B and -B. thuringiensis.
--in
addition,
also
also
and polypeptide
Copyright D 19 75 by Academic Press, Inc. All rights of reproduction in any form reserved.
In
shifted
of polyphenylalanine
contents
play
of Mg2+ by polyamines
polyamines
we have
magnesium
cells
synthesis
polypeptide
(1).
Therefore,
growth
in
for
function
that
replacement
that
of Mg2+
evident
the
spermidine
ribosomal is
a neces-
in g. thuringiensis between
(8).
polyamine
as a function
and of
Cell
Vol. 64, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS - --E. coli
Materials grown at 37' with
C in
The cells
with
the
and 0.2% casamino
acids.
a Klett-Xummerson
calorimeter
a water-immiscible et al.
cells
(lo),
acetate,
centrifuged
the
further
previously
(l)-
with
Dialyzed
in
by means of atomic
with
and
a slight
and Mg2+ contents
according
to the method
of ribosomes
and S-100,
pH 7.5,
10 mM and
as described
pre-
purification
of S-100
by Sephadex
Washed ribosomes containin&
60 mM KCl,
ribosomes
were prepared 100 mM Tris-HCl,
6 mM 2-mercaptoethanol
were obtained
(2 mg/ml)
of magnesium
were analyzed
instrument
15 min
against
by dialyzing
3 liters
of Buffer
I
C.
Measurements ions
cells
were prepared
a buffer
acetate,
10 mg of washed ribosomes 48 hr at 4'
x g for
and 6 mM 2-mercaptoethanol)
for
pH 7,,5> 10 mM magnesium
for
of the 60 filter),
I (10 mM Tris-HCl,
and S-100
ribosomes
and 500 mM NH4C10
supplemented
above.
A method
G-50 was described by washing
Buffer
were
were washed by centrifugation
preparation
60 mM NH,+Cl,
ribosomes
(11).
polyamine
of silicones
For the
as indicated
Unwashed viously
layer
were washed with
magnesium
cells
(No.
at 15,000
When the the
(9)
The growth
by centrifugation
intervals.
Berliner
medium
salt
were measured,
of Hurwits
thuringiensis
minimal
time
cells
through
of
were harvested
at appropriate of the
B and Bacillus
2 liters
0.2% glucose
was followed
AND METHODS
a
the
ions
presence
absorption
of strontium
spectrometry
hollow-cathode
modification
and of polyamines
(12)
- Magnesium
chloride
using
lamp.
Polyamines
of the
method
(1 mg/ml)
a Hitachi
208
were measured
of Dion
and Herbst
(13).
Procedures ture threitol,
(0.1
ml),
for which
polyphenylalanine contained
100 mM NH&Cl,
synthesis
80 mM Tris-HCl
1 mM ATP, 0.4
898
- The reaction (pH 7.5))
mix-
1 mM dithio-
mM GTP, 4 mM phosphoenol-
Vol. 64, No. 3, 1975
pyruvate,
BIOCHEMICAL
4 pg pyruvate
E. coli
B tRNA
Mannheim lyzed
0.5
ribosomes,
A O.O8-ml disc
A260 unit
(382
specified
(Boehringer
Biochemicals),
E. coli
phenylalanine at the
kinase
(General
GmbH),
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
S-S100
and magnesium
of each reaction
(25 mm diameter)
and the
efficiency
mixture
hot
with was 1.2
(U)
0.1
20 Vg
(Boehringer dia-
I-ICi of
acetate
("C)
and polyamines
at 30'
C for
was placed
trichloroacetic
a liquid
GmbH),
thuringiensis
was incubated
aliquot
The counting
or g.
(80 pg protein),
Wi/pmole),
was assayed
10 pg poly
of 2. &
concentrations,
radioactivity
Mannheim
15 min.
on a paper
acid
scintillation
insoluble spectrometer.
x 106 cpm/Wi.
RESULTS Polyamine,
Mg2+ and ribosome
phases per
- The putrescine,
g of wet cells
As shown in the
1, the phase
logarithmic
detected did
of g. ___ coli
Fig.
logarithmic
late
contents
not
any phase
vary
markedly
some increase
logarithmic
phase.
increase
of total
logarithmic
spermidine
of growth
phase
the
other
hand,
different
are
tabulated
of cations
At this
stage,
the
charge
in total
charge
reached
almost
50%, while
during
the
phase
of growth.
at the
early
times
as high
units:
380)
logarithmic as those (Table
phase of s. -coli
during
units:
at the
2).
899
it
the
the
increase
stationary
of
of polyaminewas about
contents 100)
middle
The
during
percentage
The ribosome (Klett
1.
to the
of polyamines.
was not
phases,
in Table
was due primarily
in the
the Mg2+ conten
growth
per g wet cells
1.
during
those
Spermine
charge
late
than
10% to 20%) occurred
The values
of growth
1 and Table contents
of growth.
On the
of growth.
charge
Fig.
were much higher phase
(about
in
and putrescine
throughout
although
B at various 2+ contents and Mg
spermidine,
are presented
or stationary
in
in E. coli
30%
of --E. coli was about 1.7 phase
(Klett
Vol. 64, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
400
300
10 x 5
200
:: .Y s 100
I
0 5
0
Time
(hr)
and polyamines E. coli time intervals a;d The=' were measured as described e----e, Klett units; o-o, t---j, spermidine.
Effect ribosome nents
of spermidine preparations
may differ
phase
protein
to study
of growth
stationary
phase
logarithmic
obtained or absence centage
synthetic
the
or not
of stimulation
cells
and RNA (16) ribosomes
activity
of growth
than
amounts
phase
of growth
of spermidine.
by spermidine
900
did
not
the
either
from
the
3, the from
of ribosomes in
the
However, vary
is
logarithmic
obtained that
phases it
2 and Table
than
compo-
growth
those
of ribosomes
was higher
the
contents,
from
As shown in Table
by various
that
at various
to spermidine
stationary
of suitable
the
(14,L5)
better
synthesis
has been reported
from
of growth.
phase
from
it
whether
respond
polyphenylalanine the
on polyphenylalanine
obtained
in their
of interest
contents in E. coli B at various B cells were Harvested at various spermidine and putrescine contents in the text. Mg2+; A---d, putrescine;
- Since
of ribosomes
20
15
10
markedly
the
presence peramong
1.
111
246
320
385
390
6
7
8
16
24
"rhese values wet cells)); (putrescine
36
Klett units
various
Charge
5
Time of harvest (hr)
Table
1.38
were calculated from the Spermidine, 3 x (spermidine olmoles/g wet cells)).
0.63
4.58
1.00
0.84 1.30
1.72
1.38
0.72
2.56
1.50
2.52
0.87
data
Putrescine
concentrationa
of polyamines
Spermidine
Charge
phases.
4.68
4.54
5.30
5.16
3.70
m2+
growth
concentrations
of Fig. (Vmoles/g
6.59
6.70
6.38
8.40
10.24
6.07
E _.
69.5
69.9
71.2
63.1
50.4
61.0
Mg2+
at
charges
30.5
30.1
28.8
36.9
49.6
39.0
Polyamines
of
B harvested
Percentage
coli
1. Mg 2+, 2 x [Mg2+(~moles/g wet cells)); Putrescine,
2+ in
Total
and Mg
2 x
Vol. 64, No. 3, 1975
Table
BIOCHEMICAL
2. Effect with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of spermidine ribosomes
on polyphenylalanine
obtained
from various
synthesis
growth
phases
coli of -0 E -* Unwashed ribosome content A260 units g wet cells >
Klettunits at time of harvest
Ions
(mM)
Mg2+ Spermidine
spermidine
(CPM)
100
488
157
3:s
8933 18391
205
200
484
157
3-5
8838 18070
204
300
373
147
3:5
8106 16085
198
380
288
147
3:s
7549 14497
192
Polyphenylalanine synthesis was carried out under conditions. Ions specified in the table were the concentrations for polyphenylalanine synthesis. *Phe; phenylalanine.
the ribosomes clearer
from different
in the ribosomes
growth from g.
standard optimal
These phenomena were
phases.
thuringiensis.
DISCUSSION It
is well
ribosome for
function.
by polyamines
E. coli
Mg"
are essential
for
The function
of Mg2+ is
partially
known that
harvested
1.7 times greater phase of growth,
than
that
phases.
concentration
of E -0 -coli
did not vary
On the other
harvested
1.6 times that
harvested
content
of
was about
at the stationary markedly
hand,
the
at the
logarithmic
of E. -coli
harvested
902
of
substituted
phase of growth
of g. -coli
the Mg2+ content
growth
was about
the ribosome
at the logarithmic
the different
growth
Although
(17-20).
maintenance
throughout
total
charge
phase of at the
station-
Vol. 64, No. 3,1975
BlOCHEMlCAL
3, Effect
Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of spermidine
with
ribosomes
on polyphenylalanine
obtained
from
Ions
(mM)
synthesis
various
growth
phases
of B. thuringiensis. Unwashed ribosome content A260 units g wet cells
Klett units at time of harvest
% Stimulatior by spermidine
$~~?~~~ated (CPM)
Mg2+ Spermidine
100
336
18 10
715
1910 10560
553
200
168
18 10
7:5
1274 6788
533
300
157
20 12
7:5
927 4838
522
380
143
12
715
873 4562
526
The conditions
ary phase
20
of the
of growth.
somes is
equal
If
to that
with
especially
by spermidine,
charge under tic cells the
total
conditions
yield
was 32% (1).
activity harvested stationary
at the phase
logarithmic starvation
the phase. in
coli
(Klett
velocity Recently
a conditional
to Mg2+ in
cells,
protein
these
charge
found
the
greatest
On the
other
in the
values
results
of polypeptide
903
incubated
polyphenylalanine those
(Klett
chain
auxotroph
in
units:
synthewhole 111)
E. & and
(Table
1) were
that
spermidine
elongation
at the
suggest
has been reported
polyamine
are
by polyamines,
ribosomes
390) of growth
These
ribo-
--in vivo. percentage of spermidine-
hand,
phase
units:
2.
synthesis the
system,
it
in Table
Mg 2+ are replaced
logarithmic
23% and 9.6"/0, respectively. may increase
g. that
cell-free
which
of polyamines
during
polycationic
were as indicated
ratio
our hypothesis
the E. coli in
the
in whole
compatible
In
experiment
that
polyamine
of E. coli
decreases
Vol. 64, No. 3,1975
the
rate
peptide hypothesis
BIOCHEMICAL
of protein chain
synthesis
elongation
that
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
by limiting
(21).
polyamines
This
the
velocity
observation
are important
of poly-
supports
during
--in vivo
the protein
synthesis.
ACKNOWLEDGEMENTS The authors would like to express their thanks to Dr. B. K. Joyce of Colorado State University for her help in preparing this manuscript. This work was supported in part by a Grant in Aid from the Ministry of Education.
REFERENCES 1. Igarashi, K., Sugawara, K., Izumi, Hirose, S. (1974) Eur. J. Biochem., to 2. Cohen, S. S. (1971) Introduction Englewood Cliffs, N. J. Takeda, Y. (1969) J. Biochem. Tokyo, Y. (1969) Biochem. Biophys. ;: Takeda, . Takeda, Y. and Igarashi, K. (1970)
68, 937-940. 6. Takeda, Y., Matsuzaki, 111, 1-6.
I.,
Nagayama,
48, 495-502.
polyamines,
C. and Prentice
Hall,
66, 345-349.
Acta, 179, 232-234. J. Biochem. Tokyo,
K. and Igarashi,
K.
(1972) J. Bacterial.,
K., Hikami, K., Sugawara, K. and Hirose, S. (1973) 7. Igarashi, Biochim. Biophys. Acta, 299, 325-330, 8. Changchien, L. M. and Aronson, J. N. (1970) J. Bacterial.,
103, 734-740. 9. Davis, B. D. and Mingioli,
10.
Hurwits,
C.,
90, 1692-1695.
Braun,
E. S. (1950) J. Bacterial., C. B. and Peabody, R. A. (1965) J.
60, 17-28. Bacterial.,
Nirenberg, M. W. and Matthaei, J. H. (1961) Proc. Natl. Acad. Sci. TJ. S., 47, 1588-1602. 12. Igarashi, K., Izumi, I., Hara, K. and Hirose, S. (1974) Chem. Pharm. Bull. Tokyo, 22, 451-454. E. J. (1970) Ann. N. Y. Acad. Sci., 13. Dion, A. S. and Herbst, 11.
171, 723-734. W. and Price, R. P. (1974) Biochem. 14. Dewitt, Res. Commun., 56, 593-598. S. and Subramanian, A. R. (1974) 15. Ramagapal, Sci. U. S., 71, 2136-2140. 16.
Mangiarotti, G., Turco, (1974) Nature New Biol., 17. Weiss, R. L. and Morris, 204, 502-511. 18. Weiss, R. L. and Morris, Kimes, B. W. and Morris, 12%. Igarashi, K., Sugawara, Tokyo, in press. 21. Jorstad, C. M. and Morris,
119, 857-860.
Biophys. Proc.
Natl.
Acad.
E., Ponzetto, A. and Altrada, F. 247, 147-148. D. R. (1970) Biochim. Biophys. Acta, D. R. (1973) D. R. (1973) K. and Hirose,
Biochemistry, Biochemistry, S. (1975)
D. R. (1974)
904
J.
12, 436-441. 12, 442-449. J. Biochem.
Bacterial.,
Vol. 64, No. 3,1975
POLYAMINE
AS A FUNCTION
Igarashil,
Seiyu
RESEARCH COMMUNICATIONS
AND MAGNESIUM CONTENTS AND POLYPEPTIDE
SYNTHESIS Kazuei
AND BIOPHYSICAL
OF CELL GROWTH
Katsuko
Hirosel,
Haral,
and Yoshifumi
Yasuhiro
Watanabel,
Takeda2
Faculty of Pharmaceutic 1 Sciences, Chiba University, Yayoi-cho, Chiba, Japan B and Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamada-Kami, Suita, Osaka, Japan2 Received
April
2,1975
SUMMARY: The polyamine and ribosome contents during the logarithmic phase of growth were much higher than those in the late lo arithmic On the other hand, the Mg 2g content or stationary phase of growth, did not vary markedly throughout the different growth phases0 These data, together with the results of a previous communication (l), suggest that increased polyamines during the logarithmic phase of growth may play significant roles not only in the neutralization of the negative charges of increased ribosomes but also in the increase of the velocity of polypeptide chain elongation by binding to the The polyphenylalanine synthetic activity of ribosomes ribosomes, from the logarithmic phase of growth was higher than that of ribosomes obtained from the stationary phase of growth in the presence of optimal concentrations of spermidine.
It in
the
that
has become growth
a partial
can occur reported
sary
of various
protein
cofactor
It
polyamines (2).
in E. coli
an important
We have proposed
not
(3-T)., only
synthesis
but
has been reported
studied
the
the
that
relationship synthesis
a97
role
previously vivo
usually
we have
optimal
recently
concentration
stimulated
synthesis
B and -B. thuringiensis.
--in
addition,
also
also
and polypeptide
Copyright D 19 75 by Academic Press, Inc. All rights of reproduction in any form reserved.
In
shifted
of polyphenylalanine
contents
play
of Mg2+ by polyamines
polyamines
we have
magnesium
cells
synthesis
polypeptide
(1).
Therefore,
growth
in
for
function
that
replacement
that
of Mg2+
evident
the
spermidine
ribosomal is
a neces-
in g. thuringiensis between
(8).
polyamine
as a function
and of
Cell
Vol. 64, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS - --E. coli
Materials grown at 37' with
C in
The cells
with
the
and 0.2% casamino
acids.
a Klett-Xummerson
calorimeter
a water-immiscible et al.
cells
(lo),
acetate,
centrifuged
the
further
previously
(l)-
with
Dialyzed
in
by means of atomic
with
and
a slight
and Mg2+ contents
according
to the method
of ribosomes
and S-100,
pH 7.5,
10 mM and
as described
pre-
purification
of S-100
by Sephadex
Washed ribosomes containin&
60 mM KCl,
ribosomes
were prepared 100 mM Tris-HCl,
6 mM 2-mercaptoethanol
were obtained
(2 mg/ml)
of magnesium
were analyzed
instrument
15 min
against
by dialyzing
3 liters
of Buffer
I
C.
Measurements ions
cells
were prepared
a buffer
acetate,
10 mg of washed ribosomes 48 hr at 4'
x g for
and 6 mM 2-mercaptoethanol)
for
pH 7,,5> 10 mM magnesium
for
of the 60 filter),
I (10 mM Tris-HCl,
and S-100
ribosomes
and 500 mM NH4C10
supplemented
above.
A method
G-50 was described by washing
Buffer
were
were washed by centrifugation
preparation
60 mM NH,+Cl,
ribosomes
(11).
polyamine
of silicones
For the
as indicated
Unwashed viously
layer
were washed with
magnesium
cells
(No.
at 15,000
When the the
(9)
The growth
by centrifugation
intervals.
Berliner
medium
salt
were measured,
of Hurwits
thuringiensis
minimal
time
cells
through
of
were harvested
at appropriate of the
B and Bacillus
2 liters
0.2% glucose
was followed
AND METHODS
a
the
ions
presence
absorption
of strontium
spectrometry
hollow-cathode
modification
and of polyamines
(12)
- Magnesium
chloride
using
lamp.
Polyamines
of the
method
(1 mg/ml)
a Hitachi
208
were measured
of Dion
and Herbst
(13).
Procedures ture threitol,
(0.1
ml),
for which
polyphenylalanine contained
100 mM NH&Cl,
synthesis
80 mM Tris-HCl
1 mM ATP, 0.4
898
- The reaction (pH 7.5))
mix-
1 mM dithio-
mM GTP, 4 mM phosphoenol-
Vol. 64, No. 3, 1975
pyruvate,
BIOCHEMICAL
4 pg pyruvate
E. coli
B tRNA
Mannheim lyzed
0.5
ribosomes,
A O.O8-ml disc
A260 unit
(382
specified
(Boehringer
Biochemicals),
E. coli
phenylalanine at the
kinase
(General
GmbH),
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
S-S100
and magnesium
of each reaction
(25 mm diameter)
and the
efficiency
mixture
hot
with was 1.2
(U)
0.1
20 Vg
(Boehringer dia-
I-ICi of
acetate
("C)
and polyamines
at 30'
C for
was placed
trichloroacetic
a liquid
GmbH),
thuringiensis
was incubated
aliquot
The counting
or g.
(80 pg protein),
Wi/pmole),
was assayed
10 pg poly
of 2. &
concentrations,
radioactivity
Mannheim
15 min.
on a paper
acid
scintillation
insoluble spectrometer.
x 106 cpm/Wi.
RESULTS Polyamine,
Mg2+ and ribosome
phases per
- The putrescine,
g of wet cells
As shown in the
1, the phase
logarithmic
detected did
of g. ___ coli
Fig.
logarithmic
late
contents
not
any phase
vary
markedly
some increase
logarithmic
phase.
increase
of total
logarithmic
spermidine
of growth
phase
the
other
hand,
different
are
tabulated
of cations
At this
stage,
the
charge
in total
charge
reached
almost
50%, while
during
the
phase
of growth.
at the
early
times
as high
units:
380)
logarithmic as those (Table
phase of s. -coli
during
units:
at the
2).
899
it
the
the
increase
stationary
of
of polyaminewas about
contents 100)
middle
The
during
percentage
The ribosome (Klett
1.
to the
of polyamines.
was not
phases,
in Table
was due primarily
in the
the Mg2+ conten
growth
per g wet cells
1.
during
those
Spermine
charge
late
than
10% to 20%) occurred
The values
of growth
1 and Table contents
of growth.
On the
of growth.
charge
Fig.
were much higher phase
(about
in
and putrescine
throughout
although
B at various 2+ contents and Mg
spermidine,
are presented
or stationary
in
in E. coli
30%
of --E. coli was about 1.7 phase
(Klett
Vol. 64, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
400
300
10 x 5
200
:: .Y s 100
I
0 5
0
Time
(hr)
and polyamines E. coli time intervals a;d The=' were measured as described e----e, Klett units; o-o, t---j, spermidine.
Effect ribosome nents
of spermidine preparations
may differ
phase
protein
to study
of growth
stationary
phase
logarithmic
obtained or absence centage
synthetic
the
or not
of stimulation
cells
and RNA (16) ribosomes
activity
of growth
than
amounts
phase
of growth
of spermidine.
by spermidine
900
did
not
the
either
from
the
3, the from
of ribosomes in
the
However, vary
is
logarithmic
obtained that
phases it
2 and Table
than
compo-
growth
those
of ribosomes
was higher
the
contents,
from
As shown in Table
by various
that
at various
to spermidine
stationary
of suitable
the
(14,L5)
better
synthesis
has been reported
from
of growth.
phase
from
it
whether
respond
polyphenylalanine the
on polyphenylalanine
obtained
in their
of interest
contents in E. coli B at various B cells were Harvested at various spermidine and putrescine contents in the text. Mg2+; A---d, putrescine;
- Since
of ribosomes
20
15
10
markedly
the
presence peramong
1.
111
246
320
385
390
6
7
8
16
24
"rhese values wet cells)); (putrescine
36
Klett units
various
Charge
5
Time of harvest (hr)
Table
1.38
were calculated from the Spermidine, 3 x (spermidine olmoles/g wet cells)).
0.63
4.58
1.00
0.84 1.30
1.72
1.38
0.72
2.56
1.50
2.52
0.87
data
Putrescine
concentrationa
of polyamines
Spermidine
Charge
phases.
4.68
4.54
5.30
5.16
3.70
m2+
growth
concentrations
of Fig. (Vmoles/g
6.59
6.70
6.38
8.40
10.24
6.07
E _.
69.5
69.9
71.2
63.1
50.4
61.0
Mg2+
at
charges
30.5
30.1
28.8
36.9
49.6
39.0
Polyamines
of
B harvested
Percentage
coli
1. Mg 2+, 2 x [Mg2+(~moles/g wet cells)); Putrescine,
2+ in
Total
and Mg
2 x
Vol. 64, No. 3, 1975
Table
BIOCHEMICAL
2. Effect with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of spermidine ribosomes
on polyphenylalanine
obtained
from various
synthesis
growth
phases
coli of -0 E -* Unwashed ribosome content A260 units g wet cells >
Klettunits at time of harvest
Ions
(mM)
Mg2+ Spermidine
spermidine
(CPM)
100
488
157
3:s
8933 18391
205
200
484
157
3-5
8838 18070
204
300
373
147
3:5
8106 16085
198
380
288
147
3:s
7549 14497
192
Polyphenylalanine synthesis was carried out under conditions. Ions specified in the table were the concentrations for polyphenylalanine synthesis. *Phe; phenylalanine.
the ribosomes clearer
from different
in the ribosomes
growth from g.
standard optimal
These phenomena were
phases.
thuringiensis.
DISCUSSION It
is well
ribosome for
function.
by polyamines
E. coli
Mg"
are essential
for
The function
of Mg2+ is
partially
known that
harvested
1.7 times greater phase of growth,
than
that
phases.
concentration
of E -0 -coli
did not vary
On the other
harvested
1.6 times that
harvested
content
of
was about
at the stationary markedly
hand,
the
at the
logarithmic
of E. -coli
harvested
902
of
substituted
phase of growth
of g. -coli
the Mg2+ content
growth
was about
the ribosome
at the logarithmic
the different
growth
Although
(17-20).
maintenance
throughout
total
charge
phase of at the
station-
Vol. 64, No. 3,1975
BlOCHEMlCAL
3, Effect
Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of spermidine
with
ribosomes
on polyphenylalanine
obtained
from
Ions
(mM)
synthesis
various
growth
phases
of B. thuringiensis. Unwashed ribosome content A260 units g wet cells
Klett units at time of harvest
% Stimulatior by spermidine
$~~?~~~ated (CPM)
Mg2+ Spermidine
100
336
18 10
715
1910 10560
553
200
168
18 10
7:5
1274 6788
533
300
157
20 12
7:5
927 4838
522
380
143
12
715
873 4562
526
The conditions
ary phase
20
of the
of growth.
somes is
equal
If
to that
with
especially
by spermidine,
charge under tic cells the
total
conditions
yield
was 32% (1).
activity harvested stationary
at the phase
logarithmic starvation
the phase. in
coli
(Klett
velocity Recently
a conditional
to Mg2+ in
cells,
protein
these
charge
found
the
greatest
On the
other
in the
values
results
of polypeptide
903
incubated
polyphenylalanine those
(Klett
chain
auxotroph
in
units:
synthewhole 111)
E. & and
(Table
1) were
that
spermidine
elongation
at the
suggest
has been reported
polyamine
are
by polyamines,
ribosomes
390) of growth
These
ribo-
--in vivo. percentage of spermidine-
hand,
phase
units:
2.
synthesis the
system,
it
in Table
Mg 2+ are replaced
logarithmic
23% and 9.6"/0, respectively. may increase
g. that
cell-free
which
of polyamines
during
polycationic
were as indicated
ratio
our hypothesis
the E. coli in
the
in whole
compatible
In
experiment
that
polyamine
of E. coli
decreases
Vol. 64, No. 3,1975
the
rate
peptide hypothesis
BIOCHEMICAL
of protein chain
synthesis
elongation
that
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
by limiting
(21).
polyamines
This
the
velocity
observation
are important
of poly-
supports
during
--in vivo
the protein
synthesis.
ACKNOWLEDGEMENTS The authors would like to express their thanks to Dr. B. K. Joyce of Colorado State University for her help in preparing this manuscript. This work was supported in part by a Grant in Aid from the Ministry of Education.
REFERENCES 1. Igarashi, K., Sugawara, K., Izumi, Hirose, S. (1974) Eur. J. Biochem., to 2. Cohen, S. S. (1971) Introduction Englewood Cliffs, N. J. Takeda, Y. (1969) J. Biochem. Tokyo, Y. (1969) Biochem. Biophys. ;: Takeda, . Takeda, Y. and Igarashi, K. (1970)
68, 937-940. 6. Takeda, Y., Matsuzaki, 111, 1-6.
I.,
Nagayama,
48, 495-502.
polyamines,
C. and Prentice
Hall,
66, 345-349.
Acta, 179, 232-234. J. Biochem. Tokyo,
K. and Igarashi,
K.
(1972) J. Bacterial.,
K., Hikami, K., Sugawara, K. and Hirose, S. (1973) 7. Igarashi, Biochim. Biophys. Acta, 299, 325-330, 8. Changchien, L. M. and Aronson, J. N. (1970) J. Bacterial.,
103, 734-740. 9. Davis, B. D. and Mingioli,
10.
Hurwits,
C.,
90, 1692-1695.
Braun,
E. S. (1950) J. Bacterial., C. B. and Peabody, R. A. (1965) J.
60, 17-28. Bacterial.,
Nirenberg, M. W. and Matthaei, J. H. (1961) Proc. Natl. Acad. Sci. TJ. S., 47, 1588-1602. 12. Igarashi, K., Izumi, I., Hara, K. and Hirose, S. (1974) Chem. Pharm. Bull. Tokyo, 22, 451-454. E. J. (1970) Ann. N. Y. Acad. Sci., 13. Dion, A. S. and Herbst, 11.
171, 723-734. W. and Price, R. P. (1974) Biochem. 14. Dewitt, Res. Commun., 56, 593-598. S. and Subramanian, A. R. (1974) 15. Ramagapal, Sci. U. S., 71, 2136-2140. 16.
Mangiarotti, G., Turco, (1974) Nature New Biol., 17. Weiss, R. L. and Morris, 204, 502-511. 18. Weiss, R. L. and Morris, Kimes, B. W. and Morris, 12%. Igarashi, K., Sugawara, Tokyo, in press. 21. Jorstad, C. M. and Morris,
119, 857-860.
Biophys. Proc.
Natl.
Acad.
E., Ponzetto, A. and Altrada, F. 247, 147-148. D. R. (1970) Biochim. Biophys. Acta, D. R. (1973) D. R. (1973) K. and Hirose,
Biochemistry, Biochemistry, S. (1975)
D. R. (1974)
904
J.
12, 436-441. 12, 442-449. J. Biochem.
Bacterial.,
