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Chromatography of Ehrlich ascites tumor cell high molecular weight ribonucleic acid on calcium phosphate
Vol. 6, No. 1,196l
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
CRRCMATOGRAPRI OF RRRLICH ASCITRS TZMOR CRLL HIGH MOLECULAR WRIGHT RIBONUCLEIC ACID
CALC1l.M PROSPMTE
ON
Giorgio Bernardi and Serge N. Timasheff* Centre de Recherches sur les Macromolecules Strasbourg, France
Received
31, 1961
July
The results
reported
in a recent
ribonucleic
acid
investigate
the chromatographic
acid
on calcium
(RNA) on this
results
bearing
phosphate
gel
present Colter
material.
was extracted
1.4 x lo6 by light ultracentrifuge, The slower
for
the
properties
samples
and Brown,
in very
communication
we.will
describe
of passage
through
The RNA sample tumor
cells
molecular
showed
two peaks
37%, the
faster with
and Brown,
1956;
of used in the of
was found
to be
in the analytical one with
one for those
a calcium
by the method
weight
s = 19 S, the faster
good agreement
of RNA (Colter
ribonucleic
ascites
the sample
for
us to
weight
columns.
average
one with
encouraged
of RNA and the possibility
Ehrlich
weight
accounted
63% of the material.
reported
Kronman,
s = 32 S.
by Brown --et al. Timasheff,
Colter
1960).
Chromatographic hydroxyapatite (1955).
Its
of deoxy-
molecular
the effect
phosphate
slower
are
of high
properties
scattering;
component
similar
namely
from
(1956).
1961)
In the present
RNA on calcium
work
on the chromatography
(Bernardi,
behavior
on two points,
and Brown
These
phosphate
on the physical
fractionating
paper
prepared
The duration
experiments according
were
carried
to the method
of each experiment
never
out at room temperature of Tiselius, exceeded
Hjertzn
on and Levin
one hour.
*Senior Post-tictoral Fellow of the National Science Foundation, U. S. A., Eastern Regional Research Laboratory, U. S. 1959-60; permanent address: Department of Agriculture, Philadelphia, Pennsylvania. 58
Vol. 6, No. 1,196l
BIOCHEMICAL
In a first calcium
series
phosphate
sium phosphate solvent,
buffer,
of the
analysis,
from
larger
large
samples
phosphate
the control
molecular
sample
that
adsorption-desorption reported
data
within
deal
with
after
solutions
were
the control of only
stored.
It
the state
M phosphate
stronger
decrease
place
molecular
after
in F about
weight
the continued
Time
These
was 1.4
out
value
prevented
were
showed
were
the
the
that all
the
(generally
found
former
and s after in
of
to those
remarkably
While
of x
Aggregation
similar
in
show unequi-
as a result
1960),
however,
scatter-
RNA was
results
to point
effects,
and Brown,
the latter
24 hours
suffici-
as described
The disytmaetry
important
samples.
in its
(20-259.).
s = 14 S.
already
when the different
showed
24 hours
in a drop
at 20'
the same conditions
phenomena
us from seeing
which whether
a much
invariably the drop
continued.
The observed mild
is
effects,
buffer),
adsorbed
of light
(prepared
sample.
at
in two
the sample
of the RNA as soon as possible
Colter
(l-57.)
sedimented
of eluted
of the RNA decreases
and the chromatographed
a few percent
0.20
weight
These
sample
sample,
buffer
one peak with
weight
of 1.4 x 106. the eluted
Timasheff,
Elution
By ultracentrifugal
sample whereas
molecular
chromatography.
(Kronman,
the starting
As a control
started.
only
H potas-
pH - 6.8.
the performance
the control
procedure.
1 hour)
reported
to permit
weight
the molecular
with
(25 mg RNA on 9x2 cm columns)
average
and 1.1 in
on the
M in phosphate
the control
showed
obtained
The weight
column
absorption.
the column
experiments
were
adsorbed in 0.005
buffer,
of RNA from to show that
scale
showed's
vocally
M phosphate
of ultraviolet
to be 0.7 x 106, whereas
above)
the
was made 0.20
calcium
ing measurements.
quite
0.20
washing
s = 20 S and s = 29 S, respectively,
Using
took
After
solution
was possible
on and eluted
(in
with
as elution
it
peaks with
found
pH - 6.8.
starting
mg of RNA were
x 6 cm) as a 0.05% solution
on the basis
the same time
ently
(1.3
RNA was desorbed
an aliquot
l-4
of experiments,
columns
was quantitative
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
degradation
conditions degradation
of RNA on the calcium
(pH 6.8, after
012 M phosphate passage 59
through
buffer) the
phosphate and, column
column
under
in particular, are
in sharp
in
Vol. 6, No. 1,196l contrast
BIOCHEMICAL
with
connection recent
the behavior
with
reports
of DNA on similar
the present of Brown
on the reversible
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dissociation
(Bernardi,
it is of interest
observation,
et al.
colums
(Brown,
Ellem
of similar
In
to recall
and Colter,
samples
1961).
1960;
the
Brown,
of ENA under
quite
1961) mild
conditions. Fractionation
of ENA was attempted
scheme shown in Fig. phate
(Fig.
line.
result
DNA (Bernardi, obtained
is
1961).
are "false"
however,
and no further
coefficients,
This
can be obtained,
The two fractions,
1).
sedimentation
Two peaks
1.
by stepwise
similar
to that
In both peaks
were work
(Hjerte'n,
it
according
at 0.15
and 0.20
found
out
quite
M phos-
along
in the fractionation is
to the
to have identical
was carried
obtained
instances
elution
possible
that
that of native
the peaks
1959).
LO-
3.0 -
2.0 -
LO-
F
3 *
1 Od 0
1
I-
20
40
:
0.25 L
0.50 1
I \-60
FRACTIONS
Fig. 1 Chromatography The stepwise increases arrows.
of Ehrlich in buffer
Ascites molarity
60
Tumor BNA on calcium phosphate. are indicated by the vertical
Vol. 6, No. 1,196l
BIOCHEMICAL
The authors making
available
wish
to thank
the sample
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Drs.
Raymond A.
of RNA used
in this
Brown and John S. Colter
for
study.
REFERENCES
Bernardi,
G., Biochem. Biophys. Research Cowmzns.5, 54 (1961)
Brown, RI A.; Proc. Natl. Acad. Sci., U. S., 47, 465 (1961). Brown, R. A., Ellem, K. A. 0. and Colter, J. S., Nature 187, 509 (1960). Coltef, J. S. and Brown, R. A., Science 124, 1077 (1956). Biophys. Acta 3l, 216 (1959). Hjerten, S., Biochim. S. N., Colter, J. S. and Brown, R. A., Biochim, Rronman, M. J., Timasheff, Biophys. Acta 4C, 410 (1960). Tiseliua, A., Hjerten, S. and Levin, O., Arch. Biochem. Biophys. 65, 132 (1955).
61
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
CRRCMATOGRAPRI OF RRRLICH ASCITRS TZMOR CRLL HIGH MOLECULAR WRIGHT RIBONUCLEIC ACID
CALC1l.M PROSPMTE
ON
Giorgio Bernardi and Serge N. Timasheff* Centre de Recherches sur les Macromolecules Strasbourg, France
Received
31, 1961
July
The results
reported
in a recent
ribonucleic
acid
investigate
the chromatographic
acid
on calcium
(RNA) on this
results
bearing
phosphate
gel
present Colter
material.
was extracted
1.4 x lo6 by light ultracentrifuge, The slower
for
the
properties
samples
and Brown,
in very
communication
we.will
describe
of passage
through
The RNA sample tumor
cells
molecular
showed
two peaks
37%, the
faster with
and Brown,
1956;
of used in the of
was found
to be
in the analytical one with
one for those
a calcium
by the method
weight
s = 19 S, the faster
good agreement
of RNA (Colter
ribonucleic
ascites
the sample
for
us to
weight
columns.
average
one with
encouraged
of RNA and the possibility
Ehrlich
weight
accounted
63% of the material.
reported
Kronman,
s = 32 S.
by Brown --et al. Timasheff,
Colter
1960).
Chromatographic hydroxyapatite (1955).
Its
of deoxy-
molecular
the effect
phosphate
slower
are
of high
properties
scattering;
component
similar
namely
from
(1956).
1961)
In the present
RNA on calcium
work
on the chromatography
(Bernardi,
behavior
on two points,
and Brown
These
phosphate
on the physical
fractionating
paper
prepared
The duration
experiments according
were
carried
to the method
of each experiment
never
out at room temperature of Tiselius, exceeded
Hjertzn
on and Levin
one hour.
*Senior Post-tictoral Fellow of the National Science Foundation, U. S. A., Eastern Regional Research Laboratory, U. S. 1959-60; permanent address: Department of Agriculture, Philadelphia, Pennsylvania. 58
Vol. 6, No. 1,196l
BIOCHEMICAL
In a first calcium
series
phosphate
sium phosphate solvent,
buffer,
of the
analysis,
from
larger
large
samples
phosphate
the control
molecular
sample
that
adsorption-desorption reported
data
within
deal
with
after
solutions
were
the control of only
stored.
It
the state
M phosphate
stronger
decrease
place
molecular
after
in F about
weight
the continued
Time
These
was 1.4
out
value
prevented
were
showed
were
the
the
that all
the
(generally
found
former
and s after in
of
to those
remarkably
While
of x
Aggregation
similar
in
show unequi-
as a result
1960),
however,
scatter-
RNA was
results
to point
effects,
and Brown,
the latter
24 hours
suffici-
as described
The disytmaetry
important
samples.
in its
(20-259.).
s = 14 S.
already
when the different
showed
24 hours
in a drop
at 20'
the same conditions
phenomena
us from seeing
which whether
a much
invariably the drop
continued.
The observed mild
is
effects,
buffer),
adsorbed
of light
(prepared
sample.
at
in two
the sample
of the RNA as soon as possible
Colter
(l-57.)
sedimented
of eluted
of the RNA decreases
and the chromatographed
a few percent
0.20
weight
These
sample
sample,
buffer
one peak with
weight
of 1.4 x 106. the eluted
Timasheff,
Elution
By ultracentrifugal
sample whereas
molecular
chromatography.
(Kronman,
the starting
As a control
started.
only
H potas-
pH - 6.8.
the performance
the control
procedure.
1 hour)
reported
to permit
weight
the molecular
with
(25 mg RNA on 9x2 cm columns)
average
and 1.1 in
on the
M in phosphate
the control
showed
obtained
The weight
column
absorption.
the column
experiments
were
adsorbed in 0.005
buffer,
of RNA from to show that
scale
showed's
vocally
M phosphate
of ultraviolet
to be 0.7 x 106, whereas
above)
the
was made 0.20
calcium
ing measurements.
quite
0.20
washing
s = 20 S and s = 29 S, respectively,
Using
took
After
solution
was possible
on and eluted
(in
with
as elution
it
peaks with
found
pH - 6.8.
starting
mg of RNA were
x 6 cm) as a 0.05% solution
on the basis
the same time
ently
(1.3
RNA was desorbed
an aliquot
l-4
of experiments,
columns
was quantitative
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
degradation
conditions degradation
of RNA on the calcium
(pH 6.8, after
012 M phosphate passage 59
through
buffer) the
phosphate and, column
column
under
in particular, are
in sharp
in
Vol. 6, No. 1,196l contrast
BIOCHEMICAL
with
connection recent
the behavior
with
reports
of DNA on similar
the present of Brown
on the reversible
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dissociation
(Bernardi,
it is of interest
observation,
et al.
colums
(Brown,
Ellem
of similar
In
to recall
and Colter,
samples
1961).
1960;
the
Brown,
of ENA under
quite
1961) mild
conditions. Fractionation
of ENA was attempted
scheme shown in Fig. phate
(Fig.
line.
result
DNA (Bernardi, obtained
is
1961).
are "false"
however,
and no further
coefficients,
This
can be obtained,
The two fractions,
1).
sedimentation
Two peaks
1.
by stepwise
similar
to that
In both peaks
were work
(Hjerte'n,
it
according
at 0.15
and 0.20
found
out
quite
M phos-
along
in the fractionation is
to the
to have identical
was carried
obtained
instances
elution
possible
that
that of native
the peaks
1959).
LO-
3.0 -
2.0 -
LO-
F
3 *
1 Od 0
1
I-
20
40
:
0.25 L
0.50 1
I \-60
FRACTIONS
Fig. 1 Chromatography The stepwise increases arrows.
of Ehrlich in buffer
Ascites molarity
60
Tumor BNA on calcium phosphate. are indicated by the vertical
Vol. 6, No. 1,196l
BIOCHEMICAL
The authors making
available
wish
to thank
the sample
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Drs.
Raymond A.
of RNA used
in this
Brown and John S. Colter
for
study.
REFERENCES
Bernardi,
G., Biochem. Biophys. Research Cowmzns.5, 54 (1961)
Brown, RI A.; Proc. Natl. Acad. Sci., U. S., 47, 465 (1961). Brown, R. A., Ellem, K. A. 0. and Colter, J. S., Nature 187, 509 (1960). Coltef, J. S. and Brown, R. A., Science 124, 1077 (1956). Biophys. Acta 3l, 216 (1959). Hjerten, S., Biochim. S. N., Colter, J. S. and Brown, R. A., Biochim, Rronman, M. J., Timasheff, Biophys. Acta 4C, 410 (1960). Tiseliua, A., Hjerten, S. and Levin, O., Arch. Biochem. Biophys. 65, 132 (1955).
61