BIOCHEMICAL
Vol. 62, No. 3, 1975
MODIFICATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF RIBOSOMAL PROTEINS DURING
LIVER REGENERATION
W. Marshall
Anderson,
Anette
Laboratories
Grundholm
of Molecular
Faculty Memorial St. Received
December
H. Sells*
Biology
of Medicine
University
John's,
and Bruce
of Newfoundland
Newfoundland
AlC 5S7 Canada
4,1974 SUMMARY
Analysis of the 2D gel electrophoretic pattern of ribosomal proteins from both the small and large subunit of rat liver were made at various times No changes were observed in the electrophoretic following partial hepatectomy. mobility of proteins from the 60s subunit during periods of 2 hr. to 72 hr. of liver regeneration. Changes were observed, however, in two proteins of the 40s subunit a short time after partial hepatectomy. Protein S6 disappeared from its normal position and a new spot appeared as a more negative form as early as 2 hr. post regeneration. This modification persisted for at least 18 hr. At 72 hr., S6 returned to its normal position. Protein S , on the other hand, underwent a different pattern of change during the ear f y stages of liver regeneration. S was observed to migrate as 2 spots at 2 hr. after partial hepatectomy and 2 his pattern was preserved at the 4 hr. period. At 8 hr., the pattern was further modified to 2 spots which was distinct from the earlier change. This pattern was similar at 12 hr. At 18 hr. only the normal S2 protein was observed. No further change in S2 migration was observed at the 72 hr. period of liver regeneration.
INTRODUCTION Following biochemical changes (2,3),
changes include
which
components itative liver
partial
hepatectomy,
occur
in the remaining
an increased appears
of the changes
capacity
to be directly
protein
synthetic
have been observed
a number
of regenerating related system
(2,3).
669
morphological
of the liver liver
to the function
in the proteins
(4,5).
Copyright o 19 75 bv Academic Press, Inc. All rights of’reproduction in asy form reserved.
portion
of rapid
for
(1). protein
and These synthesis
of the ribosomal
Qualitative
as well
as quant-
synthesized
by regenerating
Vol. 62, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The present studies were designed to determine whether modifications occur in the ribosomal protein
components during liver
dimensional (2-D) polyacrylamide
gel electrophoresis
Kaltschmidt and Wittmann (6) affords an excellent proteins from different
regeneration.
The two-
technique developed by
opportunity
sources and make qualitative
to compare ribosomal
examinations of modification
of these proteins. MATERIALSANDMETHODS Female Sprague-Dawley rats (100-300 g) used in this study were maintained as previously
described (7).
and doubly recrystallized electrophoresis unit
Acrylamide was purchased from Eastman Kodak Company
from acetone before use.
Urea solutions used in the
procedure were deionized by passage through a LD-3 Demineralizer
(Corning Glass Works). Partial
anesthesia (8).
hepatectomy was performed while the animals were under light Control rats were sham-operated.
Following surgery,
the rats
were allowed free access to food and water for 18 hr. at which time the livers were removed. The livers Buffer II
were chilled,
weighed, minced in 2.5 volumes of cold
(250 mMsucrose, 100 mMKCl, 10 mMHEPES', 0.1 mMDisodium EDTA,
5 mMMg (CH3C02J2, brought to pH 7.4 with KOH) and homogenized with a glass teflon
homogenizer.
by centrifugation aspiration
at 20,000 g for 10 min.
supernatant fraction The lipid
was prepared
layer was removed by
and 0.1 volume of 15%DOCwas added to the supernatant fraction
and centrifuged aspiration
The post mitochondrial
at 20,000 g for 10 min.
and the supernatant fraction
to obtain the crude ribosomal pellet.
The lipid
layer was again removed by
centrifuged
at 150,000 g for 1.5 hr.
This pellet
was resuspended in PRM (20
mMHEPES, 100 mMKCl, 5 mMMg(CH3C02)2, brought to pH 7.4 with KOH) to a concentration
of approximately
200 A260 units/ml and dissociated
into ribosomal
subunits with puromycin and 500 mMKC1 as described by Blobel and Sabatini (9). I The abbreviations used are: HEPES,N-2-hydroxyethylpiperazine-N-2 acid; DOC, sodium deoxycholate.
ethanesulfonic
BIOCHEMICAL
Vol. 62, No. 3,1975
This
mixture
was layered
KCl,
20 mM Tris-Xl
pH 7.5,
and centrifuged were
at 40,000
collected
Company)
over
through
prepare
at
with
g for
66% acetic
by a modification
The separation
gel
proteins
18 cm) was filled
gel
pooled
were with
separately
used directly a small
to
volume
of
(12).
and Harris
of Groves
of the Kaltschmidt
ug)
equal
17.5
hr.
with
in
1-D Buffer
onto
volume
the
et al
(room
2-D buffer
(12)
were
Black.
stained
The gels
gel.
procedure
gel
diluted
with
140 volts
space
(to
Gels were
to a 16% acrylamide for
43 hr.
and 3% acetic
destained
an equal
The cylindrical
and polymerized at
(12).
(1 cm) was 4.5%.
The remaining
temperature).
Protein
Electrophoresis
and 2% agarose.
in 7% ethanol were
(11).
spacer
were
of 1-D buffer
at 3 mA/gel
(10).
and Wittmann
spacer
cm) and electrophoresed
The gels
and Wittmann
by Waller
by the method
and layered
(18 cm x 0.2
Blue
as described
in PRM and 100 mM MgC12 and
(12)
equilibrated
0.5% Aniline
suspended
(750-950
for
temperature).
were
and the
with
electrophoresed
slab
or layered
The gradients
Specialties
subunits
(15 cm) was 5% acrylamide,
of 2% agarose
were
at 25'C.
(Instrument
These
300 mM
A260 units/gradient)
SW27 rotor)
electrophoresis
acid
was performed
gels
containing
40s and 60s subunits
(4'C).
(35 A260 units/ml)
was determined
volume
gradient
(80-100
Monitor
to the
16 hr.
for
concentration
Ribosomal
sucrose
(Beckman
16 hr.
RESEARCH COMMUNICATIONS
at -2O'C.
Subunits extracted
g for
proteins
PRM and stored
linear
and 3 mM Mg(CH3C02)2
corresponding
100,000
ribosomal
a IO-30%
an ISCO ultraviolet
and the peaks
and pelleted
AND BIOPHYSICAL
as described
acid
(room containing
by Kaltschmidt
RESULTS
(not
shown)
the large
Fingerprints
of proteins
revealed
33 resolvable
subunit.
components,
only
Since basic
our
gel
proteins
from
40s subunits
proteins
from
procedure
the
excludes
of each subunit
will
(Fig.
1) and 60s subunits
small
subunit
and 40 from
the examination be considered
of acidic in the present
study. The 60s liver partial
hepatectomy
were
ribosomal compared
proteins with
obtained
those
671
obtained
at various from
times
sham-operated
following controls.
Vol. 62, No. 3,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
^” .
Fig.
1.
Fingerprint
.“,
of basic
proteins
The electrophoretic
patterns
reveal
any of the
proteins
throughout
Several
40 basic
modifications
were
proteins
were
compared
in Fig.
2A-G,
changes
partial
removal
modification for
gel.
that
is
normally
At 2 hr.
and a new protein position
These
(Fig. spot
For
presented appear
2B),
in the
the amount
appeared
of S2 in the gel
pattern.
with
no significant period
partial
changes
of normally
This
pattern
672
following pattern
migrating
second
was duplicated
portion
in any of the dimension
protein
charge
of
regeneration
the upper
observed
of the
a more positive
As indicated
of liver only
were
portion
of the 40s
as 2 hr.
the period
of
regeneration.
a different
the sake of clarity,
lower
changes
of liver
as early
no changes
liver.
hepatectomy.
revealed
over
since
of rat
when fingerprints
two proteins
mobility
each of the two proteins.
proteins
in
were
the 72 hr.
following
noted
the 40s subunit
there
controls
of the liver.
pattern
that
however,
of electrophoretic
of each gel
from
observed,
with were
‘
slab
S2 was decreased
to the right for
of the
S2 at 4 hr.
BIOCHEMICAL
Vol. 62, No. 3, 1975
Fig.
post
2.
mobility
at
(Fig.
pattern
2C),
just
following
protein
however,
of the modified
at a position
12 hr.
S2 appears to the
in the
electrophoretic
at 8 hr.
2D),
the
electrophoretic
changed to another form which migrated in 2 the normal S2. This pattern remained constant
hepatectomy
to have reverted
same position
(Fig.
S
below
partial
migrated
(Fig.
RESEARCH COMMUNICATIONS
Upper portion of fingerprint of proteins from 40s subunit at various times following partial hepatectomy. Arrows indicate the positions of the two proteins of interest. A. control 875 up protein; B. 2 hr. 875 ug; C. 4 ht. 900 ug.; D. 8 hr. 875 ug.; E. 12 hr. 860 ug; F. 18 hr. 700 ug.; G. 72 hr. 850 ug.
hepatectomy
the gel
AND BIOPHYSICAL
(Fig.
to its
as S 2 from
mobility
2E),
normal
but
at 18 hr.
form
(of only
sham-operated
of S2 was noted
(Fig. one spot
controls).
at 72 hr.
2F) which
No modification
after
partial
hepatectomy
2G). Modification
subunit
during
liver
modification
ification
By 72 hr.
the normally
(Fig.
migrating
possibly
presented as 2 hr.
mobility
throughout
post
than
the first
an indication
although that
a small the
of change.
hepatectomy
the normal 18 hr.
S6.
of liver
mobility amount
reversion
673
pattern
S6 of the 40s
(Fig.
This
The
2B),
of S6 was to a form
2G) the electrophoretic form,
of protein
a simpler
in S2, the modification
electrophoretic
was maintained
mobility
as early
to the change
a more negative
the gel,
regeneration
of S6 occurred
in contrast
3B-F).
of electrophoretic
however,
which
type
of mod-
regeneration
(Fig.
of S6 had reverted
of streaking
was still
had
not
to
was observed entirely
in
complete.
Vol. 62, No. 3, 1975
BIOCHEMKAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION A number of post translational reported.
Of these modifications,
modifications
of proteins have been
both phosphorylation
(13-16) and acetylation
(17) of ribosomal proteins from various sources have been described. phosphorylation regenerating result
or acetylation
liver,
could account for the modification
since insertion
positively
a positive
charged protein.
of S6 in
of a phosphate group into protein would
in a more negative charge, while addition
tend to neutralize
Either
of an acetyl group would
charge on an amino group and produce a less A comparison of our gel pattern for the 40s subunit
with that of Welfle et al (18) reveals that protein S in our numbering system 6 appears to be identical to S9 in their system, and it is interesting to note that Stahl et al (15) observed that Sq (our S6) could be extensively in vitro,
phosphorylated
whether as an isolated protein or as a component of the 40s subunit
or 80s monomer. In the case of protein
S2, the modification
appears to be more complex
S2 appears to undergo a pattern of cyclic change during the than that of S 6' first 18 hr. of liver regeneration. The first change observed at 2 hr. results in the disappearance of someof the normally migrating S2 (Fig. 3B) and the appearance of another component which migrates as a protein more positively
charged
than normal S2. Although we cannot state categorically
that the second spot is
a modification
in the quantitative
product of S2, our gel procedure results
of protein from the first the decrease in staining
dimension gel into the second dimension slab gel. intensity
and experimental gels
suggest that the new spot is indeed a modified S2. Since
the new component migrates more rapidly than normal S2, the initial
Thus,
of the normal S2 and appearance of a new spot
when equal amounts of protein are applied to the control (Fig. 3A and B) strongly
migration
modification
toward the cathode in the first of S2 could be either
dimension
a dephosphorylation
or deacetylation. Between 4 and 8 hr. post hepatectomy, a second modification previously
occurs to the
modified S2, since this spot disappears from the gel pattern
674
(Fig. 3D)
BIOCHEMICAL
Vol. 62, No. 3, 1975
and a new spot This a,
appears
migration
described
except
for
second
changes.
As with
also
short
this
second
group
paper
following
partial
in protein
S , 2'
hepatectomy.
This
Wool's
interesting
the ribosome
size
particular
early
during
dramatically,
proteins. regeneration (25)
although
compared
of normal
and regenerating
18-20
hours
specific
partial
a larger
is
the major hepatectomy
(Fig.
for
these is
3E and F) S2 remains.
that
the
of phosphorylation. Our recent
as early
indicate
as 2 hrs.
as 2 hrs.
a modification
following
to normal
changes
is
partial
by 18 hrs.,
not yet
in the 40s subunit
clear.
Lt
proteins
may
of mRNA and preferentially
have obtained of protein
evidence
changed
alteration.
types
groups
liver, liver
this
changes
the rate
is
to normal
of albumin, after
these
presented
there
also
earlier
modification
the normal
S occurs 6
as early
of these
Several
ribosomes
the synthesis
that
in
of L7
basis
hepatectomy.
Our studies
do not document
second
the result
of S2 had returned
significance
Scornik
to show that
modification
again
of the
has demonstrated
partial
identical
residue
regeneration
is
&
are
the chemical
and only
(22)
after
liver.
to recognize
liver
hrs.
pattern
to speculate
post
hepatectomy
occurred
results
The physiological
permit
of the
change
which
terminal
of S2, this
and Wool
observed
removal
of L7 and L12 for
two proteins
disappears
partial
The migration
consequently
is
of S2 also
the
of S2.
in the amino
12 and 18 hr.
made 22-24
that
for
modification
of S6 following
indicate
to that
to establish
however,
by Gressner
were
position
of S2 may be a deacetylation
between
form
observations
studies
of an acetyl
required,
since
modified
modification
(19,20)
the previous
lived,
A recent
Their
is
similar
group
modification
work
the normal
appears
by Wittmann's
Further
3.
below
pattern
the presence
This
(21).
directly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthesis
- with
proportion
results
suggesting (23,24)
regenerating
of ribosomes
synthethat
increases mouse liver
present
as poly-
the --in vivo rate of translation of polyribosomes same. Hill et al (26) has noted that
the
export
protein
relative
of the liver,
to other
is
reduced
proteins.
ACKNOWLEDGEMFNCS These
studies
were
supported
-
by grants
675
from
the Medical
Research
Council
Vol. 62, No. 3, 1975
BIOCHEMKAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of Canada, the DamonRunyon Memorial Fund and the National Foundation-March of Dimes. We are grateful
to Mrs. M. Cooper for technical
assistance.
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