Vol. 75, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PHOSPHORYLATION OF NUCLEAR MATRIX PROTEINS IN ISOLATED REGENERATING RAT LIVER NUCLEI Steven
L. Allen,
Department
Ronald
Berer.ney*
of Pharmacology
Reproductive
The Johns
December
Therapeutics,
Laboratory
and the Oncology
University
Baltimore, Received
Brady
Biology,
Hopkins
S. Coffey
and Experimental
The James Buchanan for
and Donald
School
Maryland
Center
of Medicine
21205
13,1976
Summary. The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an --in vitro system of isolated nuclei and Y-~~P-ATP. Phosphorylation of the nuclear matrix proteins was 2-3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis. Recently, liver
we reported
nuclei
proteins
(1).
with
The nuclear
daltons
Upon investigating we reported
that
the nuclear
matrix
synthesis. in these matrix
*
Present
isolation
over
90% of newly
may be involved
processes. proteins
address:
fractions
intranuclear
(2).
Specific
consists
and represents the
This
regard,
largely
site
than
10% of the
synthesized that
DNA is this
weight total
residual
we have
phosphorylation
--in
determined vitro
rat
nuclear
of proteins.
in regenerating
nuclear
with
structural such as DNA
proteins the
liver,
associated
may be involved
ability
of these
in the presence
Division of Cell and Molecular Biology, State of New York, Buffalo, New York 14214.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
range
functions
matrix
from
nonhistone
initially
of nuclear
of the nuclear
matrix
of acidic
of DNA replication
in the regulation
In this
protein
in the molecular
less
suggests
modifications
to undergo
of a nuclear
matrix
3 main polypeptide
60,000-70,000
framework
the
of y-
32
nuclear P-ATP.
University
111 ISSN
0006-291X
Vol. 75, No. 1, 1977
Much interest phosphorylation function
in recent of acidic
BIOCHEMICAL
years
AND BIOPHYSICAL
has been focused
nonhistone
proteins
RESEARCH COMMUNICATIONS
on the possible in
the regulation
role
of the
of nuclear
(3,4,5). MATERIALS
AND METHODS
Male Sprague-Dawley rats (400-450 g) underwent partial hepatectomy by surgical removal of 213 of the liver following the methods of Higgins and Anderson (6). Animals were maintained in a controlled light environment and surgery was carried out under ether anesthesia between 8-10 a.m. Liver nuclei were isolated through high molarity sucrose by the procedure of Berezney and Coffey (1). The isolated nuclei were phosphorylated by incubation with y- 32 P-ATP utilizing a modification of the method of Johnson --et al. (7). Incubations were carried out at 37" C for 5 minutes in 6 ml containing: 3 x 10m8 moles Y-~~P-ATP (5OpCi); 5 mM MgC12; 115 mM NaCl; 30 mM Tris-HCl, pH 7.4 with the addition of nuclei equivalent to 60 mg of total nuclear protein. The reaction was terminated b the addition of 6 ml of the reaction buffer at 4°C containing 6 mM ATP. The 3I P labeled nuclei were isolated and washed 3 times by centrifugation at 780 x g for 15 minutes. The labeled nuclei were then processed for the isolation of the nuclear matrix proteinsas described by Berezney and Coffey (1). The final step for the removal of tightly bound RNA and DNA employing nuclease digestion was omitted since SDS-acrylamide gel electrophoresis demonstrated no measurable difference in the polypeptide pattern before or after nuclease action. The nuclear pellet was subjected to the following extractions which were carried out at 4'C in 10 ml solutions containing 10 mM Tris buffer pH 7.4: (A) Three extractions with 0.2 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 30 min; (B) Three extractions with 2 M NaCl containing 0.2 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 60 min.; (C) 1% Triton X-100 in 5 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 30 min. and two washes with 0.25 M sucrose, 0.05 mM Tris, pH 7.4 containing 5 mM MgC12. Protein bound phosphate was isolated by modification of the procedures of Kleinsmith Removal of the nuclear lipid was not necessary since --et al. (8). initial experiments revealed that extraction with chloroform:methanol (2:l; v:v) did not remove 32P-containing material from the pellet. Nucleic acids were removed by the method of Munro and Fleck (9). The fractions were precipitated with 0.2 E PCA on ice for 10 minutes. After centrifuging at 27,000 x g for 10 minutes the pellets were washed two additional times in 0.2 E PCA and then The samples were resuspended in 0.8 N PCA and heated at 70°C for 20 minutes. cooled on ice, centrifuged at 27,000 x g, and dried with 10 ml of cold ether. the pellets were heated at 100°C for 15 To release protein bound phosphate, minutes in 0.5 ml of 18 NaOH, cooled and then 0.49 ml of 70% perchloric acid was added to yield a final concentration of 0.2 g PCA. The suspensions were centrifuged at 27,000 x g for 10 minutes and the supernatants analyzed for inorganic phosphate by the method of Chen et al. (10). Radioactivity was determined by liquid scintillation counting in 10 ml of Hydromix (Yorktown Research Inc.). Protein was determined by the Lowry assay (11). SDS (sodium dodecyl sulfate) acrylamide gel electrophoresis was performed according to the procedure of Weber and Osborn (12) and consisted of 10% acrylamide gels in 0.1% SDS, 0.05 M Tris, pH 7.4 which were run at 4 ma per gel with a solution of 0.1% SDS, 0.05 M Tris, pH 7.4 in both lower and upper chambers.
112
2
Standard
Averages
error
of five
Nuclei
Proteins
3.57
5.73
involving
100 2 1.84
5 0.97
between
pooled
of Total Protein (z>
100
of differences
experiments
of the mean Significance
individual
Protein Proteins
Liver
Total Nuclear Nuclear Matrix
Regenerating (12 hours)
Matrix
Protein
Nuclei
Nuclear
Liver
Nuclear
Total
Normal
Percent Nuclear
groups:
liver
5
9 6
4 rats
per
AvsB 4 0.005 E vs F 4 0.05 C vs D C 0.025 G vs H C 0.05
from
68+ 67+
70 +_ 24
73+
x lo5
(mg PO4/mg Protein)
Total Phosphate in Protein
PROTEINS
+ 1.13 + 9.40
+ 1.07
+ 0.41
AvsE CvsG BvsFc DvsHc
C 0.005 C 0.05 0.05 0.10
(E) (F)
(B)
(A)
32P04/mg
determination.
8.20 30.94
8.46
4.01
Protein)
(ppmole
Specific Activity of Protein
PHOSPHORYLATION OF TOTAL NUCLEAR AND NUCLEAR MATRIX
TABLE I
x 10
4
32P04/mg
4.25 13.81
5.15
2 1.12 + 3.66
+_ 1.39
1.60 +_ 0.18
(mg
(G) (H)
(D)
(c)
PO4)
Specific Activity of Phosphate
H
c za G m 9
5 9 2
:
c
3
,’
s v
5 .
.i
< 0
Vol. 75, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAI. RESEARCH COMMUNICATIO’QS
T
X PROTEIN 2 HRREGENRmM LIVER
SPECIFIC ACTPill
SPECIFIC KTIL NoRk(pL LIVER
16
ri N
32 of isolated nuclei and 7- P-ATP phosphorylation specific activity of the total nuclear proteins isolated nuclear matrix proteins (M). Data are normal liver nuclei and nuclei isolated 12 hours partial hepatectomy.
FIGURE 1.
the resulting (N) and the presented for following
Samples for electrophoresis were dissolved as lyophylised powders in 5% SDS, 0.05 M Tris, pH 7.4 containing 2% $-mercaptoethanol by heating at 70°C for 20 min. followed by incubation at room temperature for three hours. RESULTS AND DISCUSSION --In vitro 12 hour with if
phosphorylation
regenerating
normal
rat
the specific
protein
approximately
nuclear
nuclei
was 2*3 fold
liver
unit
This
phosphate nuclear
liver
nuclei.
5% of the
total
(Table matrix
The matrix
higher
as incorporation
I,
Fig.
1).
After
were
isolated
matrix
proteins
proteins,
however,
114
that
proteins
per
observed
unit
amount
phosphorylation
from both
normal
represent essentially
as was observed were
in
and was observed
--in vitro
and contained
per mg of protein
protein
than
either
The recovered
residues
nuclear
was significant
proteins
nuclear
total
difference
was expressed
of phosphate
proteins.
32 P-ATP of the
nuclei.
nuclei,
and regenerating
same amount
liver activity
or per
the isolated
rat
by y-
differentially
the in the
total
phosphory-
of of
BIOCHEMICAL
Vol. 75, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNKATIONS
am.
-A.-&43-&-$?--o--$?‘E---S
RI
IO
Radioactivity isolated from hepatectomy. on 100 pg of
FIGURE 2.
lated
in both
higher
than
protein
normal total
and regenerating
nuclear
was markedly
from both 2).
which
corresponded
in the nuclear the
during
specific
matrix nuclear
by a factor
(I.);
comparable matrix
labeled
proteins
high (l), matrix
molecular the resolution polypeptides
115
(Fig.
solubilized
nuclear
which
of the nuclear
regeneration
of the
reported
the phosphate
protein
liver
electrophoresis
to the major
nuclei
Phosphorylation
and regenerating
Although
(Fig.
identify
gel
as previously
normal
liver
proteins.
inoreased
SDS acrylamide was performed
associated with the nuclear matrix proteins normal livers and 12 hours following partial SDS acrylamide gel electrophoresis was performed protein according to Beresney and Coffey (4).
were
weight
proteins
of protein
on each gel in the Rf zones
proteins
was not
matrix
(100 pg)
resolved
appeared
matrix
1).
nuclear
amounts
was 2-3 fold
previously
sufficient
phosphorylated.
A-C, identified
to definitively
Vol. 75, No. 1, 1977
A time
study
regenerating partial
14-16
hours
by ATP was performed
newly
replicated
in regenerating
of specific
reviewed
preceded
hepatectomy.
was not measured
proteins in the the
in
(2),
of the nuclear regulation
isolation,
in vitro
these
liver
is
in
alt 12 hours
of DNA synthesis
important
to note
and that
the extent
which
that
ooour?
the
of dephos-
experiments.
it
is
associated
is
conceivable
matrix
preceding
of DNA synthesis
structure
protem
of phosphorylation
the onset It
DNA in vivo
rat
of the nuclear
a maximum level
This
partial
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of phosphorylation
demonstrated
after
phorylation
importance
onset
hepatectomy.
phosphorylation
matrix
of the
liver
after
Since
BIOCHEMICAL
with
the isolated
that this
and functions
the phosphorylation
event
in the nucleus. of the nuclear
nuclear
could
be of
We have recently protein
matrix
Grant
&13745.
(13).
ACKNOWLEDGEMENTS This
work
was supported
by the National
Cancer
Institute,
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13.
Berezney, R. and Coffey D.S. (1974) Biochem. Biophys. Res. Comm. 60, 14101417. Berezney, R. and Coffey, D.S. (1975) Science 189, 291-293. Kleinsmith, L. (1975) J. Cell Physiol. 85, 459-476. Stein, G.S., Spelsberg, T.C. and Kleinsmith, L. (1974) Science 183, 817824. Allfrey, V.G., Inoue, A., Kain, J., Johnson, E.M. and Vidali, G. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 785-801. Higgins, G.M. and Anderson, R.M. (1931) Arch. Pathol. l2, 186-202. Johnson, E.M., Vidali, G., Littan, V.C., and Allfrey, V.G. (1973) J. Biol. them. 248, 7595-7600. Kleinsmith, L., Allfrey, V.G. and Mirsky, A.E. (1966) Proc. Natl. Acad. Sci. 55, 1182-1189. Munro, H.N. and Fleck, A. (1965) Meth. Biochem. Anal. 14, 113-176. Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. Chen, P.S., Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Weber, R. and Osbom, M. (1969) J. Biol. Chem. 244, 4406-4412. Berezney, R. and Coffey, D.S. (1976) Adv. Enzyme Reg. 2, 63-100.
116
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PHOSPHORYLATION OF NUCLEAR MATRIX PROTEINS IN ISOLATED REGENERATING RAT LIVER NUCLEI Steven
L. Allen,
Department
Ronald
Berer.ney*
of Pharmacology
Reproductive
The Johns
December
Therapeutics,
Laboratory
and the Oncology
University
Baltimore, Received
Brady
Biology,
Hopkins
S. Coffey
and Experimental
The James Buchanan for
and Donald
School
Maryland
Center
of Medicine
21205
13,1976
Summary. The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an --in vitro system of isolated nuclei and Y-~~P-ATP. Phosphorylation of the nuclear matrix proteins was 2-3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis. Recently, liver
we reported
nuclei
proteins
(1).
with
The nuclear
daltons
Upon investigating we reported
that
the nuclear
matrix
synthesis. in these matrix
*
Present
isolation
over
90% of newly
may be involved
processes. proteins
address:
fractions
intranuclear
(2).
Specific
consists
and represents the
This
regard,
largely
site
than
10% of the
synthesized that
DNA is this
weight total
residual
we have
phosphorylation
--in
determined vitro
rat
nuclear
of proteins.
in regenerating
nuclear
with
structural such as DNA
proteins the
liver,
associated
may be involved
ability
of these
in the presence
Division of Cell and Molecular Biology, State of New York, Buffalo, New York 14214.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
range
functions
matrix
from
nonhistone
initially
of nuclear
of the nuclear
matrix
of acidic
of DNA replication
in the regulation
In this
protein
in the molecular
less
suggests
modifications
to undergo
of a nuclear
matrix
3 main polypeptide
60,000-70,000
framework
the
of y-
32
nuclear P-ATP.
University
111 ISSN
0006-291X
Vol. 75, No. 1, 1977
Much interest phosphorylation function
in recent of acidic
BIOCHEMICAL
years
AND BIOPHYSICAL
has been focused
nonhistone
proteins
RESEARCH COMMUNICATIONS
on the possible in
the regulation
role
of the
of nuclear
(3,4,5). MATERIALS
AND METHODS
Male Sprague-Dawley rats (400-450 g) underwent partial hepatectomy by surgical removal of 213 of the liver following the methods of Higgins and Anderson (6). Animals were maintained in a controlled light environment and surgery was carried out under ether anesthesia between 8-10 a.m. Liver nuclei were isolated through high molarity sucrose by the procedure of Berezney and Coffey (1). The isolated nuclei were phosphorylated by incubation with y- 32 P-ATP utilizing a modification of the method of Johnson --et al. (7). Incubations were carried out at 37" C for 5 minutes in 6 ml containing: 3 x 10m8 moles Y-~~P-ATP (5OpCi); 5 mM MgC12; 115 mM NaCl; 30 mM Tris-HCl, pH 7.4 with the addition of nuclei equivalent to 60 mg of total nuclear protein. The reaction was terminated b the addition of 6 ml of the reaction buffer at 4°C containing 6 mM ATP. The 3I P labeled nuclei were isolated and washed 3 times by centrifugation at 780 x g for 15 minutes. The labeled nuclei were then processed for the isolation of the nuclear matrix proteinsas described by Berezney and Coffey (1). The final step for the removal of tightly bound RNA and DNA employing nuclease digestion was omitted since SDS-acrylamide gel electrophoresis demonstrated no measurable difference in the polypeptide pattern before or after nuclease action. The nuclear pellet was subjected to the following extractions which were carried out at 4'C in 10 ml solutions containing 10 mM Tris buffer pH 7.4: (A) Three extractions with 0.2 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 30 min; (B) Three extractions with 2 M NaCl containing 0.2 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 60 min.; (C) 1% Triton X-100 in 5 mM MgC12 for 10 min. followed by centrifugation at 780 x g for 30 min. and two washes with 0.25 M sucrose, 0.05 mM Tris, pH 7.4 containing 5 mM MgC12. Protein bound phosphate was isolated by modification of the procedures of Kleinsmith Removal of the nuclear lipid was not necessary since --et al. (8). initial experiments revealed that extraction with chloroform:methanol (2:l; v:v) did not remove 32P-containing material from the pellet. Nucleic acids were removed by the method of Munro and Fleck (9). The fractions were precipitated with 0.2 E PCA on ice for 10 minutes. After centrifuging at 27,000 x g for 10 minutes the pellets were washed two additional times in 0.2 E PCA and then The samples were resuspended in 0.8 N PCA and heated at 70°C for 20 minutes. cooled on ice, centrifuged at 27,000 x g, and dried with 10 ml of cold ether. the pellets were heated at 100°C for 15 To release protein bound phosphate, minutes in 0.5 ml of 18 NaOH, cooled and then 0.49 ml of 70% perchloric acid was added to yield a final concentration of 0.2 g PCA. The suspensions were centrifuged at 27,000 x g for 10 minutes and the supernatants analyzed for inorganic phosphate by the method of Chen et al. (10). Radioactivity was determined by liquid scintillation counting in 10 ml of Hydromix (Yorktown Research Inc.). Protein was determined by the Lowry assay (11). SDS (sodium dodecyl sulfate) acrylamide gel electrophoresis was performed according to the procedure of Weber and Osborn (12) and consisted of 10% acrylamide gels in 0.1% SDS, 0.05 M Tris, pH 7.4 which were run at 4 ma per gel with a solution of 0.1% SDS, 0.05 M Tris, pH 7.4 in both lower and upper chambers.
112
2
Standard
Averages
error
of five
Nuclei
Proteins
3.57
5.73
involving
100 2 1.84
5 0.97
between
pooled
of Total Protein (z>
100
of differences
experiments
of the mean Significance
individual
Protein Proteins
Liver
Total Nuclear Nuclear Matrix
Regenerating (12 hours)
Matrix
Protein
Nuclei
Nuclear
Liver
Nuclear
Total
Normal
Percent Nuclear
groups:
liver
5
9 6
4 rats
per
AvsB 4 0.005 E vs F 4 0.05 C vs D C 0.025 G vs H C 0.05
from
68+ 67+
70 +_ 24
73+
x lo5
(mg PO4/mg Protein)
Total Phosphate in Protein
PROTEINS
+ 1.13 + 9.40
+ 1.07
+ 0.41
AvsE CvsG BvsFc DvsHc
C 0.005 C 0.05 0.05 0.10
(E) (F)
(B)
(A)
32P04/mg
determination.
8.20 30.94
8.46
4.01
Protein)
(ppmole
Specific Activity of Protein
PHOSPHORYLATION OF TOTAL NUCLEAR AND NUCLEAR MATRIX
TABLE I
x 10
4
32P04/mg
4.25 13.81
5.15
2 1.12 + 3.66
+_ 1.39
1.60 +_ 0.18
(mg
(G) (H)
(D)
(c)
PO4)
Specific Activity of Phosphate
H
c za G m 9
5 9 2
:
c
3
,’
s v
5 .
.i
< 0
Vol. 75, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAI. RESEARCH COMMUNICATIO’QS
T
X PROTEIN 2 HRREGENRmM LIVER
SPECIFIC ACTPill
SPECIFIC KTIL NoRk(pL LIVER
16
ri N
32 of isolated nuclei and 7- P-ATP phosphorylation specific activity of the total nuclear proteins isolated nuclear matrix proteins (M). Data are normal liver nuclei and nuclei isolated 12 hours partial hepatectomy.
FIGURE 1.
the resulting (N) and the presented for following
Samples for electrophoresis were dissolved as lyophylised powders in 5% SDS, 0.05 M Tris, pH 7.4 containing 2% $-mercaptoethanol by heating at 70°C for 20 min. followed by incubation at room temperature for three hours. RESULTS AND DISCUSSION --In vitro 12 hour with if
phosphorylation
regenerating
normal
rat
the specific
protein
approximately
nuclear
nuclei
was 2*3 fold
liver
unit
This
phosphate nuclear
liver
nuclei.
5% of the
total
(Table matrix
The matrix
higher
as incorporation
I,
Fig.
1).
After
were
isolated
matrix
proteins
proteins,
however,
114
that
proteins
per
observed
unit
amount
phosphorylation
from both
normal
represent essentially
as was observed were
in
and was observed
--in vitro
and contained
per mg of protein
protein
than
either
The recovered
residues
nuclear
was significant
proteins
nuclear
total
difference
was expressed
of phosphate
proteins.
32 P-ATP of the
nuclei.
nuclei,
and regenerating
same amount
liver activity
or per
the isolated
rat
by y-
differentially
the in the
total
phosphory-
of of
BIOCHEMICAL
Vol. 75, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNKATIONS
am.
-A.-&43-&-$?--o--$?‘E---S
RI
IO
Radioactivity isolated from hepatectomy. on 100 pg of
FIGURE 2.
lated
in both
higher
than
protein
normal total
and regenerating
nuclear
was markedly
from both 2).
which
corresponded
in the nuclear the
during
specific
matrix nuclear
by a factor
(I.);
comparable matrix
labeled
proteins
high (l), matrix
molecular the resolution polypeptides
115
(Fig.
solubilized
nuclear
which
of the nuclear
regeneration
of the
reported
the phosphate
protein
liver
electrophoresis
to the major
nuclei
Phosphorylation
and regenerating
Although
(Fig.
identify
gel
as previously
normal
liver
proteins.
inoreased
SDS acrylamide was performed
associated with the nuclear matrix proteins normal livers and 12 hours following partial SDS acrylamide gel electrophoresis was performed protein according to Beresney and Coffey (4).
were
weight
proteins
of protein
on each gel in the Rf zones
proteins
was not
matrix
(100 pg)
resolved
appeared
matrix
1).
nuclear
amounts
was 2-3 fold
previously
sufficient
phosphorylated.
A-C, identified
to definitively
Vol. 75, No. 1, 1977
A time
study
regenerating partial
14-16
hours
by ATP was performed
newly
replicated
in regenerating
of specific
reviewed
preceded
hepatectomy.
was not measured
proteins in the the
in
(2),
of the nuclear regulation
isolation,
in vitro
these
liver
is
in
alt 12 hours
of DNA synthesis
important
to note
and that
the extent
which
that
ooour?
the
of dephos-
experiments.
it
is
associated
is
conceivable
matrix
preceding
of DNA synthesis
structure
protem
of phosphorylation
the onset It
DNA in vivo
rat
of the nuclear
a maximum level
This
partial
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of phosphorylation
demonstrated
after
phorylation
importance
onset
hepatectomy.
phosphorylation
matrix
of the
liver
after
Since
BIOCHEMICAL
with
the isolated
that this
and functions
the phosphorylation
event
in the nucleus. of the nuclear
nuclear
could
be of
We have recently protein
matrix
Grant
&13745.
(13).
ACKNOWLEDGEMENTS This
work
was supported
by the National
Cancer
Institute,
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13.
Berezney, R. and Coffey D.S. (1974) Biochem. Biophys. Res. Comm. 60, 14101417. Berezney, R. and Coffey, D.S. (1975) Science 189, 291-293. Kleinsmith, L. (1975) J. Cell Physiol. 85, 459-476. Stein, G.S., Spelsberg, T.C. and Kleinsmith, L. (1974) Science 183, 817824. Allfrey, V.G., Inoue, A., Kain, J., Johnson, E.M. and Vidali, G. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 785-801. Higgins, G.M. and Anderson, R.M. (1931) Arch. Pathol. l2, 186-202. Johnson, E.M., Vidali, G., Littan, V.C., and Allfrey, V.G. (1973) J. Biol. them. 248, 7595-7600. Kleinsmith, L., Allfrey, V.G. and Mirsky, A.E. (1966) Proc. Natl. Acad. Sci. 55, 1182-1189. Munro, H.N. and Fleck, A. (1965) Meth. Biochem. Anal. 14, 113-176. Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. Chen, P.S., Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Weber, R. and Osbom, M. (1969) J. Biol. Chem. 244, 4406-4412. Berezney, R. and Coffey, D.S. (1976) Adv. Enzyme Reg. 2, 63-100.
116
