Download Purification and Characterization of Cytosoi Protein 45/7.8 Present

[CANCER RESEARCH 40. 1623-1629.
0008-5472/80/0040-OOOOS02.00
May 1980]
Purification and Characterization
in Rapidly Growing Hepatomas1
of Cytosoi Protein 45/7.8
Present
Katari S. Raju, Harold P. Morris, and Harris Busch
Department of Pharmacology, Baylor College of Medicine, Houston. Texas 77030 [K. S. P.. H. B.J. and Department of Biochemistry,
Medicine. Washington. D. C., 20059 [H. P. M.]
ABSTRACT
Protein 45/7.8
MATERIALS
(molecular weight x 10~3/isoelectric
point)
was found in the cytosol of several rapidly growing hepatomas
including Morris hepatomas 3924A and 9618A2 and Novikoff
hepatoma. It was not found in Morris hepatomas 7794A and
8999, which have intermediate growth rates, or in the slowgrowing Morris hepatoma 9618A, normal rat liver, 18-hr regen
erating rat liver, or livers of fetal rats. Protein 45/7.8 was
isolated under nondenaturing conditions from Morris hepato
mas 3924A and 9618A2 and Novikoff hepatoma in high purity
by purification in three steps: ammonium sulfate fractionation;
diethylaminoethyl cellulose chromatography; and hydroxylapatite chromatography. The protein was highly purified as shown
by two-dimensional, isoelectric-focusing
sodium dodecyl sulfate:polyacrylamide gels. The amino acid composition of pro
tein 45/7.8 from the three tumors studied was very similar; the
acidic:basic amino acid ratio was 1.4. The NH2 terminus amino
acid of protein 45/7.8 was proline, and its COOH terminus
amino acid was tyrosine. Protein 43/8.1 found in normal liver
is different from hepatoma protein 45/7.8 by amino terminus,
amino acid composition, and tryptic peptides.
INTRODUCTION
Previous reports have demonstrated many biochemical sim
ilarities of slow-growing Morris hepatomas and regenerating
and normal liver (23). There are many similarities between fastgrowing tumors and fetal livers, both of which differ from normal
and regenerating rat liver (21-23). Quantitative and some
possible qualitative differences were found in the mRNA pop
ulation of the Novikoff hepatoma and in normal and regener
ating rat liver (4, 8). In 2-dimensional gel comparisons of
cytoplasmic proteins (7) and nuclear proteins (21, 22) of sev
eral hepatomas with different growth rates, several proteins
were found in some hepatomas but not in nontumorous tissues.
Of these, cytosol protein 64/7.2 which is common to several
hepatomas and whose concentration increased with growth
rate was isolated earlier (14).
The present study reports the purification and partial char
acterization of a cytosol protein 45/7.8 (molecular weight x
10~3/pl) which was found only in rapidly growing hepatomas
and not in medium- and slow-growing hepatomas, normal liver,
18-hr regenerating rat liver, or fetal liver. Inasmuch as it could
be a "marker" for fast-growing tumors (3), it was isolated and
partially characterized.
1These studies were supported by Cancer Research Center Grant CA-10893
awarded by the National Cancer Institute. Department of Health, Education, and
Welfare; the OeBakey Medical Foundation; the Pauline Sterne Wolff Memorial
Foundation; a generous gift from Mrs. Jack Hutchins; and the Bristol-Myers
Fund.
Received May 7, 1979; accepted February 5. 1980
Howard University. College of
AND METHODS
DEAE-cellulose (DE-52) and hydroxylapatite
were pur
chased from Whatman, Inc., Clifton, N. J. and Bio-Rad Labo
ratories, Richmond, Calif., respectively. Ampholines (pH 3.5 to
10) were purchased from LKB Instruments, Inc., Rockville, Md.
All other chemicals were of analytical grade.
Animals. Male Holtzman rats (175 to 200 g) were used as
normals and for implanting Novikoff hepatoma cells. For fetal
livers, pregnant Holtzman rats were sacrificed after 19 days of
gestation. Morris hepatomas 9618A, 7794A, 8999, 9618A2
(maintained in Buffalo rats), and 3924A (maintained in ACI rats)
were used (11 ).
Buffers. Buffer A [50 mM Tris-HCI (pH 7.4) and 5 rriM
MgCI2]; Buffer B (130 mM NaCI, 5 mM KCI, and 8 mM MgCI2);
Buffer C [10 mM Tris-HCI (pH 8.0) and 1 mM 2-mercaptoethanol]; Buffer D [2.5 mM Tris-HCI (pH 8.5) and 1 mM 2-mercaptoethanol]; Buffer E [50 mM potassium phosphate (pH 6.2)];
Buffer F [100 mM potassium phosphate (pH 6.2)]; Buffer G
[200 mM potassium phosphate (pH 6.2)]; and Buffer H [250
mM potassium phosphate (pH 6.2)] were used.
Preparation of Cytosol Proteins. Normal and 18-hr regen
erating livers from 200-g normal male Holtzman rats were
perfused (12) and homogenized in Buffer A containing 1 mM
PMSF2 (1). With fetal livers and Morris hepatomas in which
perfusion could not be carried out, the tissue was rinsed in
Buffer B containing 1 mM PMSF and homogenized in Buffer A
containing 1 mM PMSF. Novikoff hepatoma ascites fluid was
collected. The cells were washed with Buffer B containing 1
mM PMSF, suspended in 10 volumes (w/v) of Buffer A con
taining 1 mM PMSF, and homogenized with a tight pestle. The
homogenate was centrifuged at 27,000 x g for 20 min. The
postmitochondrial
supernatant was further centrifuged at
142,000 x g for 3 hr to remove the microsomes.
DEAE-cellulose Separation. All operations were conducted
at 4°. To the cytosol, solid ammonium sulfate was added to
50% saturation, and the mixture was centrifuged at 27,000 x
g for 15 min. The precipitated residue was discarded. To the
supernatant, solid ammonium sulfate was added to 60% satu
ration, and the mixture was centrifuged at 27,000 x g for 15
min. The precipitated residue was dissolved in Buffer C to a
concentration of 12 to 15 mg protein per ml, dialyzed against
Buffer C, and centrifuged at 27,000 x g for 10 min. Ten ml of
supernatant were loaded onto a DEAE-cellulose column (2.0
x 30 cm) equilibrated with Buffer C. Elution was carried out
with Buffer C, and the flow-through fraction was collected and
concentrated by Ultrafiltration.
Hydroxylapatite Chromatography for Purification of Hep
atoma Protein 45/7.8. The concentrated DEAE-cellulose flow2 The abbreviations
used are: PMSF, phenylmethylsulfonyl
fluoride; pi, isoe-
lectric point.
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1623
K. S. Raju et al.
through fraction was dialyzed against Buffer E and loaded on
a hydroxylapatite column (1x10
cm) equilibrated with Buffer
E. When the bound fraction was eluted with Buffer F, the eluate
contained protein 45/7.8 as the main component on 2-dimensional gels. Following elution with Buffer F, no additional protein
45/7.8 was eluted from the column with solutions of higher
ionic strength.
Quantitation of Protein 45/7.8. Ammonium sulfate fractions
were analyzed by 2-dimensional gel electrophoresis (13). Pro
tein spots were stained with Coomassie brilliant blue and cut
from the gel, the protein was eluted (9, 14), and its absorbance
at 540 nm was determined. For quantitation, protein spot 45/
7.8 was eluted into one tube, and the rest of the protein spots
were eluted into another tube. Protein 45/7.8 was recovered
almost quantitatively in the DEAE-cellulose and hydroxylapatite
purification steps. An overall loss of 12 to 15% of protein
occurred during the isolation procedure. The purification at
each step was determined by protein analysis and by quanti
tative analysis of the spot in the gels.
Purification of Liver Protein 43/8.1.
The concentrated
DEAE-cellulose flow-through fraction (Fig. 2/4) was dialyzed
against Buffer D [2.5 HIM Tris-HCI (pH 8.5) and 1 mw 2mercaptoethanol] and loaded on a DEAE-cellulose column (1
x 10 cm) equilibrated with Buffer D. The flow-through fraction
(protein 43/8.1, the major protein) was concentrated, dialyzed
against Buffer E [50 HIM potassium phosphate (pH 6.2)], and
loaded on a hydroxylapatite column (1x10
cm) that was
equilibrated with Buffer E. The bound fraction was first eluted
with Buffer G and subsequently with Buffer H [250 HIM potas
sium phosphate (pH 6.2)]. The fraction eluted with Buffer H
contained protein 43/8.1 as the main component on 2-dimen
sional gels.
Two-Dimensional
Gel Electrophoresis,
Molecular Weight
Determination and Protein Estimation. Two-dimensional polyacrylamide gel electrophoresis was carried out according to
the method of O'Farrell (13) as modified in this laboratory (7).
Molecular weights for purified proteins were determined by the
method of Weber and Osborn (24). Protein was estimated by
the method of Lowry ef al. (10).
Amino Acid Composition and Terminal Amino Acid Deter
mination. Protein 45/7.8 was hydrolyzed with 5.7 N HCI at
110° for 22 hr and analyzed on a Beckman Model 121-M
amino acid analyzer according to the method of Spackman ef
al. (18). The method of Weiner era/. (25) was used for aminoterminal determination. The amino acid sequence of the NH2
terminus was determined by using an updated Beckman Model
890 B automatic sequencer. The Edman degradation products
were analyzed by gas chromatography, acid hydrolysis, and
subsequent amino acid analysis (17). The COOH-terminal
amino acid was determined after hydrazinolysis at 85°for 28
hr (6).
Peptide Maps. Peptide maps of the tryptic digestion prod
ucts were obtained by using the method described previously
(2). Tryptic digestion was carried out with 1.0 mg of protein in
1 ml of 0.2 M ammonium bicarbonate. A total enzyme:protein
ratio of 1:25 (w/w) was used. Trypsin was added in 2 equal
portions over a 12-hr period. The digests were lyophilized,
dissolved in 25 /¿I
of 30% acetic acid, and applied onto What
man No. 3 filter paper. Chromatography of the peptides was
carried out in butanol:acetic acid:water (4:1:5; v/v/v) for 16 to
17 hr, and electrophoresis was at pH 3.6 (2) at 3000 V for 35
1624
min in pyridine:acetic
mium:ninhydrin
acid:water (1:10:189;
v/v/v).
The cad-
stain was used to visualize the peptides (20).
RESULTS
Comparison of Cytosol Proteins
Fig. 1 presents the 2-dimensional,
isoelectric-focusing
so
dium dodecyl sulfate patterns obtained for the cytosol proteins
of normal liver in comparison with 3 fast-growing tumors, Morris
hepatoma 3924A and 9618A2 and Novikoff hepatoma. Patterns
of the fastest growing tumors, Morris hepatoma 3924A (Fig.
16), Morris hepatoma 9618A2 (Fig. 1C), and Novikoff hepa
toma (Fig. 1D), are quite similar but differ significantly from the
pattern of normal liver (Fig. 1/4). Protein spot 56/8.3 (X)
comigrated with the EF-1 (16) elongation factor of protein
synthesis; it was found in largest amounts in the rapidly growing
Morris hepatomas 3924A and 9618A2 and in Novikoff hepa
toma. The arrowheads point to proteins that are present in
higher concentrations in tumors. The protein spot marked V,
protein 64/7.2 (14), was present in all hepatomas, and its
concentration was highest in fast-growing hepatomas. Protein
spot 45/7.8 is shown by an arrow in the rapidly growing Morris
hepatoma 3924A (Fig. 1B), Morris hepatoma 9618A2 (Fig. 1C),
and Novikoff hepatoma (Fig. 1D). It was not found in normal
liver (Fig. 1/4, broken arrow), fetal liver, 18-hr regenerating
liver, in the slow-growing Morris hepatoma 9618A, or in the
intermediate-growing
Morris hepatomas 8999 or 7794A (7,
14).
Purification of Protein 45/7.8
The first step of purification, ammonium sulfate fractionation,
was based on the finding that protein 45/7.8 was precipitated
mainly in the 50 to 60% ammonium sulfate fractions.
DEAE-cellulose Flow-through Fraction
Most cytosol proteins bound to DEAE-cellulose, but protein
45/7.8 did not. Fig. 2 shows 2-dimensional gel patterns of
proteins of normal liver (A), Morris hepatoma 3924A (B), and
9618A2 (C) of the flow-through fraction of the DEAE-cellulose
chromatograms of the 50 to 60% ammonium sulfate fraction
eluted with Buffer C. The tumor patterns are very similar (Fig.
2, B and C); only 2 major spots were present, including protein
45/7.8 (arrow). In the liver, there was no spot for protein 45/
7.8 (Fig. 2A, broken arrow). Liver fraction mixed with purified
hepatoma protein 45/7.8 showed (Fig. 2D) that hepatoma
protein 45/7.8 is distinct from normal liver protein 43/8.1.
Hydroxylapatite Chromatography
Purification of Hepatoma Protein 45/7.8.
Fig. 3 is a 2-
dimensional gel showing the purity of the protein 45/7.8 eluted
from a hydroxylapatite column with Buffer F. Protein 45/7.8
was completely eluted from the column with Buffer F. The
purification steps and yields of protein 45/7.8 from Morris
hepatomas 3924A and 9618A2 and Novikoff hepatoma are
shown in Table 1.
Purification of Liver Protein 43/8.1. Liver protein 43/8.1,
isolated by hydroxylapatite chromatography as described in
"Materials and Methods," migrated as a single component on
2-dimensional gels.
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Characterization
of Protein 45/ 7.8
Table 1
Fold purification
Purification of protein 45/7.8 from hepatomas
and percentage of recovery were determined as described in "Materials and Methods.
method of Lowry era/. (10), and the 45/7.8
Normal
liver
g)g)Protein
(1 2
Morris hepatoma 3924A (28.5 g)
coveryTotal
0050 cytosol
65Buffer
to 60% ammonium sulfate
55Protein
C eluate of DEAE-cellulose
47hydroxylapatite)Comparison
45/7.8 (buffer F eluate of
Protein content of the fraction was determined by the
spot was cut out of the gels for quantitative protein analysis.
Protein(mg)Protein
(mg)
1110
103
11.4
Protein45/7.8 of Liver Protein 43/8.1
AnalysisTable
by Chemical
1040
68
6.1
0.96
Fold purification
Morris hepatoma 961 8A2 (30.5 g)
Novikoff hepatoma (75
% of re- re-cation5.843148covery100705950Comparison
Proteincation
purifi- % of re-
purifi-
% of
covery
(mg)100
10306.3
13262
18396
1
10.2
100
562
with Hepatoma
ofliver 2 shows the comparison of amino acid composition
protein 43/8.1 and hepatoma protein 45/7.8 from Novi
koff hepatoma and Morris hepatomas 3924A and 961 8A?.
Theamino
acid composition (4 separate preparations and analyses
for
thatNovikoff
each sample) consistently showed in each analysis
ofproline hepatoma protein 45/7.8 has a higher content
liverprotein
by 1.7 mol % and of glycine by 1.5 mol %. The
andleucine
43/8.1 has a higher content of valine (1 .6 mol %)
theprimary
(1 mol %). This result supports the suggestion that
aminoacidstructure of these 2 proteins is different. The
remarkablysimilar
composition of hepatoma protein 45/7.8 is
bothproteins
for the 3 tumors. The most plentiful amino acids for
andglycine.
were alanine, glutamic acid, glutamine, leucine,
phenylalanine,and
The content of cysteine, methionine,
5-dimethylaminonapthalene-1-sulfonyl
histidine was low. By the
beproline chloride method, the NH2 terminus was found to
sequencefor
in hepatoma 45/7.8; a partial NH2 terminus
Pro-Thr-Tyr-Tyr-Ala-Leu.
the first 6 amino acid residues was
liverprotein
The amino terminus was found to be blocked in
5-dimethylaminonapthalene-l-sul-fonyl
43/8.1 , by both the
theEdman
chloride method and by sequence analysis using
Bautomatic
degradation method with a Beckman model 890
betyrosine sequencer. The COOH terminus was found to
43/8.1.(mg)94510910.31.6Fold
in both hepatoma protein 45/7.8 and liver protein
Peptide Maps
The tryptic peptide maps of liver protein 43/8.1 and hepa
toma protein 45/7.8 are shown in Fig. 4, A and ß,respectively.
The tryptic peptide fingerprints of liver protein 43/8.1 con
tained approximately 29 readily visible peptides. The corre
sponding tryptic peptide fingerprints of the hepatoma protein
45/7.8 also contained 30 readily visible peptides as shown in
Fig. 4 (closed, blunt arrowhead); few peptides were similar in
these 2 proteins. Thirteen peptides (Fig. 4B, arrow) were
present in the tumor protein 45/7.8 but not in the liver protein
43/8.1. Twelve peptides (Fig. 4A, V) were present in the liver
protein 43/8.1 but not in the tumor protein 45/7.8.
DISCUSSION
The 2-dimensional gel electrophoresis patterns for the abun
dant cytosol proteins of 3 rapidly growing tumors shown in this
70
60
51
52Table
2Fold
of amino acid composition of liver protein 43/8. 1 with hepatoma
45/7.8Mol
protein
recoveredAmino
of amino acid/ 100 mol
Rat liver protein43/8.1a
45/7.8°6.6
hepatoma protein
acidLysineHistidineArginineAspartic
0.072.5
±0.03C
0.04.9
±0.02
0.068.4
±0.08
acid
±0.2
ThreonineSerineGlutamic0.26.9
5.7 ±0.26
0.2612.2
±0.21
acid
0.13
ProlineGlycineAlanineHalf-cystine
4.1
0.358.50.05
±0.2312.0
0.14
0.721.20.12
0.2
6.0
0.351.0
±0.17
ValineMethionineIsoleucineLeucine
0.124.2
±0.25
0.3210.1
±0.1
±0.04
TyrosinePhenylalaninertAcid/baseNH2
2.9 ±0.06
0.063.0
0.221.4
±0.1
protein
protein
45/7.8b7.42.54.28.0
45/7.
8b7.52.64.08.26.56.811.0
7.4 ±
2.6
4.3
8.4
6.2
7.0
11.5
5.8
10.0
11.2
1.4
4.4
0.9
4.0
9.1
3.1
3.0
±
±
±0.06
±
±
±0.16
±
±
±0.21
±
±
±
±0.26
±
±
6.16.511.4
5.99.311.71.0
5.710.110.51.2
4.71.04.38.2 5.20.94.79.2
2.73.81.4ProlineTyrosineMorrishepatoma
2.82.91.4ProlineTyrosine
1.4Blocked
ProlineTyrosine
terminusCOOH
Tyrosineexperiments.experiments.+
terminusa
4"Average of
Average of 2
c Mean ±
S.E"
Ratio of (GlxNovikoff
Asx):(Lys + His + Arg).Morrishepatoma3924A
study were quite similar and differed significantly from the
pattern of normal liver. Previously, it was found that cytosol
proteins of slow-growing Morris hepatoma 9618A and inter
mediate-growing Morris hepatomas 8999 and 7794A were
similar to that of normal liver and 18-hr regenerating liver (7,
14). These results are in agreement with previous reports of
many biochemical similarities of slow-growing Morris hepato
mas with normal liver and similarities among fast-growing tu
mors (23). It is also apparent from the present and previous
studies from this laboratory (7, 14) that increasing growth rate
is associated with marked alteration in the abundance of spe
cial proteins. The protein spots marked with filled arrowheads,
V (64/7.2), and arrows (45/7.8) are such proteins.
Evidence has been presented that indicates that protein 45/
7.8 is present only in rapidly growing hepatomas, Morris hep
atomas 3924A and 9618A2, and Novikoff hepatoma (Figs. 1
and 2, 8 to D) but not in normal liver (Figs. 1/4 and 2A). In the
50 to 60% ammonium sulfate and DEAE flow-through fractions
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1625
K. S. Raju et al.
in which this protein is concentrated, it was not found in 2dimensional gel maps of fetal liver, 18-hr regenerating liver, or
slow-growing Morris hepatomas 9618A, 8999, and 7794A
(14).
This study describes the isolation and characterization of
protein 45/7.8 from cytosol of Morris hepatoma 3924A and
96182 and Novikoff hepatoma. The isolation procedure devel
oped with the Novikoff hepatoma was satisfactory for each of
these tumors. This protein was highly purified under nondenaturing conditions as shown by its migration as a single dense
spot on 2-dimensional, isoelectric-focusing
sodium dodecyl
sulfateipolyacrylamide
gels. The trailing shape of the protein
45/7.8 on 2-dimensional gel may be due to some partial
deamidation of the protein. Protein 45/7.8 that was isolated
from the 3 tumors had a similar amino acid composition, the
same single NH2-terminal proline, and single COOH-terminal
tyrosine, indicating that it is highly purified. The acidic:basic
amino acid ratio of 1:4 indicates that theoretically protein 45/
7.8 should be an acidic protein. However, the actual pi value
of 7.8 shows that this is an overall basic protein which indicates
that approximately 7 to 8% of the glutamic and aspartic acid
residues are amidated.
When carried through the same purification steps, the liver
sample contained a single major spot with an approximate
molecular weight of 43,000 and a pi value of 8.1 (Fig. 2A).
This protein has a tyrosine COOH terminus, like hepatoma
protein 45/7.8. However, this protein differs from the hepa
toma protein on the basis of molecular weight, pi, and behavior
on hydroxylapatite chromatography (hepatoma protein élûtes
at 50 to 100 rriM ionic strength, whereas liver protein élûtes
at
200 to 250 mM ionic strength), and it has a blocked NH2
terminus. The hepatoma protein had a proline NH2 terminus.
The amino acid composition of hepatoma protein consistently
showed a higher content of proline by 1.7 mol % and of glycine
by 1.5 mol %, and liver protein 43/8.1 showed a higher content
of valine by 1.6 mol % and of leucine by 1 mol %. This suggests
that these 2 proteins are different. The majority of the tryptic
peptides of hepatoma protein are different from liver protein
and vice versa, suggesting that these 2 proteins are different.
The possibility was considered that protein 45/7.8 is an
enzyme or ¡soenzymethat becomes expressed in the neoplastic state (5, 23), but none of the enzymes or isoenzymes
reported thus far have properties similar to those of protein
45/7.8. It remains to be learned whether the expression of
hepatoma protein 45/7.8 is specifically related to special
events in increased cell cycling or to other accelerated reac
tions that occur more rapidly in faster-growing tissues than in
the slower-growing cells (15, 19).
ACKNOWLEDGMENTS
The authors thank R. K. Busch for the transplantation of tumors and Dr. M. O.
J. Olson and K. Guetzow for assistance in some of the experiments.
1626
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Characterization
I50
IOO
80
1A
of Protein 45/ 7.8
—¿
I50
1B
IOO
80
—¿
60
—¿
40
t
8.5
8.0
7.0
60
i
X
.
—¿
40
s
—¿
30
—¿
30
6.0 5.0
8.5 8.0
I
7.0 6.0 5.0
PH
pH
—¿
150
1D
1C
- 100
- 80
-
60
1
I
—¿
40
40
—¿
30
—¿
30
I
8.5
I
8.0
I
7.0 6.0 5.0
pH
I
I
8.5 8.0
7.0
I I
6.0 5.0
pH
Fig. 1. Separation of cytosol proteins on 2-dimensional. isoelectric-focusing sodium dodecyl sulfate:8% gels. In each of the dimensional gel pictures, the ordinate
is approximate molecular weight in thousands as determined by migration of oligomeric mixtures of molecular weight standards (BDH. Gallard-Schlesinger Chemical
Manufacturing Corp.. Carle Place. N. Y.) for sodium dodecyl sulfate:polyacrylamide
gel electrophoresis, and the abscissa is pH range obtained from pH measurement
of sliced one-dimensional gels. The dark haze on the lower one-third of some gels is caused by staining of residual ampholines. Approximately 100 to 120 |ug protein
were separated, x. protein elongation factor 1-<>;filled arrowhead, proteins present in hepatoma in higher concentrations; solid arrow, protein 45/7.8; V, protein 64 /
7.2; broken arrow, position of protein 45/7.8 in liver. This designation is used throughout. A, liver cytosol proteins. B, Morris hepatoma 3924A (rapidly growing)
cytosol proteins; C, Morris hepatoma 9618A2 (rapidly growing) cytosol proteins; D, Novikoff hepatoma cytosol proteins
MAY 1980
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1980 American Association for Cancer Research.
1627
—¿
150
—¿100
—¿
150
2A
—¿
100
2B
—¿
80
—¿
80
—¿
60
—¿
60
co
o
I
I
8.5 8.0
I
I
7.0
PH
—¿
40
—¿
40
—¿
30
—¿
30
i
I
6.0
8.5
5.0
8.0
7.0
6.0
x
5.0
pH
—¿150
2C
—¿
150
—¿
100
20
—¿100
—¿
80
—¿
60
—¿60
r<l
I
o
i—i
—¿40
X
-40
¿
-
5
30
—¿
30
I
I
8.5 8.0
I
7.0
pH
6.0
5.0
I
I
I
8.5
8.0
7.0
6.0
5.0
PH
Fig. 2. Separation of DEAE-cellulose flow-through proteins on 2-dimensional, isoelectric-focusing
sodium dodecyl sulfate:gels. Approximately 30 to 35 fig of
protein were separated (see Fig. 1) A, liver cytosol DEAE-cellulose flow-through; 8, Morris hepatoma 3924A cytosol DEAE-cellulose flow-through; C. Morris
hepatoma 9618A2 cytosol DEAE-cellulose flow-through; D, liver cytosol DEAE-cellulose flow-through (A) mixed with 10 fig of purified hepatoma protein 45/7.8.
1628
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1980 American Association for Cancer Research.
Characterization
of Protein 45/7.8
—¿ 150
—¿ 100
—¿80
J
—¿60
¿èia6È*1*
-"i°ti
-12-.
-.<•',•
—¿40
"
jo
-
—¿30
I
I
I
I
I
8.5
8.0
7.0
6.0
5.0
I**
i:
-'"
«k
:
«
-
U
JU
u
US
LU
Chrom
t)
Chrom
PH
Fig. 3. Two-dimensional,
isoelectric-focusing
sodium dodecyl sultate:gel electrophoresis
of purified protein 45/7.8
(10 /jg).
Fig. 4. A. tryptic peptide maps of liver protein 43/8.1 ; ß.similar maps of Novikoff hepatoma protein 45/7.8. One mg of each protein was digested simultaneously
with trypsin and chromatographed simultaneously. V, peptides in the liver but not in the hepatoma protein (A); arrows, peptides which are present in the hepatoma
but not in the liver protein (ß);closed blunt arrowhead, peptides with similar mobility.
MAY 1980
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1629
Purification and Characterization of Cytosol Protein 45/7.8
Present in Rapidly Growing Hepatomas
Katari S. Raju, Harold P. Morris and Harris Busch
Cancer Res 1980;40:1623-1629.
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