Download Isolation and Purification of RP2-L, a Nuclear Protein Fraction of the

Isolation and Purification of RP2-L, a Nuclear Protein
Fraction of the Walker 256 Carcinosarcoma*
HARRISBUSCH,LUBOMIRS. HNILICA,SU-CHENCHIEN,! JOSEPHR. DAVIS,Õ
ANDCHARLESW. TAYLOR
(Departments
of Biochemistry
and Pharmacology,
Baylor University
College of Medicine,
Houston,
Texas)
SUMMARY
One hour after the injection of 5 fie. of L-lysine-U-C14 into each of a group of rats
bearing the Walker 256 carcinosarcoma, the acid-soluble proteins were extracted from
nuclear preparations of the tumor. The proteins of these extracts were chromatographed on carboxymethylcellulose, with formic acid as the eluting agent.
Rechromatography of 150 mg. of RP2-L1 on carboxymethylcellulose resulted in
further purification of the proteins as determined by starch gel electrophoresis, amino
acid analysis, N-terminal amino acids, and increase in specific activity. Since lysine
comprises 14 per cent of the total amino acid residues, the proteins were classifiable
as "slightly lysine-rich" histones. As the proteins were purified, the percentage of Nterminal proline increased. From the amino acid composition, sedimentation velocity,
and diffusion constant, the molecular weight of the proteins in purified RP2-L was
found to be 33,000. Starch gel electrophoresis of the purified product reveals the pres
ence of one major and five minor protein bands, emphasizing the need for further im
provement in methods for subfractionation of the bands.
Recent studies from this and other laboratories
(3, 4, 6, 15, 22, 26) have shown that an important
pathway in the utilization of amino acids by transplantable rat tumors is the biosynthesis of nuclear
proteins, particularly histones, or chromosomalbound nuclear proteins. In the Walker tumor and
other tumors 30-55 per cent of the isotope of
radioactive lysine which was incorporated into
nuclear proteins was found in a cationic nuclear
protein fraction tentatively coded as RP2-L (10,
11). This fraction was the second radioactive peak
eluted by a gradient increasing to l N formic acid
from carboxymethylcellulose columns to which
solutions of prelabeled acid-soluble nuclear pro
teins had been added. This peak, found initially
* These studies were supported in part by grants from the
U.S. Public Health Service, the Jane Coffin Childs Fund for
Medical Research, and the American Cancer Society.
t Predoctoral Trainee in Pharmacology.
} Postdoctoral Fellow of the American Cancer Society.
Present address: Stritoli College of Medicine, Chicago, Illinois.
1RP2-L was defined in previous studies as the second radio
active peak emerging from carboxymethyloellulose columns
when a gradient increasing to l N formic acid was added; in
these experiments L-lysine-U-C" was used as the tracer (10,
11).
Received for publication January 20, 1962.
in the Walker tumor, was later shown to be present
in chromatograms of the Jensen sarcoma, FlexnerJobling carcinoma, the Ehrlich ascites tumor, and
Sarcoma 180, as well as a human malignant mela
noma (11). RP2-L was not found in functionally
active or growing nontumor tissues, such as re
generating rat liver or embryonic rat tissues.
Later studies indicated that RP2-L was bound to
DNA in the Walker tumor and, hence, was de
scribed as a "histone" fraction (7).
The possibility that the components of this
peak might represent proteins which were uniquely
formed in the tumors (1, 9, 11) led to the present
studies on isolation and purification of this peak
and attempts to characterize the components in
terms of amino acid composition, N-terminal
amino acids, and molecular weight. In early stud
ies on the proteins of the nucleus of tumor cells,
it was found that each gram of tumor (wet weight)
contained approximately 13 mg. of acid-soluble
nuclear proteins and that RP2-L comprised ap
proximately 14 per cent of the total weight. Since
studies of the properties indicated above require
30-50 mg. of purified protein, initial experiments
were carried out on the preparation of large
amounts of RP2-L.
637
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638
Cancer Research
MATERIALS AND METHODS
Animals.—The animals used in these experi
ments were male rats weighing 180-220 gm.,
obtained from the Holtzman Rat Company, Hous
ton, Texas, and fed ad libitum on Purina Labora
tory Chow. The tumor studied was the Walker
256 carcinosarcoma. Each rat was given inocula
tions in eight subcutaneous sites which developed
approximately 6-10 gm. wet weight of tumor per
animal. One hour after injection of 5 /LÕC.
of Llysine-U-C14, the animals were anesthetized and
exsanguinated;2 the tumors were rapidly excised
and dissected free of any hemorrhagic or necrotic
tissue in the cold (4°C.). In these experiments,
60-200 gm. of tumor were used.
Extraction of cationic nuclear proteins.—In ini
tial experiments, nuclear preparations were ob
tained by the method previously described (4, 5).
Since essentially the same results were obtained
with the 600 X g precipitate (3), this simpler
method was used in studies with larger quantities
of tumors. The acid-soluble nuclear proteins were
extracted with 10 ml. of 0.25 N HCl/gm tissue in
the cold for 30 minutes with stirring. The extract
was then dialyzed against 0.005 M acetic acid.3
The sample was clarified by centrifugation prior
to chromatography.
Chromatograph]/.—Samples ranging from 1 to
4 gm. of the acid-soluble nuclear proteins were
chromatographed on carboxymethylcellulose. A
slurry of Selectacel CM-S (Brown Company, Ber
lin, New Hampshire) with a capacity of 0.9 meq/
gm was made by stirring 250 gm. of adsorbent in
10.5 1. of distilled water for 1 hour; 0.5 N NaOH
(approximately 375 ml.) was added to bring the
pH to 10.0. The slurry was blended in a Waring
Blendor for 1 minute and immediately poured
into glass columns which were 48 cm. in length
and 9 cm. in diameter. The adsorbent was packed
with air pressure (15 p.s.i.) until a column 45 cm.
in height was obtained. Eight liters of 0.05 M ace
tate buffer at pH 4.0 were passed through the col
umn with the aid of air pressure (10 p.s.i.); the
final effluent pH was 4.0. The mixing chamber, an
1In early experiments, 40 ftc. of L-lysine-T monohydrochloride (Volk Radiochemical Company, Chicago) with a spe
cific activity of 22 mc/mmole was injected intraperitoneally
into each of ten tumor-bearing rats. Because of experimental
problems in accurate determination of the specific activities of
proteins labeled with tritium by the plating technics employed,
all experiments in the present report were carried out with pro
teins obtained from rats given injections of 5 /nc. L-lysine-UC».
3To minimize enzymatic hydrolysis of the proteins, dialysis
was carried out at pH 8.O.Evidence that substantial enzymatic
degradation did not occur is the absence of fast-moving bands
on starch gels and the similarity of fractions obtained under
these conditions in the text and when DFP was added to the
original extracts.
Vol. 22, June 1962
Erlenmeyer flask, was completely filled with 3 1.
of distilled water; 4 1. of l N formic acid were ini
tially added to the reservoir. The flow rate was
maintained at 6.0 ml/min by a Minipump®and
by adjusting an Ultramax®valve at the bottom
of the glass column. Effluent fractions were col
lected every 4 minutes with the aid of a fraction
collector. Essentially identical chromatograms were
also obtained with columns of the same height
and a diameter of 5.5 cm.; the mixing flask and
flow rate were modified in proportion to the crosssectional area of the column (10, 11).
Rechromatography. —For rechromatography,
the fractions comprising the various peaks were
pooled, dialyzed against 0.005 Macetic acid in the
cold for 16 hours, and lyophilized. The dry protein
(150 mg.) was dissolved in 10 ml. of cold 0.005 M
acetic acid and was added to columns of Selec
tacel CM-S, 15 cm. in height and 2 cm. in diame
ter. These columns were prepared in the same
manner as described above but contained 9 gm.
of adsorbent in 600 ml. distilled water; the mixing
flask contained 500 ml. of distilled water, and the
reservoir contained 1 1. of l N formic acid. The
flow rate was 0.5 ml/min. Fractions were collected
every 12 minutes.
Determination of protein and radioactivity.—
Proteins in the eluate were detected spectrophotometrically at 280 m¿t.Aliquots of each fraction
were plated as previously described (10), and the
radioactivity was determined in an automatic
gas-flow counting apparatus (Nuclear-Chicago).
N-terminal amino acid analysis.—Samples of
the various fractions (20—30mg.) were reacted
with l-fluoro-2,4-dinitrobenzene for 12-14 hours
at room temperature, according to the technic of
Sanger (23) as modified by Phillips (19). Hydrol
ysis of the DNP-protein was carried out at 100°
C. for 4£hours in 11.4 HC1. The DNP-amino
acids were identified by paper chromatography;
the moving phases were 2-butanol saturated with
0.05 M phthalate (pH 6) and 1.5 M phosphate at
pH 5 (17). For quantitative determinations, the
spots were eluted with warm water (40°C.) and
measured as described by Fraenkel-Conrat et al.
(12).
Amino acid analysis.—-The protein samples
were hydrolyzed in sealed tubes with 5.7 N HC1
at 110°C.for 22 hours (8). Quantitative assays
for amino acids were made with the aid of an auto
matic amino acid analyzer (25); tryptophan was
determined after alkaline hydrolysis of the pro
teins.4 The values for serine and threonine were
4In the alkaline hydrolysis, approximately 40 per cent de
struction of tryptophan was found when known quantities of
tryptophan were added to protein hydrolysates. Less than 0.5
per cent of tryptophan was found in samples of RP2-L, and,
hence, the values are not included in the tables.
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BUSCHet al.—RP2-L, a Historie Fraction from Tumors
corrected 10 per cent for destruction during acid
hydrolysis (20). The number of amino acid resi
dues per minimal molecular weight was obtained
by dividing the molar fractions by that number
which gave the closest approximation to whole
numbers for the various amino acids. From this
value and the molecular weight determined by
ultracentrifugation,
the number of amino acid
residues was calculated to the nearest integer per
mole of protein.
Starch gel eledrophoresis.—Twoprocedures were
employed for starch gel electrophoresis of the
cationic nuclear proteins—i.e., vertical electro
phoresis at various pH ranging from 2 to 5 (18)
and horizontal electrophoresis at pH 2-3 (17). In
some of these experiments, the gels were cut longi
tudinally in half, and the upper halves were
stained to show the positions of the proteins. The
lower halves were cut into a number of segments
corresponding to the protein bands, and the
proteins were eluted with 0.25 N HC1. The
small amount of solubilized starch was pre
cipitated with ethanol sufficient to make a final
concentration of 40 per cent. The sample was
centrifuged at 30,000 X g for 20 minutes, and the
supernatant protein solution was used for deter
mination of C14and protein concentration in each
of the bands, as well as for amino acid analysis of
some of the bands.
Sedimentation velocity studies.—Measurements
of sedimentation constants of preparations of
RP2-L were carried out in a Spinco Model E ana
lytical ultracentrifuge at 59,780 r.p.m. The pro
teins were in solution in acetate buffers at pH 5,
T/2 0.2. The temperature of the rotor was main
tained at 20°C.
Diffusion measurements.—Reproducible diffu
sion constants were obtained by determination of
diffusion of protein boundaries in the Spinco
Model H electrophoresis apparatus (13). Diffusion
constants were also estimated from the boundary
spreading observed in the ultracentrifuge and cal
culated by the maximal ordinate-area method (14).
The buffer used was the same as that for the sedi
mentation studies.
Molecular weight.—The molecular weights of
the proteins were calculated from the Svedberg
and Pedersen equation (24). The partial specific
volume5 was assumed to be 0.74.
RESULTS
Chromatography of the initial extract.—-Thechromatographic pattern for the acid-soluble nuclear
proteins of the Walker tumor obtained on a pre
paratory column is shown in Chart 1. Protein peak
6 For most proteins, partial specific volume has ranged be
tween 0.70 and 0.78 ml/gm (34).
639
B, the major protein peak eluted with a gradient
increasing to l N formic acid, was found in maxi
mal concentration between fractions 270 and 350.
The radioactive peak, designated as RP2-L, was
found between fraction 270 and 450; this peak
followed protein peak B. Another radioactive
peak, designated as RP1-L, was eluted before pro
tein peak B. For further analyses, the contents of
tubes numbered 270-325 were pooled and desig
nated as protein peak B; the contents of tubes
numbered 326-450 were pooled and designated
as crude RP2-L. Table 1 presents the distribution
250
150
WALKER 256 CARCINOSARCOMA
PREPARATORY CHROMATOGRAM
E280
200
100
ISO
100
50 Z
90
100
200
300
4OO
500
FRACTION NUMBER
600
TOO
CHART1.—Chromatographie pattern of radioactivity and
protein concentration for the whole acid extract of nuclei of 400
gm. of Walker tumor. The carboxymethylcellulose columns
employed were 9 cm. in diameter and 45 cm. in height. Radio
activity and protein concentration were assayed in every fifth
effluent fraction, and hence points are omitted from the
graphs. The conditions for chromatography are described in
the text. The data are averages of two experiments.
of protein and radioactivity in various fractions
obtained by chromatography of acid-soluble nu
clear proteins of the Walker tumor. RP2-L con
tained approximately 35 per cent of the C14and
32 per cent of the weight of proteins recovered.
The specific activity of RP2-L was greater than
that of the other fractions when the specific ac
tivity was determined as counts/min/mg protein.
Rechromatography.—Chart 2 presents the chromatographic pattern obtained by rechromatography of crude RP2-L on carboxymethylcellu
lose, when similar conditions of elution were em
ployed (see "Materials and Methods"). Following
a small "breakthrough" peak, a single protein
peak was eluted by a gradient increasing to l N
formic acid with a maximum at fraction 95. The
distribution of isotope in the chromatogram in
dicates that at least two protein fractions were
eluted by a gradient increasing to l N formic acid,
and another by 8 N formic acid. Table 2 presents
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640
Vol. 22, June 1962
Cancer Research
the results of chromatograms
carried out under
these conditions and shows that each of the frac
tions has been considerably
enriched in radio
activity by comparison with the fractions obtained
in the initial chromatography.
The separation
between peak B and RP2-L was more difficult to
RECHROMAT06RAPHY
0120
0060
IOO
200
300
FRACTION
NUMBER
40O
CHART2.—Chromatographie pattern of radioactivity and
protein concentration for 150 mg. of crude RP2-L on carboxymethylcellulose columns 2 cm. in diameter and 15 cm. in
height. The conditions for chromatography are described in
the text. Fractions B, RP2-L, and C include fractions num
bered 76-100, 100-160, and 160-220, respectively.
define in this chromatogram
(Chart 2). About 85
per cent of the total isotope was eluted in essential
ly a single peak which was divided arbitrarily into
fractions B, RP2-L, and C, which included frac
tions 76-100, 100-160, and 160-220, respectively.«
Table 2 indicates that the specific activity of RP2L is not so great as that of peak E in terms of
counts/min/mg.
When the specific activities were
determined on the basis of counts/mi n//¿mole of
lysine, the values for peak E, B, RP2-L, and orig
inal extract were 1300, 800, 800, and 650, re
spectively.
Starch gel electrophoresis of the fractions obtained
from initial
and rechromatography.—Figure
1
shows that the initial preparations of RP2-L con
tained eight reproducible
bands on starch gel
electrophoresis at pH 2.3 (17). Peak A contained
one band which did not move and a diffuse protein
8It is not probable that all the proteins of the fractions ob
tained by rechromatography of RP2-L are identical to those
obtained initially. The high specific activity of peak E suggests
that other proteins are appearing in this region. However, the
mobility in starch gel for both the proteins of peak B and peak
E obtained by rechromatography was similar to that of the
proteins obtained in the initial chromatograms.
TABLE 1
SPECIFICACTIVITIESANDRECOVERY
OFPROTEINSIN
LARGE-SCALE
CHROMATOGRAPHIC
EXPERIMENTS
The conditions for chromatography are described in the text. The table presents the aver
ages of recoveries of various fractions and the percentages of the total radioactivity and weight
recovered in the various fractions. In these experiments recovery of proteins from the columns
ranged from 30 to 40 per cent. The data are averages of two experiments. The results for indi
vidual experiments are presented in parentheses. Specific activities are counts/min/mg lyophilized protein.
DO.OriginalAB2
Fraction
mg.4.7(3.9,5.5)14
cent total
counts2.6(2.2,3.0)12.6
cent total
(counts/min/mg)530
580)283
(480,
E or 3Mg.36
39)103
(33,
19)32
(9,
114)252
(92,
(211, 293)
(29, 35)
334 (242, 390)Per 39.7(39,40.3)Per
390)446
(176,
14.1)34.7(34,35.4)
(11.0,
572)580
(320,
(572, 587)
36.6 (35,38.2)S.A. 417 (400, 445)
TABLE 2
SPECIFICACTIVITIESANDPER CENTTOTALRECOVERY
ON RECHROMATOGRAPHY
OFCRUDERP2-L OBTAINEDFROMLARGE-SCALE
CHROMATOGRAPHY
The conditions for the experiment are presented in the text. The table presents the averages
of recoveries of various fractions and the percentages of the total radioactivity and weight re
covered in the various fractions. The data are averages of two experiments. The results for
individual experiments are presented in parentheses. Specific activities are counts/min/mg
lyophilized protein.
Fraction
no.B
mg.18.5
cent total
counta16.4
cent total
(11.6, 16)
RP2-L
33.2(26.5,40)
14.5 (14, 15)
CE
or 3Mg.13.8
12 (11, 13)Per
(17.9, 19)
45 (41, 49)
20 (18.4, 21.6)
16.5(13.5,
19.5)Per
(15.3, 17.2)
41.5(40,43)
16 (14, 18)
22 (21, 23.9)S.
A.
(counts/min/mg)710
(700, 720)
855 (830, 880)
648 (595, 700)
1010 (980, 1032)
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BUSCHet al.—RP2-L, a Historie Fraction from Tumors
area which was indistinctly defined. Peak B con
tained bands which moved farther than those of
crude RP2-L (Fig. 1), suggesting either that some
denaturation
of protein occurred, that some of the
protein was freed from components
which im
peded electrophoretic
mobility, or that B was en
riched in fast-moving components.
The electro
phoretic pattern for RP2-L contained one very
heavy band and several smaller bands which were
more diffuse and less intense. RP3-L or peak E
contained five slow-moving bands, of which the
most marked was that which moved the least into
the starch gel and approximated
the mobility of
slow-moving bands of the original starting mate
rial. These data along with the data indicated be
low with regard to the N-terminal amino acids
indicate that purification
of RP2-L has been
achieved but further procedures are required for
641
jected to re-electrophoresis,
a single band was
found in the same region (Fig. 2). The results of
analytical studies on amounts and specific activi
ties of proteins in various fractions of the gels are
shown in Table 3. The band coded as RP2-LY
contained the largest amount of protein with the
highest specific activity of the various fractions
studied. Tentatively,
it would appear that this
Jraction contains the main protein components of
RP2-L. The analytical data on amino acids (v.i.)
support this suggestion.7
Amino acid analysis.—The amino acid analyses
for a number of fractions are shown in Table 4. By
the nomenclature employed by Johns et al. (17),
RP2-L would be classified as a slightly lysine-rich
histone fraction (F2), since lysine comprises ap
proximately 14 per cent of the total amino acids
present. Glutamic and aspartic acid comprised
TABLE3
RECOVERY
OFRADIOACTIVITY
ANDPROTEIN
FOLLOWING
STARCH
GELELECTROPHORESIS
Two to 4 mg. of protein were subjected to electrophoresis on starch gel at pH 5.0. At the end of the run,
gels were cut in half. In one half, the protein was stained with Amido Black 10B to position the bands. The
bands were cut out of the other half, as described in the text, and the protein and radioactivity were deter
mined. Average data for two experiments are presented. Actual values are shown in parentheses. Specific
activities are counts/min/mg lyophilized protein.
Band1234567CodeRP2-LXRP2-LYRP2-LZMg.
protein0.24(0.22,0.25)0.30
protein6.0(5.3,6.6)7.3
cent total
protein213
286)249
(140,
cent total C"
recovered3.4(2.8,
4.0)5.2(3.9,6.4)10.4(8.2,
(0.27,0.32)0.5
(7,7.6)13.0(10.5,
318)282
(180,
(0.4,0.6)1.92
15.4)48.3
313)417
(250,
12.6)60
1.92)1.02
(1.92,
(46.2,50.4)25.5
433)291
(400,
(57,62.8)21.2
(0.97, 1.06)Per
(20.2, 22.2)
(25.4,25.5)Counts/min/mg
(280, 303)Per
improvement
of the purity of the products ob
tained thus far. Although the purification was not
completely satisfactory, the gel patterns show that
purified RP2-L lacks some of the faster moving
bands of peak B and some of the slower moving
bands of RP3-L.
Starch gel electrophoresis at pH 5.O.—The con
ditions employed by Neelin and Neelin (16) were
used in an effort to obtain sufficient protein for
direct amino acid analysis and determination
of
specific activity of the protein in the bands. The
electrophoretic pattern is shown in Figure 2. Only
five bands were noted, one very close to the origin,
a second faint band which moved at an inter
mediate rate, and three rapidly moving bands.
The three fast-moving bands were referred to as
RP2-LX, RP2-LY, and RP2-LZ, of which RP2LZ moved the farthest. The RP2-LY band was
the largest of all the fractions subjected to starch
gel electrophoresis.
When this fraction was ex
tracted from several similar experiments and sub-
approximately
13 per cent of the total amino acids,
and arginine comprised 8 per cent of the total
amino acids. These data suggest that the RP2-L
peak is different in amino acid composition from
the F2 peak obtained by Johns et al. from calf
thymus (17), but, since both groups of proteins
are mixtures, it is difficult to establish whether the
differences represent the presence of different pro
teins, or different compositions within the mixture.
Because of the smaller amount of arginine in this
peak, the lysine/arginine
ratio is 1.71 compared
with the 1.34 found for calf thymus (17). On the
whole, the similarities of the amino acid analysis
of this fraction to the slightly lysine-rich fraction
from calf thymus are greater than the differences.
Fractions A, B, and RP3-L contained less lysine
and arginine than the RP2-L fraction. Peak B con
tained more aspartic acid, leucine, and histidine
7Similar studies were not carried out with bands obtained
by electrophoresis at pH 2.3 because of the multiplicity and
proximity of the bands.
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Cancer Research
642
than RP2-L. RP3-L contained more aspartic acid,
glutamic acid, and leucine than RP2-L. The low spe
cific activity of peak B and the relatively large amount of histidine suggested the possibility of conCHROMATOGRAPHY
OF RAT GLOBIN
0.240
0.120
8N
so
IOO
FRACTION
ISO
NUMBER
A
200
CHART3.—Chromatographie pattern for 150 mg. of rat
globin on carboxymethylcellulose columns. The conditions for
chromatography were the same as those described for Chart 2,
with the exception that a gradient of 8 N formic acid was
begun at fraction 170.
Vol. 22, June 1962
tamination by proteins of the blood such as globin.
Accordingly, the amino acid analysis, Chromato
graphie elution pattern, and N-terminal amino
acid analysis were determined for rat globin pre
pared by the method of Rossi-Fanelli and An
tonini (21). The amino acid analysis which is
presented for comparison in Table 4 shows that
the arginine content is low and the histidine con
tent is high by comparison with RP2-L and the
whole acid extract. The high level of aspartic
acid, leucine, and phenylalanine support the pos
sibility that peak B is contaminated with globin.
Evidence that proteins other than globin and RP2L are present in peak B emerges from the content
of glutamic acid, which is higher in peak B than
in either rat globin or RP2-L; other proteins, con
taining high concentrations of glutamic acid, must
be present. The Chromatographie pattern (Chart
3) and the end-group analysis (v.i.) also support
the possibility that globin is present in peak B.
The amino acid analysis for the band coded as
TABLE 4
AMINOACIDANALYSES
OFVARIOUS
FRACTIONS
OBTAINED
BYCHBOMATOGRAPHY
ANDRECHROMATOGRAPHY
OFACID-SOLUBLE
NUCLEAR
PROTEINS
OFTHEWALKER
TUMOR
The table presents the percentages of total moles of amino acids recovered by chromatography of protein hydrolysates on
Spinco automatic amino acid analyzer. The values are averages of two to five analyses on three separate biological experimeDts.
The ranges are presented in parentheses.
PeakAlanineArginineAspartic
Extract9.6(9.2-9.9)5.6(5.5-5.7)8.6(8.5-8.6)05(0.1-0.9)12.3(11.4-13.2)7.9(7.8-7.9)3.0(2.9-3.1)3.8(3.6-3.9)8.7(8.6-8.7)10.4(10.2-10.5)0.7(0.6-0.8)3.1(3.0-3.3)5.4(5.4-5.5)6.6(6.6-6.6)5.7(5
acid£
CystineGlutamic
3(9.8-12.8)7.8(7.3-8.4)2.1(1.8-2.5)4.2(4.0-4
acidGlycineHistidineIsoleucineLeucineLysineMethioninePhenylalanineProlineSerineThreonineTyrosineValineOrigina]
4)8.5(8.4-8.5)9.3(7.8-10.9)0.6(0.4-0.8)3.4(3.0
6)A9.5(8.4-10.7)4.6(4.4-4.9)8.8(8.0-9.5)0.3(0.2-0.5)11.2(10.0-12.4)7.5(7.2-7.9)3.1(2.4-3.8)4.5(4.1-4.9)9.4(8.8-10.0)10.0(
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BUSCHet cd.—RP2-L, a Histone Fraction from Tumors
RP2-LY is also presented in Table 4. The analysis
for leucine and methionine differed from those of
purified RP2-L, but the analyses for most of the
amino acids were essentially identical. The band
coded as RP2-LZ contained more arginine, gly
cine, and methionine and less lysine, serine, and
valine than the corresponding RP2-LY band.
N-terminal amino acids.—The results of Nterminal amino acid analyses for the various frac
tions isolated from the Walker tumor are presented
in Table 5. In the crude extract, ten N-terminal
amino acids were found. The lack of equality in
quantity indicates that it is unlikely that any
single protein species containing two end groups
643
amino acids in this peak would appear to be proline and alanine, with lesser amounts of valine and
other amino acids. A correlation between these
data and those of starch gel electrophoresis at pH
5 exist when data on percentages of protein are
compared with the percentages of N-terminal
amino acid. Proline comprised 48.6 per cent of
total N-terminal amino acids, and RP2-LY com
prised 48.3 per cent of the total protein recovered
from the gel. In addition, alanine comprised 33 of
the total N-terminal amino acids, and RP2-LZ
comprised 25.5 per cent of the total protein. Valine
comprised 9.1 per cent of the N-terminal amino
acids and RP2-LX comprised 12.9 per cent of the
TABLE 5
N-TERMINALAMINOACIDSIN PROTEINSOFTHEVARIOUS
FRACTIONS
RECOVERED
BYCHROMATOGRAPHY
ANDRECHROMATOGRAPHY
OFACID-SOLUBLE
NUCLEARPROTEINSOFTHE WALKERTUMOR
The conditions used were those employed by Phillips (17). The table presents average values of from three to
five determinations. The ranges are shown in parentheses. The values are percentage of total moles recovered.
PeakAlanineAspartic
Purified33.3(27.1-40.7)2.4(2.2-2.7)2.9(0.0-5.3)0.9(0.0-2.8)48.6(42.9-54.2)2.4(0.0-4.1)9.1(7.4-10.7)R
Crude32.1(27.7-37.2)4.6(4.0-5.5)2.5(2.3-2.8)2.4(2.9-4.3)3.9(2.3-7.0)28.2(21.9-31.9)2.4(1.6-3.8)0.8(0.0-2.3)22.3(19.4-2
extract32.6(30.9-33.6)8.8(6.6-11.5)4.0(2.0-6
B27.0(23.5-30.4)5.0(4.9-5.1)6.7(4.5-8.8)8.2(6.4-10.0)2.8(2.1-3.5)2.2(0.0-4.59.4(7.9-10.9)4.4(0.5-5.5)1.0(0.6-1.3)33.8(28.7-39.0)RP2-L
4(36.0-46.8)5.3(4.6-6.0)5.0(1.3-7.06.1(5.2-7.1)2.6(0.0-6.2)0.0(0.0-2.0)22.8(20.5-2
andglutamic
acid
acidGlycineLeucine
.3)5.6(3.0-8.8)4.8(1.9-7.5)0.6(0.0-1
andisoleucineLysinePhenylalanineProlineSerineThreonineValine
9)17.6(13.1-21.0)13(0.0-3.8)1.6(0.0-4.7)23.0(18.8-25.5)Peak
andmethionineWhole
on two separate peptide chains accounts for the
presence of any two of the N-terminal amino acids
found. In the whole acid extract, alanine was the
major N-terminal amino acid and, together with
proline and valine, comprised almost three-fourths
of the N-terminal amino acids present.
In peak B, the distribution of N-terminal amino
acids was similar to that of the whole extract, al
though more glycine, leucine, serine, and valine
and less proline, lysine, and aspartic acid were
found. In RP2-L, considerably less aspartic acid,
glycine, leucine, lysine, phenylalanine, and valine
were found by comparison with the whole extract
or with peak B. Although the percentage of alanine
as an end group was not markedly different in
RP2-L from the values for the whole extract, the
values for proline were progressively increased as
the RP2-L was purified. The major N-terminal
total protein recovered. These correlations sug
gest that the major component of RP2-L is pro
tein in which the N-terminal amino acid is proline.
A direct analysis of N-terminal amino acids on
band RP2-LY has not been possible because of
the low recovery of protein from the gels.
PHYSICALCONSTANTS
Table 6 presents the results of determinations
of the molecular weight and other physical con
stants for RP2-L. A homogeneous peak was ob
served on ultracentrifugation. The average molec
ular weight of the proteins of RP2-L is approxi
mately 33,000 on the basis of physical constants
and the amino acid analysis.
DISCUSSION
The proteins containing the radioactivity of
peak RP2-L have been purified by rechromatog-
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644
Cancer Research
Vol. 22, June 1962
raphy on carboxymethylcellulose. The product
consists of a mixture of proteins of high specific
activity which are of the general group denoted as
"slightly lysine-rich" histones (16, 17). One major
band and several minor bands were noted on
starch-gel electrophoresis of the product. The Nterminal amino acid of the major proteins is
probably proline, as suggested by the increasing
concentration of this N-terminal amino acid as
the purity of the proteins increased. One of the
acids. It is not possible to state whether the in
dividual proteins are qualitatively different from
those found in nuclei of normal cells or whether
other factors account for the differences which
have been found. It is possible that the proportion
of the same proteins differs from that found in
normal nuclei. It is also possible that the proteins
with which histones may complex in nontumor
tissues are reduced in amount in tumor tissue and,
hence, the proteins of RP2-L emerge earlier in the
chromatograms than do corresponding proteins of
TABLE6
nontumor tissue.
The critical problem confronting such studies is
PHYSICAL
CONSTANTS
ANDMOLECULAR
WEIGHTFORRP2-L
the need for methods for improvement of the
fractionations within this group of proteins. Al
The methods for determination of the sedimentation and
diffusion constants are described under "Materials and Meth
ods." The data are averages of two experiments. The diffusion though it would seem that the simplest approach
and sedimentation constants were determined with solutions to this problem would be the development of
containing 0.5 per cent and 1 per cent protein in each case. starch-gel technics employing large-scale proce
The molecular weight was derived with the aid of the diffusion dures, efforts in this direction in this and a number
constant determined in the Tiselius apparatus because of the
of other laboratories have been disappointing,
greater accuracy of this method.
thus far, in the resolution of the proteins in the prod
ucts obtained. Another possibility which could
ConstantaDSC,
be anticipated is the development of ion exchangers
with improved resolving power; the possibility
w (by free diffusion in Tiselius
cm/sec
apparatus)DM,
exists that weaker exchangers with phenolic hycm/sec1.32X10-'Vsec32,70032,390
2.68X10~7sq
droxyls might be useful for this purpose.
ultracentrifugation)Sa,,
w (by
In view of the difficulties in subfractionation
WAverage
of these proteins by Chromatographie technics,
recent studies have been directed toward the use
molecular weight by physi
constantsAverage
cal
of a combination of chemical and Chromatographie
technics (14, 16). Starch gel electrophoresis of
molecular weight from
fractions indicates that further purification of the
amino acid compositionRPi-L3.32X10-'sq
histones may be achieved by this combined pro
cedure. At the moment, one fraction which is ap
problems in the detection of dinitrophenylproline
parently of a higher order of purity has been ob
is its relatively great destruction on hydrolysis of tained in this laboratory,8 but further studies are
the protein, and hence there is a need to employ a necessary to establish the purity unequivocally.
large amount of protein to find the end-group;
The need for structural analysis of these pro
teins is apparent. If neoplastic cells contain pro
this fact accounts for earlier reports from this lab
oratory which did not indicate the presence of teins which are different from those of other tis
proline as an N-terminal amino acid (2). The pro
sues, their structure must be determined not only
teins of RP2-L are moderately small in view of for comparative purposes but also for the purpose
the molecular weight of approximately 33,000 of directing chemotherapeutic endeavors. It is
possible that structural differences could be uti
which has been obtained. The primary structure
should be analyzable by "fingerprinting" and lized to suggest the synthesis of polypeptide ana
other technics once the proteins are isolated in logs containing either cytotoxic groups or amino
acids in altered sequences. Such compounds might
pure form.
These data indicate that RP2-L is a mixture of interfere with templates involved in the biosyn
proteins which resemble proteins of calf thymus in thesis of nuclear proteins of tumor cells.
amino acid analysis and in N-terminal amino
1L. S. Hnilica and H. Busch, unpublished.
FIG. 1.—Starch gel electrophoresis of various fractions ob
tained by rechromatography of crude RP2-L. The conditions
employed were those used by Johns et al. (17). The letters on
the patterns refer to the following: (a) peak B, (6) purified
RP2-L, (c) peak C, (d) RP3-L, (e) crude RP2-L, the source
material.
FIG. 2.—Starch gel electrophoresis of RP2-L at pH 5.0
according to the conditions described by Neelin and Neelin
(18). The letters on the patterns refer to the following: (a)
pattern of purified RP2-L and (6) pattern for re-electrophoresis
of RP2-LY.
Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research.
CJ
CD
O
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BUSCH et al.—RP2-L, a Histone Fraction from Tumors
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
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Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research.
Isolation and Purification of RP2-L, a Nuclear Protein Fraction of
the Walker 256 Carcinosarcoma
Harris Busch, Lubomir S. Hnilica, Su-Chen Chien, et al.
Cancer Res 1962;22:637-645.
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