Download Purification and Immunological Characterization of Human

[CANCER RESEARCH 42, 4836-4841,
0008-5472/82/0042-OOOOS02.00
November 1982]
Purification and Immunological Characterization
of Human Pancreatic
Ribonuclease
Minoru Kurihara,1 Michio Ogawa, Toshiyuki Ohta, Eiji Kurokawa,
Takeshi Kitahara, Goro Kosaki, Takehiko
Watanabe, and Hiroshi Wada
Second Department of Surgery, Osaka University Medical School. Fukushima-ku, Osaka 553 [M. O., T. O., E. K., T. K., G. K.]. and Second Department
Pharmacology, Osaka University Medical School, Kita-ku, Osaka 530 ¡T.W., H. W.¡,Japan
ABSTRACT
Three alkaline RNases were purified from human pancreatic
juice to near homogeneity as judged by sodium dodecyl sulfate
and native polyacrylamide gel electrophoreses. The molecular
weights of these RNases determined by sodium dodecyl sulfate
electrophoresis were 27,000, 19,000, and 13,000. The activ
ities of these three RNases were increased by addition of Mg2+,
Ca2+, Co2*, K+, Na+, spermine, and spermidine and decreased
by the addition of heparin, Cu2+, and Zn2+. These RNases
showed higher hydrolytic activity toward polycytidylic acid than
toward polyguanylic acid, polyuridylic acid, or polyadenylic
acid.
The three human pancreatic RNases were immunologically
identical but differed from human liver RNase, as shown by
Ouchterlony double-diffusion test. Radioimmunoassay of hu
man pancreatic RNase showed that immunologically similar
RNases are present in human saliva, urine, and serum.
INTRODUCTION
RNase activity has been observed to be elevated in the
serum of patients with malignant tumors (4, 12, 32). Recently,
Reddi and Holland (21, 23) reported that pancreatic RNase
activity in human serum could be measured in a specific way
with poly(C)2 as substrate and could serve as a biochemical
marker in the diagnosis of carcinoma of the pancreas. More
recently, however, Peterson (20) found that patients with pan
creatic cancer could not be distinguished in this way from
those with pancreatitis or with nonpancreatic disease, although
the RNase activity in all cases of pancreatic cancer was differ
ent from that in normal individuals. Moreover, he reported that
poly(C)-specific RNase activity did not decrease in the serum
of patients after total pancreatectomy.
These conflicting results could be because serum RNase
with preference for poly(C) may originate from other organs
beside the pancreas or could be due to variation in the stage
of disease and secondary pathology of patients from whom
sera were obtained.
In the present study, we purified RNase from human pan
creatic juice, examined its properties, and developed a RIA for
human pancreatic RNase. By this sensitive and specific
method, we investigated the immunoreactivity of pancreatic
of
RNase and compared it with those of RNases in various body
fluids.
MATERIALS
AND METHODS
Materials
Pancreatic juice was obtained by surgical cannulation of the pan
creatic duct of patients undergoing resection of the head of the pan
creas and stored at -20° until use. These patients had a periampullar
neoplasm or gastric cancer with pancreatic infiltration. Human serum,
urine, and saliva used for dilution curves were obtained from a healthy
33-year-old man. The following reagents were used: poly(C) (Yamasa
ShöyuCo., Japan); yeast RNA, poly(G), poly(U), poly(A), SDS electro
phoresis protein markers, and bovine RNase A (Sigma Chemical Co.,
St. Louis, Mo.); Sephadex G-75 superfine, Sephadex G-25, Sepharose
4B (Pharmacia, Uppsala, Sweden); phosphocellulose and ampholine
(Brown Co., England); cellulose dialysis tubing (Nakarai Chemicals,
Japan); Freund's complete adjuvant (Wako Pure Chemical Industries,
Ltd., Japan); Nali5l (New England Nuclear); goat anti-guinea pig IgG
serum and normal guinea pig serum (Eiken Chemicals Co., Japan).
Human liver RNase was purified in our laboratory (19). Poly(G) agar
was prepared by the method of Schmukler ef al. (25). Heparin Sepharose-CL-6B was purchased from Pharmacia. The gel was allowed to
swell in distilled water and washed for about 15 min on a sintered glass
filter with 0.05 M potassium phosphate buffer, pH 8.0, before being
packed into a column for use.
Enzyme Assay
RNase activity was assayed by the method of Reddi (21). The
standard assay system was as follows. The sample was diluted 500fold with 0.1 M potassium phosphate buffer, pH 6.8. Then, 0.05 ml of
diluted solution, 0.05 ml of poly(C) (4 mg/ml), and 0.15 ml of 0.1 M
potassium phosphate buffer, pH 6.8, were mixed and incubated at 37°
for 15 min. The reaction was stopped by adding 0.25 ml of cold 12%
perchloric acid containing 20 mM lanthanum nitrate. The solution stood
in an ice bath for 15 min and was centrifuged in the cold at 12,000 x
g for 15 min. Then, 200 /il of supernatant was diluted with 1 ml of
distilled water, and its absorbance at 260 nm was measured. The
RNase activity that caused an increase in absorbance of 1.0 under
these assay conditions was defined as 1 unit.
Protein Determination
Protein concentration was determined by the method of Lowry ef al.
(13) or from the absorbance at 280 nm.
Purification of Human Pancreatic RNase
Received February 4, 1982; accepted July 30. 1982.
' To whom requests for reprints should be addressed, at The Second Depart
ment of Surgery, Osaka University Medical School, 1-1-50 Fukushima, Fuku
shima-ku, Osaka 553, Japan.
2 The abbreviations used are: poly(C), polycytidylic acid; RIA, radioimmunoassay; poly(G), polyguanylic acid; poly(U), polyuridylic acid; poly(A), polyadenylic
acid; SDS, sodium dodecyl sulfate.
Received February 4, 1982; accepted July 30, 1982.
4836
All procedures
were carried out at 4°unless otherwise stated.
Step 1. Acid Treatment. Frozen pancreatic juice (2,500 ml) was
brought to room temperature before use. The pH was adjusted to 3.5
with 35% HCI, and the solution was kept in an ice bath for about 1 hr.
The solution was centrifuged at 10,000 x g for 10 min, and the
precipitate was discarded.
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VOL. 42
Human Pancreatic RNase
Step 2. First Phosphocellulose Column Chromatography. The
supernatant (2510 ml) was applied to a phosphocellulose column using
a glass filter (10 cm in diameter x 5 cm in height) previously equilibrated
with 0.05 M potassium phosphate-HCI buffer, pH 3.5. The column was
washed with 1500 ml of 0.05 M potassium phosphate buffer, pH 6.8,
and RNase was eluted with the same buffer containing 1.5 M NaCI.
The solution was dialyzed twice against 5 liters of the same buffer.
Step 3. Second Phosphocellulose Column Chromatography. The
sample (670 ml) was applied to a phosphocellulose column (2.5 x 52
cm) previously equilibrated with 0.05 M potassium phosphate buffer,
pH 6.8. The column was washed with 300 ml of the buffer, and then
protein was eluted with 2 liters of a linear gradient 0 to 0.8 M NaCI in
the same buffer (Chart 1,4). The flow rate was 40 ml/hr, and fractions
of 20 ml were collected. Fractions with activity were combined (500
ml) and dialyzed twice against 5 liters of the above buffer.
Step 4. Third Phosphocellulose Column Chromatography. The
sample was applied to a smaller phosphocellulose column (1.5 x 45
cm) under the same conditions as before, and the protein was eluted
with 800 ml of a linear gradient of 0.1 to 0.7 M NaCI (Chart M3). The
flow rate was 20 ml/hr. Fractions of 10 ml were collected, and those
with activity were combined (420 ml) and dialyzed against 4 liters of
0.05 M potassium phosphate buffer, pH 8.O.
Step 5. Poly(G) Affinity Chromatography. The sample was applied
to a poly(G) affinity column (5.5 x 1.5 cm) equilibrated with 0.05 M
potassium phosphate buffer, pH 8.O. The column was washed, and
then RNase was eluted with the same buffer containing 1 M NaCI (Chart
1C). The sample (72 ml) was dialyzed against 1 liter of the same buffer
and concentrated by lyophilization.
Step 6. Sephadex G-75 Gel Filtration. The concentrated prepara
tion (2 ml) was applied to a Sephadex G-75 superfine column (1.8 x
113 cm) equilibrated with 0.05 M potassium phosphate buffer, pH 8.0,
containing 0.3 M NaCI. The flow rate was 2 ml/hr, and fractions of 2.5
ml were collected. Three peaks of RNase activity were found (Chart
1D) and Peak 1 (RNase I), Peak 2 (RNase II), and Peak 3 (RNase III)
were collected separately. RNase I was purified by an additional step.
Step 7. Isoelectric Focusing. The eluate of Peak 1 was dialyzed
against 0.05 M potassium phosphate buffer, pH 8.0, and applied to an
isoelectric focusing column (pH 9 to 11). Fractions of 1 ml were
collected. The RNase activity was located at pH 10.0 (Chart 1£).
Heparin affinity Chromatography could be used instead of isoelectric
focusing. When heparin affinity Chromatography was used, the sample
from Step 6 was dialyzed extensively against 0.05 M potassium phos
phate buffer, pH 8.0, and applied to a heparin affinity column (1.5 x 20
cm) equilibrated with the same buffer. The column was washed, and
4.0
I
2.0
50
Fraction
0
No
50
Fraction No.
Chart 1. Purification of RNase from human pancreatic juice. A, second phosphocellulose column Chromatography. The eluate from the first phosphocellulose
column was applied to a second phosphocellulose column (2.5 x 52 cm) equilibrated with 0.05 M potassium phosphate buffer (pH 6.8). The protein was eluted with
a linear gradient of NaCI (0 to 0.8 M). Fractions of 20 ml were collected, and those with activity (Tube Nos. 55 to 80) were pooled. Bars, fractions collected for further
purification. B, third phosphocellulose column Chromatography. The sample shown in A was applied to a third phosphocellulose column (1.5 x 45 cm) equilibrated
with 0.05 M potassium phosphate buffer (pH 6.8). The protein was eluted with a linear gradient of NaCI (0.1 to 0.7 M). Fractions of 10 ml were collected and active
fractions (Tube Nos. 25 to 65) were pooled. C. poly(G) affinity column Chromatography. The sample shown ¡nB was applied to a poly(G) affinity column (1.5 x 5.5
cm) equilibrated with 0.05 M potassium phosphate buffer (pH 8.0). The protein was eluted with the same buffer containing 1 M NaCI. Fractions of 5 ml were collected.
D, Sephadex G-75 gel filtration. The eluate from the poly(G) affinity column was concentrated as described in the text and applied to a Sephadex G-75 superfine
column (1.8 x 113 cm) previously equilibrated with 0.05 M potassium phosphate buffer (pH 8.0) containing 0.3 M NaCI. RNase was separated into 3 peaks of activity,
which are referred to as RNases I, II, and III, in order of elution. E, isoelectric focusing. The RNase I fraction in D was applied to an LKB 8011 column. Electrophoresis
was carried out for 48 hr at 0.3 ma and 600 V in 0.5% ampholine (pH 9 to 11) with a sucrose gradient. Fractions of 1 ml were collected.
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M. Kuhhara et al.
then material was eluted with a linear gradient of 0 to 0.8 M NaCI.
RNase activity was eluted with 0.45 to 0.55 M NaCI.
fraction in the absence of the unlabeled
measured in duplicate.
Electrophoresis
RESULTS
Polyacrylamide gel electrophoresis was performed by the method of
Shapiro ef al. (26) using 10% polyacrylamide gel in the presence of
0.1% SDS. Polyacrylamide gel electrophoresis
at pH 4.5 was per
formed by the method of Reisfeld ef al. (24) using 10% polyacrylamide
gel.
Purification
Preparations of Antiserum
Antiserum against human pancreatic RNase was produced in guinea
pigs. The animals were given injections into multisites in the skin with
a mixture of 0.25 ml of human RNase I (0.25 mg/ml) and an equal
amount of complete Freund s adjuvant. The injection was repeated
after 3 weeks and 6 weeks and, 10 days after the last injection, blood
was collected by heart puncture, and the serum was separated and
stored at -20°.
Ouchterlony Analysis
Double diffusion in agar was performed by the procedure
terlony (19).
of Ouch
RIA Procedure
All samples were
RNase was purified from human pancreatic juice as de
scribed in "Materials and Methods." On Sephadex G-75 gel
filtration, RNase was separated into 3 peaks (Chart 1D): RNase
I, RNase II, and RNase III, in order of elution. RNase II and
RNase III were essentially homogeneous at this step. RNase I
was purified further by isoelectric focusing. Changes in specific
activity and recovery during the purification procedure are
summarized in Table 1. With this purification procedure, the
yield of enzymic activity was 8.9% with about 1000-fold in
crease in specific activity.
Purity
The purities of the RNase preparations were examined by
polyacrylamide gel electrophoresis. Each of the 3 human pan
creatic RNases moved as a single, broad band on 10% poly
acrylamide gel at pH 4.5, and the activity coincided in the
position with the protein band (Fig. 1). On polyacrylamide gel
The purified human pancreatic RNase I was labeled with radioiodine
following the method of Hunter and Greenwood (10). The procedure
used for radioimmunoassay for human pancreatic RNase was a modi
fication of the double-antibody technique of Morgan and Lazarow (14).
Bovine
Serum Albumin
iJDvalbumin
The reaction mixture consisted of 0.4 ml of 0.9% NaCI solution
buffered with 0.01 M phosphate, pH 7.6, containing 0.2% bovine serum
albumin and 0.1% sodium azide (NaN3), 0.1 ml of standard solution or
samples, 0.1 ml of labeled RNase I (about 3x10"
cpm) as tracer
•¿Â£
3
Chymotrypsinogen
RNase I t
antigen, and 0.1 ml of diluted antiserum (1:10,000). The mixture was
mixed well and stood overnight at 4°.After this first incubation, 0.1 ml
RNase n
of diluted normal guinea pig serum (1:100) and 0.1 ml of diluted antiguinea pig IgG serum (1:20) were added with thorough mixing. The
mixture stood overnight at 4°and then was centrifuged at 3,000 rpm
at 4°for 30 min. The supernatant was decanted, and the radioactivity
of the precipitate was counted in a Packard auto gamma counter. The
percentage of binding of the tracer antigen was expressed as the ratio
of the radioactivity of the bound fraction to the radioactivity of the same
antigen.
RNase
0.5
0
Rf.
Chart 2. Determination of the molecular weight of RNases I, II, and III. Molec
ular weights of RNases I, II, and III were determined by electrophoresis
as
described in "Materials and Methods." Cyt. C. cytochrome C.
Table 1
Summary of enzyme purification
Purification was started from 2500 ml of human pancreatic juice. The RNase activity was measured as
described in the text. Protein concentration was determined by measuring the absorbance at 280 nm.
ProcedurePancreatic
protein
activ
activity
(%)0.057
Yield8
ity
(units)772.51096.87650.6510394172.844.2
(A280)1357514106.21742406.5157.853.350.8
juiceAcid
1000.078
treatment1st
1420.37
phosphocellulose2nd
841.25
phosphocellulose3rd
662.54
phosphocellulosePoly(G)
513.24
affinitySephade.
220.87
3-75 gel filtration
RNase 1
RNase II
IIIIsoelectric
RNase
5.72
14
0.62
71.68
5.7275'47
44.16
13.26.5Total 14.949.62Total 0.20NO6Specific
8.91%ND
focusing RNase 1Volume250025106705004207223.5
184
1.25
The yield was calculated from the enzymatic activity.
1 ND, not determined because the sample contained ampholine.
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Human Pancreatic RNase
Table 2
Effects of ions and chemicals on enzyme activity
Assays were carried out in 0.1 Mpotassium phosphate buffer, pH 6.8, containing 5 mw ions or chemicals.
Activity in the absence of ions or chemicals was taken as 100%.
CUM)RNase
Ions and chemicals (5
dine204
1
115
100
137
115
189102Spermine141
126
RNase II
115
7
33
82
141Mg2*172
114Co2*154
115Spermi 265Cu2*40 15Zn2*36
RNase IIINone100
100Na*114
112K*110
30Heparin33
14
50
l"g ">'
lOng ml
I00ng/ml
l ug ml
lOngml
SOngml
Chart 3. Cross-reactivities of bovine RNase A with antiserum with human pancreatic RNase and human liver RNase. The conditions for incubation and separation
of bound antigens are described in "Materials and Methods." Data are shown on a semilog plot.
Table 3
Substrate specificity
Assays were carried out as described in the text. The concentration of
substrate was 4 mg/ml. The activity with poly(C) as substrate was taken as
100%.
SubstrateRNase
I
RNase II
100
RNase III
100
RNase APoly(C)100100Poly(A)6
4.3
5
0.5
14.7Poly(G)2.2 0
0
0.6Poly(U)8.6 17.8
containing 0.1% SDS, RNase I, RNase II, and RNase III mi
grated as single bands, and their molecular weights were
estimated as 27,000, 19,000, and 13,000, respectively (Chart
2).
Enzymological Properties
Effects of Ions and Chemicals on Enzymatic Activity. The
enzymatic activities of the 3 RNases were increased in the
presence of 5 rriM of Na+, K+, Mg2+, Co2+, spermine, and
spermidine and decreased by 5 mw of Cu2+, Zn2+, and heparin
(Table 2). There were no remarkable differences in the effects
of these ions and chemicals on the 3 RNases, although RNase
II gave slightly different results from RNase I and RNase III with
spermidine, Cu2+, and heparin.
Substrate Specificity. These RNases cleaved poly(C) better
than poly(A), poly(G), or poly(U). Their substrate specificity
was similar to that of bovine RNase A (Table 3).
Immunological Properties
Ouchterlony Test. Single precipitation lines were formed
between anti-human pancreatic RNase serum and the 3 pan
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1982
creatic RNases, and all the lines fused. No precipitation line
was formed between antihuman pancreatic RNase I serum and
liver RNase (Fig. 2).
Radioimmunoassay.
The standard curve is shown in Chart
3. Using this standard curve, the concentration of human
pancreatic RNase could be measured in the range 10 to 500
ng. As shown in Chart 3, slight cross-reactivity was detected
between human pancreatic RNase and human liver RNase
(about 10% at the point of 50% 8/S0), but there was no crossreactivity between human pancreatic RNase and bovine RNase
A. The dilution curves of human serum, urine, saliva, and
pancreatic juice were parallel with the standard curve for
human pancreatic RNase (Chart 4). The samples used for
dilution curves were not from the patients whose pancreatic
juice was used for purification of RNase. The amounts of
RNase, calculated from the dilution curves, were 480 ng/ml in
serum, 200 ng/ml in saliva, 240 ng/ml in urine, and 6400 ng/
ml in pancreatic juice.
DISCUSSION
RNases are widely distributed in various human tissues, and
their purifications have been reported by several groups (6,11,
16, 18, 22, 28, 29). Since the report of Reddi (21), pancreatic
RNase has been of interest because of its possible diagnostic
value as a marker of pancreatic cancer. At present, however,
little is actually known about human pancreatic RNase in rela
tion to the origin of human serum RNase. In the present study,
we purified RNase from human pancreatic juice and examined
its properties. Using this purified enzyme, we developed a RIA
for determination of the immunoreactivities of various RNases
in human biological fluids.
The pancreatic RNase that we have purified has 3 isozymes
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M. Kurihara et al.
sample dilution
I 256
I 128
I 64
I 32
I 16
18
100
human pancreatic
RNase standard
pancreatic juice
m
m
I
U
50
I
IO
100
Pancreatic RNase (ng/ml)
1000
Chart 4. Standard curve for human pancreatic RNase and dilution curves for
serum, urine, saliva, and pancreatic juice. These human body fluids were diluted
with 0.01 M phosphate:0.9% NaCI solution containing 0.2% bovine serum albu
min and 0.1 % sodium azide.
with different molecular weights. Weickmann ef al. (31 ) purified
an RNase from human pancreas. Their RNase gave a single
band of protein on polyacrylamide gel electrophoresis in the
presence of SDS, but on electrophoresis in 10% polyacryl
amide at pH 4.5 it gave at least 3 protein bands. They purified
their enzyme to the step of affinity chromatography, but we
have purified RNase further by Sephadex G-75 gel filtration
and isoelectric focusing. In the present study, pancreatic
RNase activity was separated into 3 fractions by gel filtration.
We also purified pancreatic RNase from human pancreas in a
separate investigation, to be published,3 and found that it was
also separated into at least 2 distinct fractions of activity with
different molecular weights on gel filtration. Although we could
not analyze the amino acid compositions or sugar compositions
of these fractions because of the limited quantity of purified
enzyme available, we tentatively conclude from the present
results that human pancreatic RNase has at least 3 isozymes,
consisting of the same polypeptide chain and differing only in
their degree of glycosylation, as shown for bovine pancreatic
RNase (2). However, it is still possible that some of these 3
RNases might be artifacts formed on acid treatment (29), that
they are allotypes, not isozymes, because pancreatic juice was
collected from several individuals, or that their difference might
result from disease, although the pancreatic juice was collected
from patients with periampullary neoplasm and gastric cancer.
Previous papers described slight cross-reactivity between
human pancreatic RNase and bovine RNase A (31) and no
cross-reactivity between pancreatic RNase and human liver
RNase (1 7). However, our results were different; we found no
cross-reactivity between human pancreatic RNase and bovine
RNase A by RIA. However, the variation in results may simply
be due to differences in the methods used. Moreover, slight
cross-reactivity was demonstrated between human liver RNase
and human pancreatic RNase by RIA, although no precipitation
3 M. Kurihara. N. Ogawa. T. Ohta. E. Kurokawa.
Watanabe, and H. Wada, manuscript in preparation.
4840
T. Kitahara. G. Kosaki. T.
line was formed in an Ouchterlony double-diffusion test be
tween human liver RNase and antiserum to human pancreatic
RNase.
Human serum contains many types of acidic RNases and
alkaline RNases originating from various tissues (1, 3, 15).
Alkaline RNase has been of interest because its activity is
thought to be elevated in the serum of patients with malignant
tumors. Houck and Berman (9) first reported the serum RNase
levels in patients with various diseases, indicating that conges
tive heart failure, myocardial infarction, and primary kidney
disease with uremia were associated with an elevated serum
RNase level. Zykto and Cantero (32) and Chretien ef al. (4)
reported that the serum RNase level was increased in patients
with carcinoma. Serum RNase was also reported to increase in
malnourished children (27). Akagi et al. (1) reported that there
are at least 5 alkaline RNases in human serum and that 4 of
them have properties similar to those of the bovine pancreatic
enzyme.
Recently, Reddi (21) reported that human serum RNase was
a good biochemical marker in diagnosis of carcinoma of the
pancreas because of its unique specificity for poly(C). Warshaw
ef at. (30) also reported that increased poly(C)-specific serum
RNase was of adjunctive value in the diagnosis of pancreatic
carcinoma. On the contrary, Peterson (20) reported that the
level of serum RNase activity toward poly(C) of patients with
pancreatic cancer was not different from that of patients with
nonpancreatic diseases. Doran ef al. (5) and Hölbling ef al. (8)
also reported that serum RNase with preference for poly(C)
was not a specific marker of carcinoma of the pancreas.
Furthermore, Peterson (20) reported that after total pancreatectomy, serum RNase activity remained in the normal range.
Recently, we also experienced a case in which serum RNase
activity with poly(C) as substrate was normal after total pancreatectomy.
In the present study, dilution curves of saliva were parallel
with the standard curve of human pancreatic RNase in RIA,
suggesting that salivary RNase is immunologically very similar
to pancreatic RNase. Bovine RNase from the salivary gland
has also been reported to be very similar to bovine pancreatic
RNase (7). Thus, human salivary gland may be a source of
serum RNase in some patients. The previous reports and
present results suggested that further study is needed to clarify
the reasons for elevation of RNase in the serum of patients with
malignant tumors.
ACKNOWLEDGMENTS
We thank Prof. Charles A. Dekker, University of California, and Prof. Masachika Irie, Hoshi College of Pharmacy, for valuable advice during preparation of
the manuscript.
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RNaseI
RNaseII
Fig. 2. Immunodiffusion of RNases. The center well contained anti-RNase I
serum. Peripheral wells contained RNases I, II. III. and human liver RNase. Well
I. RNase I; Well II, RNase II; Well III. RNase III; Well IV, human liver RNase.
«Naselli
Fig. 1. Gel electrophoresis of RNases I, II, and III on 10% acrylamide gels at
pH 4.5. Approximately 3 to 5 fig of enzyme was applied per tube, and electro
phoresis was carried out for 8 hr at 2 ma per tube. Electrophoresis was done in
duplicate. One was stained with Coomassie brilliant blue. The other was cut into
2-mm sections with a gel slicer, and RNase activity was assayed. Migration was
from left (anode) to right (cathode). Arrow, direction of electrophoretic migration.
NOVEMBER
1982
4841
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
Purification and Immunological Characterization of Human
Pancreatic Ribonuclease
Minoru Kurihara, Michio Ogawa, Toshiyuki Ohta, et al.
Cancer Res 1982;42:4836-4841.
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