Vol. 48, No. 5, 1972
BINDlNG
BIOCHEMICAL
OF RADIOACTIVELY AND RICINUS
TO RAT LIVER-
LABELED
Hiroshi
RESEARCH COMMUNICATIONS
CONCANAVALIN
A
COMMUNJS AGGLUTININ
AND RAT ASCITES
Isao Kaneko, Faculty
AND BIOPHYSICAL
HEPATOMA-NUCLEI
Satoh and the late Tyunosin
Ukita
of Pharmaceutical Sciences, University of Tokyo, Bunkyo- ku, Tokyo and Institute Sasaki Foundation, Chiyoda-ku, Tokyo, Japan
Received
August
3,
1972
The presence of exposed oligosaccharides on the surface of rat Summary: liver- and rat ascites hepatoma (AH 108A)-nuclei was demonstrated by use of 3H-labeled concanavalin A (Con A) and Ricinus communis agglutinin (RCPHA). Rat liver- and AH 108A-nuclei have considerably more receptor sites for Con A than for RC-PHA. With respect to the RC-PHA receptor site, the AH 108A nuclei have about ten fold more sites than the rat liver nuclei. The densities of these plant agglutinins receptor sites on either of the nuclear surfaces were lower than that of ascites hepatoma (AH 13) cell surface. Recently oligosaccharides
extensive
studies
using plant agglutinins
is known about the presence of cellular
organelles
we report nuclei
of 3H-labeled
binding
agglutinins
and Ricinus
of exposed saccharides
and mitochondoria.
to rat liver; concanavalin
communis
on the cell surface
On the other hand,
(PHA)lm3).
or the nature
such as nuclei
the differential
like residues)
have been performed
agglutinin
little
on surfaces
In this communication,
and rat ascites A (specific (specific
hepatoma-
for z;-mannose for g-galactose
like residues).
Materials
and Methods
Concanavalin by the method (RC-PHA) subsequent preparations 3
A (Con A) was obtained from of Agrawal and Goldstein 4) . Ricinus
was purified Bio-Gel
P-150
Con A with 3H-acetic
affinity
gel filtration
were electrophoretically
H-Acetyl-concanavalin
Copyright All rights
by Sepharose
A i3H-Con anhydride
@ 1972 by Academic Press, Inc. of reproduction in any form reserved.
column
as described
jack bean meal (Sigma) communis
chromatography and previously 5) . These
and ultracentrifugally A) was prepared
(100 mCi/mmol)
1504
agglutinin
homogeneous.
by acetylation
6) and further
of native
purified
by
Vol. 48, No. 5, 1972
BIOCHEMICAL
Sephadex G-50 column 3
H-Acetyl-Ricinus
the native agglutinins
from
buffer
of Donryu
method
homogenized M CaC12. pellet
sedimented rpm.
suspended
P-150
into female
were isolated
homogenizer
The
in 2 M sucrose-10 -3M CaC12. The suspension -3 M CaC12 and the nuclei were over 2.3 M sucrose-10
the nuclei
for 60 min in a Hitachi
were washed with cold-PBS-
as possible
and used immediately.
RPS-25
at 0°C.
assay was performed
of different
were washed three
scintillation
at 20,000
All steps were carried
at 0°C as follows
in the presence
with SDS.
rotor
4 x 10m3M CaC12,
nuclei/ml
Mi+
rats
by a slightly
at 700 x g for 10 min.
in 0.4 ml PBS- 4 x 10W3M CaC12 at a concentration
by liquid
albino
or AH 108A cells were -3 in 0.3 M sucrose- 4 x 10
suspended
solubilized
column.
homogenized
The binding
min the nuclei
and 120, 000
rat liver
was centrifuged
in the same solution
out as rapidly
55,000
on Bio-Gel
and AH 108A-nuclei
by centrifugation
Finally
A and ‘H-RC-PHA
of 3H-Con
inoculation
et al. 7) Fresh
of Lynch
The homogenate
was then layered
weights
intraperitoneal
Rat liver-
By SDS-polyacryl-
cells AH 13 and AH 108A were used ‘7 and 10
with a loose Teflon
was further
by
were also indistinguishable
by gel filtration
hepatoma after
strain.
modified
was also prepared
activity.
(PBS) were approximately
as determined
respectively,
of agglutination
Molecular
saline
Rat ascites days,
of 0.1 M mannose.
13H-RC-PHA)
these preparations
native agglutinins.
respectively,
in the presence
agglutinin
in respect
gel electrophoresis
in phosphate
RESEARCH COMMUNICATIONS
technique and purified by Sepharose affinity column 3 H-Con A and ‘H-RC-PHA were found to be identical with
chromatography.
amide
chromatography
communis
the same acetylation
AND BIOPHYSICAL
free PBS was used instead Results
of 5 x lo7
of 3H-agglutinin.
After
times with the same cold buffer,
The nuclei-bound spectrometer.
amounts
; The nuclei were
radioactivity
and
was then determined
In the case of cell suspension,
Ca++-
of PBS- 4 x 10e3M CaC12. and Discussion
Table I shows the sugar specificities
1505
60
of the labeled
agglutinins,
Vol. 48, No. 5, 1972
TABLE
BIOCHEMICAL
I Inhibition of 3H-Con with sugars.
AND BIOPHYSICAL
A and ‘H-RC-PHA
binding
AH 13 cell (4.0 x 105)
to cells
[ 3~1 -RC-PI-IA
[ 3H1 -Con A bound Sugar (20 mM)
RESEARCH COMMUNICATIONS
AH108A nuclei (2.0 x 106)
and nuclei bound
AH 13 cell AHI08A nuclei (4.0 x 105) (2.0 x 106)
g-galactose
87.5%
104.6%
14.0%
54.5%
lactose
76.4
102.6
10.3
60. 0
melibiose
90.0
104.8
10.4
45.7
g-glucose
31.8
63.1
54.2
91.2
g-mannose
14.8
31.6
57.2
83.0
methyl-o-gglucoside
16.9
26.0
77.2
102.7
N-acetyl- DglucosamEe
70.1
50.6
72.9
94.5
N- acetyl- DgalactosaZine
89.8
98.0
33.1
89.3
sucrose
37.2
50.0
93.1
114.0
none
100.0 (9,387
dpm)
100.0 (9,178
100.0 (3,814
dpm)
100.0 (1,432
dpm)
dpm)
Table I Sugar was added to the PBS solution containing 6 pg of 3H-agglutinin, and the solution was stood for I hour at 0°C. The cells or nuclei suspension was then added and binding assay was performed.
3 H-Con
A and ‘H-RC-PHA,
indicate
that these agglutinins
as to the cells ; The binding and methyl-cu-g-glucoside, acetyl-galactosamine. galactose, mannose
lactose
determined
inhibition.
of 3H-Con
A was inhibited
but not by galactose.
and melibiose,
The amount
but little
melibiose
was inhibited
The sugar specificities
A or ‘H-RC-PHA
1506
byg-mannose
by methyl-cY-&glucoside,
well with those in agglutination of ‘H-Con
to the nuclei
strongly
lactose,
On the other hand ‘H-RC-PHA
agreed
The results
have the same sugar specificity
or N-acetyl-glucosamine.
in the binding
by binding
or Nby
glucose,
of these agglutinins
of cells reported bound to the nuclei
previously was
8,9
BIOCHEMICAL
Vol. 48, No. 5, 1972
directly
proportional
x 107/ ml,
to the nuclei
and the binding
concentration
was completed
of ‘OH-agglutinin
added was varied,
until
but little
saturation,
of competitive
sugars
were plotted
Saturated
amount
1).
within
30 min.
When the amount
of agglutinin
agglutinins
to the method
bound increased
were bound in the presence
The data of sugar specific
according
of agglutinin
RESEARCH COMMUNICATIONS
over the range of 0.5 - 1.5
the amount
radioactive
(Fig.
the nuclei
AND BIOPHYSICAL
binding
to both of
of Steck and Wallach “)(Fig.
bound to a nucleus
was determined
by repeating
experiments as those of Fig. 2. Assuming a molecular weight of 55,000 for 3 H-Con A and 120,000 for 3H-RC-PHA, we calculated the average number of agglutinin (K) (Table
receptor
sites per nucleus
association
constant
II). The results
indicate
that both nuclei
sites for Con A than for RC-PI-IA. site,
and the apparent
the AH 108A nuclei
have considerably
With respect
more
to the RC-PI-IA
have about ten fold more
receptor
receptor
sites than the rat liver
n
nuclei.
The K values for ‘H-Con
to rat liver- and AH 108A-nuclei -1 the same (1.21 - 1.43 x lo6 M ) and that for ‘H-RC-PHA -1 . - 8.9x106M AH 108A nuclei are considerably
were approximately toAH108Anucleiwas7.7 larger
in size than rat liver
are calculated nuclei
rat liver
nuclei
outer
even when compared
surfaces
of the density
rat liver-
of Con A receptor receptor
by density
in unit area.
and AH I3 cell surface.
fold higher
more
On the than
Table II also
receptor
The density
and AH 106Asites.
sites g-fold
of these agglutinin
was several
site numbers
sites on the
of these receptor
than that on either
of the
surfaces. Recently
nuclei
outer surface,
the same number
sites on AH 13 cell surface nuclear
so that when the receptor
AH 108A nuclei have RC-PHA
shows the comparison nuclear
nuclei
per unit area of nuclear
have approximately
other hand,
A binding
by several
agglutination
Nicolson
et al.
plant agglutinins
of rat liver-
reported
the agglutination
11) . In our experiment,
and AH 106A-nuclei
PHA was very weak and the agglutinin
titer
1507
by either
of bovine liver however,
the
of Con A and RC-
did not show reproducibility.
2).
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0
0.04
0.08
0.12
0.16
0.20
a24
0.28
032
1 (w-‘.ml ) cPHA)
0
3
6
9
12
3H-Agglutinm
added
15
18
21
Fig. 2.
(119)
Fig. 1. Fig.
1. Binding nuclei.
of 3H-Con
A and ‘H-RC-PHA
to rat liver-
and AH 108A-
2, Con A to AH 108A nuclei ; b, RC-PHA to AH 108A nuclei ; c, RCPHA to rat liver nuclei. The specific activity of the 3H-Con A and 5HRC-PHA was 9,000 dpm/pg and 5,660 dpm/pg respectively. The binding assay was as described in text. w , total dpm bound ; , dpm bound in -the presence of 0.1 M methyl-cu-D-glucoside (2) or !!Pgalactose (b and ~1 ; 0-0 , specific dpm bound, i. e., [total dpml - [ dpm in the presence of competitive sugar] Fig.
2. Sugar specific binding and AH 108A-nuclei. The data of sugar specific C 1 =PHA bound Kxn ’
of 3H-Con binding 1 [PHAI
A and ‘H-RC-PHA were plotted + L n
according
, [PHAI
to rat liverto the equation 10):
= concentration
of
free PHA (pg/ml) ; n = number of PHA binding sites per nucleus ; C = number of nuclei ; K = the apparent affinity constant of PHA ; PHA bound = amount of the sugar specific binding to nuclei expressed in dpm. yT , Con A to AH 108A nuclei ; M , Con A to rat liver nuclei ; O--O , RC-PHA to AH 108A nuclei
1508
BIOCHEMICAL
Vol. 48, No. 5, 1972
TABLE
Cell
II
Number of Con A and and to AH 13 cells.
Surface
or
Rat
molecules
Number molecules
of Con A bound
per cell or nucleus
(run21
AH
RC-PHA
bound
RESEARCH COMMUNICATIONS
to rat
liver-
and
AH
108A-nuclei,
Number of RC-PHA molecules bound
area
nuclei
AH
AND BIOPHYSICAL
13 cell
698
9.8
eO.4)
108A nuclei
415
l.6~0.1)x107
liver nuclei
208
8.0
per
x lo7
e2.0)
x lo6
per cell or nucleus
pm2
1.4
x lo5
1.4
&O.
4.0
x lo4
2.1
3.8
x lo4
1.8
per
pm2
1) x lo7
2.0
x lo4
c+o.11
x 106
5.1
x 103
bO.2)
x 10 5”
8.6
x lo2
Table II molecules bound was calculated by the method of Steck value was the average of two or three determinations surface area was calculated from its average diameter.
:tidm&l:3 “p: O The Cell or nucleus
JEThis value was calculated at saturating concentration 1-c.
The weakness
of agglutination
than cell surface topological
from the specific counts bound to the nuclei of agglutinin in experiments as that of Fig.
of the nuclei may be due to the lower
of the receptor
distribution
sites or to some difference
of the receptor
The role of saccharides However, cell
nuclei
the difference may be related
replication
sites between
of nuclear
of surface
to the difference
and macromolecule
of surface
cell and nucleus.
outer membrane
saccharides
between
of nuclear
density
is not known.
normal
function
and tumor such as DNA
transport.
Acknowledgements We thank Dr. Hikoya Hayatsu of the Faculty of Pharmaceutical Sciences, University of Tokyo, for his helping us in the preparation of the manuscript. Dr. Toshiaki Osawa is also acknowledged for valuable discussions. References 1. Kornfeld, (1971)
S., Rogers,
J. and Gregory,
1509
W. : J. Biol.
Chem.,
246 : 6561
Vol. 48, No. 5, 1972
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2. Cline, M. J. and Livingston, D. C. : Nature New Biology, 232 : 155 (1971) 3. Ozanie, B. and Sambrock, J. : Nature New Biology, 232 : 156 (1971) 4. Agrawal, B. B. L., and Goldstein, I. J. : Biochem. J. 23C (1965) 5. Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N., Osawa, T. and 28 : 84 (1972) Ukita, T. : Experientia, 6. Agrawal, B. B. L., Goldstein, I. J., Hassing, G. S., and So, L. L. : Biochemistry, 7 : 4211 (1968) 7. Lynch, W. E., Brown, R. F., Umeda, T., Langreth, S. G. and Lieberman, I. : J. Biol. Chem., 245 : 3911 (1970) 8. Nicolson, G. L. and Blaustein, J. : Biochim. Biophys. Acta, 266 : 543 (1972) 9. Inbar, M. and Sachs, L. : Proc. US Nat. Acad. Sci., 63 : 1418 (1969) 10. Steck, T. L. and Wallach, D. F. H. : Biochim. Biophys. Acta, 97 : 510 (1965) 11. Nicolson, G. L., Lacorbikre, M. and Delmonte, P. : Exptl. Cell Res., 71 : 468 (1972)
1510