Binding of radioactively labeled concanavalin A and Ricinus communis agglutinin to rat liver- and rat ascites hepatoma-nuclei

Binding of radioactively labeled concanavalin A and Ricinus communis agglutinin to rat liver- and rat ascites hepatoma-nuclei

Vol. 48, No. 5, 1972 BINDlNG BIOCHEMICAL OF RADIOACTIVELY AND RICINUS TO RAT LIVER- LABELED Hiroshi RESEARCH COMMUNICATIONS CONCANAVALIN A C...

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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)

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