BIOCHEMICAL
Vol. 79, No. 2, 1977
INCREASE MEMBRANES
OF A HIGH
OF BHKZI
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MOLECULAR
CELLS
TRANSFORMED
A. Lage Davila* Viral Received
Oncology
October
WEIGHT
Unit,
PROTEIN
IN
BY HAMSTER
PLASMA
SARCOMA
VIRUS
and L. Montagnier
Institut
Pasteur,
Paris,
France
5,1977
SUMMARY - Electrophoretic analysis of plasma membrane fractions from '-3 cells non-transformed and transformed by the B34 strain of Hamster Sarcoma Virus showed a reproducible pattern of 18 main bands among which a band corresponding to a polypeptide of nominal molecular weight 177,000 daltons, is considerably increased in membranes of transformed cells. Labelling and solubilization studies suggest that it is an integral glycoprotein. INTRODUCTION The changes
that
occur
transformation
by oncogenic
recent
see (1).
review,
such as the loss cytoskeleton, which
in transformed which
proteins) density
BHK21.
are possible,
we chose
hydrophobic
viral
results
with
obtained of a large,
weight
of approximately were
the BHK21 cell
presumably 177,000
obtained
in
investigation We report
showing
in a transformed 100,000
systems of the
to
here
some
a significant with
the electrophoretic the
cell
observation
to be the main components
polypeptide
daltons
by optimalizing
of polypeptides
system,
using
of intramembranous
of this
(7 - 9).
integral,
the
bilayer
laboratory in three
are likely
fractures
known about
increase
a biochemical
a
or the endo-
the phospholipid
3-fold
(for
and microtubules
is
from our
transformation
which
(2),
cables
interpretations
to undertake
proteins
crease
MATERIALS
with
malignant
the exocytoskeleton,
Much less
studies
several
seen in freeze
the resolution
4).
associated
after
Although
affect
of actin
(3,
Previous
of the particles
results
changes
(6) had shown a 2-to
(IMP)
including
following documented
of the LETS protein
cells are
(5).
freeze-fracturing
integral
of these
or the diminution
proteins
(integral particle
Most
periphery
have been widely
such as the dissociation
occur
membrane
at the cell
viruses
300,000
in-
a molecular clone. system dalton
These for range.
AND METHODS
An asparagine-dependent subclone of BHK21/13 cells was used. It had the same growth properties as the untransformed population of BHKZI cells (IO). Following infection of C13/8 cells with Hamster *
Permanent
Copyright All rights
address:
0 1977 by Academic of reproduction in any
Instituto National de Oncologia y Radiobiologia, 29 y E, Vedado, La Habana, Cuba. Press, Inc. form reserved.
577 ISSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sarcoma Virus (II) and cloning in agarose medium containing dextran sulphate (12), several transformed clones were isolated. One of these, HS5, was chosen for further study. It was found to be non-virus producing and highly malignant: less than 100 cells were sufficient to induce an invasive and metastasing tumor in the Syrian hamster, whereas the 50% tumorigenic dose of its untransformed counterpart was 3.104 cells (13). Conditions were established to obtain a purified plasma membrane fraction from both types of cells, beginning with Warren's Zn-fixation method (14). In each experiment, approximately 2.108 subconfluent cells were removed, either by trypsinisation or by scraping from large plastic plate, washed twice in saline and kept for 8 min at room temperature in I mM ZnC12. After centrifugation, the cells were allowed to swell in distilled water and were broken in a Dounce Homogenizer in medium containing 3 mM MgC12, 10 mM Na phosphate buffer, pH 8.0. Nuclei were removed by centrifugation and the ghosts in the supernatant were concentrated on a 65% (w/w) sucrose layer. A linear sucrose density gradient (1.23 - 1.15 g/cm3) was then formed over the ghost containing sucrose layer and centrifuged to equilibrium at 81,000 X g for 15 hours in a SW 27.1 Beckman rotor. Fractions were collected with an Isco fractionator with continuous W absorbance recording. The electrophoretic profile of membrane polypeptides was analyzed in polyacrylamide gels containing sodium dodecyl sulphate (SDS), using the discontinuous Tris-buffered system of Laemmli (15) with minor modifications. Separation gels were 7.5% polyacrylamide which were 0.57% crosslinked, containing 0.1% SDS.
RESULTS cation
The final
isopycnic
procedure
produced
centrated than
for
1.196
in
the first
Cl3/8
cells:
+ 0.005
obtained brane
fraction 0.75M
it
according
adsorbed
files,
types
18 major
molecular
weight
increased
quantities
was obtained in untreated cells. from
Cl318
177,000
material,
yielded
daltons
(Band
appeared
in
but
+ 0.005
g/cm3,
of the final
state
of large
they
described
could for
replicas in
transformed using experiments,
ghosts.
showed
the ghosts
band
cells. either
from both pro-
of nominal found
in
The same result trypsinised or band
(Band
in HS5 membranes
out
of non-
electrophoretic
migrating peak
mem-
be washed
removal
IO) was consistently
from
578
con-
HS5 cells
of
preparation
A prominent
as a double
were
for
respectively. controls
reproducible
a more rapidly
the purifi-
ghosts
denser density
Freeze-fracture
(Fig.]).
in membranes
10 independent In addition,
membranes,
experiments,
(16).
bands
Cell
was 1.186
microscopy
in an aggregated
of membranes
containing
density
to the conditions
ribosomes
peaks.
had a buoyant
ghost
in this
used
was somewhat
to be a homogeneous
present
intramembranous particles cell types (17). Both
ghosts
and electron
showed were
KCl,
specifically
which
16 and 9 independent
Some riboscmes with
of three
peak,
HS5 cell
contrast
centrifugation
a profile light
g/cm3 and the Cl318
from Phase
equilibrium
10) due to
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. I Electrophoretic patterns of the membrane proteins of C13/8 and H'S5 cells. Staining was with Coomasie Blue R 250. Direction of migration in the figure is downward. Gel A: membrane fraction of Cl318 cells. Gel B: membrane fraction of HS5 cells. Gel C contained standard proteins, from top to bottom: chicken muscle myosin, rabbit muscle phosphorylase B and bovine serum albumin. Mobilities of standard proteins were reproducible within + 4% and were linearly related to the logarithm of their molecular Geights.
the appearance 94,000
of a second
daltons. Three different
protein
criteria
having were
a nominal
used to quantitate 579
molecular
weight
of
the
increase
of
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
s 3.
, 2.
, 1.1
-0
60
90
Yolecrler
w6illL:b
Irll-31
250 Cl 6 I
B
*30
.2.0
.ltl
ac
0
580
BIOCHEMICAL
Vol. 79, No. 2, 1977
Band 10. Direct fold
increase.
sliced gels
UV scanning
(18)
Band
incubated
collagenase related
(Advance
polypeptides 10 could
to the conditions vesiculation
is also
for
components from
the membrane of actin
from human erythrocytes
ghosts
proteins
fairly
was
175 units
of
the same
that
collagen-
of the membrane
fraction.
preparation
by a
performed
according
and myosin
with
(19).
treatment
This
from Sarcoma
conco-
180 ascitic
(20).
Finally, HS5 cells were grown in culture 14 C-fucose in order to label their carbohydrate membranes
which
yielded
showing
M) treatment
the extraction
non-integral
of
Corporation) sample,
M) and KC1 (0.45
know to extract
ghosts
Biofacture
not be removed
used
from myosin
HS5 membranes
at 37°C in presence
are not major
EDTA (0.001
(Fig.ZB).
distinguished
as an untreated
dye from
of double-labelled
increase
of purified
two hours
profile
Band
cell
for
Form III
sequential
distribution
be electrophoretically A sample
of a three-
at 605 nm of eluted
of a two-fold
components.
electrophoretic
to the estimation
profile
and the radioactivity
10 could
previously
led
density
to the estimation
and collagen
mitant
(Fig.2A)
The optical
gels led
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
obtained
simple
corresponding and 90,000
from cells
electrophoretic to nominal
daltons
without
radioactivity molecular
(Fig.3).
to Band 10. When cells peak, as well electrophoretic
detached
initially
as the 200,000
peak peak,
gave a
with
of 200,000,
four
major
175,000,
in stained
detached
daltons
Fucose-labelled
trypsinisation pattern
weights
The second
were
medium containing chains.
gels
corresponded
by trypsinisation,
was not
found
peaks
115,000 this
in the
"C'fucose
pattern.
DISCUSSION These brane daltons
experiments
fucose-containing occurs
indicate protein
that
a significant
of apparent
in the virus-transformed
increase
molecular
cell
clone.
weight
This
of a mem177,000
protein
is
Quantitation of the electrophoretic patterns of Cl318 and Fig. 2 HS5 cell membrane proteins. A: Direct UV scanning of stained gels. Abcissa: mobility relative to bovine serum albumin, taken as 1.000. Ordinate: optical density at 280 run relative to that of band 9 (unvarying in both cell types) taken as an internal standard. Solid line: C13/8; dotted line: HS5. B: Double-labelling experiment: C13/8 and HS5 cells were grown for res48 hours in the presence of 14C and 3H labelled amino acids, pectively. Labelled membranes were isolated separately, then mixed and co-electrophoresed in the same gel. Direction of migration is from right to left. Ordinate: cpm of each gel slice as the percentage of total counts recovered. The
arrows
indicate
the
two
581
major
differences.
BIOCHEMICAL
Vol. 79, No. 2, 1977
30
II
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10
Sld”mmbw
0x( (WA ( Enlpnttnrr
I HI1
(I
14 Electrophoretic patterns of C fucose-labelled membrane from HS5 cells. After electrophoresis, the gel was stained and sliced and the radioactivity in each slice was determined. The diagram below the abcissa indicates the correspondence between slices and stained bands. Continuous line (m) corresponds to a membrane fraction purified from scraped, non-trypsinised cells. Protection against proteolysis was achieved by adding 0.2% diisopropyl-fluorophosphate to the collection and homogenisation media. Discontinuous line (A---A) corresponds to a membrane fraction purified from trypsinised cells. The arrow indicates the position of Band 10. Fig. 3 fractions
distinguishable
from myosin
association its
of this
enrichment
with
the
with
in a purified
total
cell
component
represents
proteins,
in the That
protein
and collagen
the plasma
plasma
homogenate.
components.
membrane
protein
is
labelled
is
indicated
preparation,
5% of the
case of the transformed
this
membrane
Densitometry
approximately
The specific by
compared
data
indicate
total
plasma
that
this
membrane
clone.
in
the presence
of radioactive
precursors
indicates that it is a product of cellular meta--in vitro bolism and is not a serum component which is adventitiously bound to the membrane. In addition, its labelling with fucose suggests that it
is
a glycoprotein.
significantly a small
portion
to be exposed Finally, protein,
Since
change
since
fucose
the migration
of the peptide
removal
upon
of the protein sequence
bound
is
not
removed
by trypsin,
582
band,
does not
apparently
to the sugar
at the cell surface. this protein has the characteristics it
trypsinisation chain
only seems
of an integral
EDTA and KC1 treatments.
BIOCHEMICAL
Vol. 79, No. 2, 1977
Although
we have no evidence
intramembranous
particles
quantitative
increase
transformed
clone
membranes
is
of the
protein/lipid
striking.
of membrane
a component
The increased cells
is
in
buoyant
also
of the
the parallel
and particles
the virus-
density
consistent
proteins
cell
of plasma
with
a higher
proteins certain
phenotype.
from
in
suggested
disorganization. transport it
in altered
is
it
could
for
in This
(21)
the change
and Isaka
17),
alter
could
some integral
their
the
the anchorage
which
or metabolites that
the increased
of transformed
which
may
transformed
of components
ultimately
result
proteins are
quantitative
insertion cells
are
critical increase
in
involved for
cell
may result
properties. a similar
clones
of BHKZI cells
rently
investigating
of virally
clone.
accompanying
if
Moreover, of ions
We found
(6,
alterations
conceivable
growth
optimal
daltons
by Stone
membrane
and the endocytoskeleton
in active
94,000
reported
the plasma
structural
division,
not
we nevertheless
the transformed
previously
For instance,
of the exo-
were
proteins,
systems.
As we have previously of integral
conditions weight
band of approximately
to those
in other
their
is
freeze-fractures,
the electrophoretic
an additional
explain
protein
the protein
of low molecular
may be similar (22)
seen in of both
transformed
Although
profile
this
ratio.
the resolution detected
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
177K protein
transformed
by hamster
sarcoma
the possibility
that
and chemically
increase
transformed
this
change
in virus.
two other We are cur-
is a general
feature
cells.
ACKNOWLEDGEMENTS One of us (A. L.D.) was Government (C.I.E.S.). We thank examination of plasma membrane B. Collandre, C. Axler-Blin and assistance.
supported by a fellowship of the French G. Torpier for the electron microscopy preparations and Mrs. J. Gruest, G. Jonkman for their skilful technical
REFERENCES I. 2. 3. 4. 5. 6. 7.
Nicolson, G.L. (1976) Biochim. Biophys. Acta 458, 1-72. Hynes, R.O. (1976) Biochim. Biophys. Acta 458, 73-107. Weber, K., Lazarides, E., Goldman, R.D., Vogel, A. & Pollack, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 2, 363-369. Pollack, R., Osborn, M. & Weber, K. (1975) Proc. Natl. Acad. Sci. USA 72-, 994-998. Singer, S.J. & Nicolson, G.L. (1972) Science 175, 720-731. Torpier, G., Montagnier, L., Biquard, J.M. & Gier, P. (1975) Proc. Natl. Acad. Sci. USA 72, 1695-1698. Branton, D. (1966) Proc. Natl. Acad. Sci. USA 55, 1048-1056.
583
Vol. 79, No. 2, 1977
8. 9. IO.
Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Marchesi, V.T., Tillack, T.W., Jackson, R.L., Segrest, J.P. & Scott, R.E. (1972) Proc. Natl. Acad. Sci. USA 2, 1445-1449. Pinto da Silva, P. & Nicolson, G.L. (1974) Biochim. Biophys. Acta 363, 311-319. M. (1971) & "La L-asparaginase", MontagnG, L., Gruest, J. & Boccara, Colloque International CNRS n0197, pp. 159-164. Torpier, G., Gruest, J. & Montagnier, L. (1974) Exp. Cell Res. E, 437-442. Zavada, J. & MacPherson, I. (1970) Nature 225, 24-26. Montagnier, L. (1968) C. R. Acad. Sci. (Paz) D 267, 921-924. Torpier, G. 8 Montagnier, L. (1970) Int. .J. Cancer a, 529-535. 3, Warren, L., Glick, M.C. & Nass, N.K. (1966) J. cell Physiol. 269-287. Laemmli, U.K. (1970) Nature 227, 680-685. D.C. & Blobel, G. (1973) J. Cell Biol. 56, Aldeman, M.R., Sabatini, 206-229 * Montagnier, L. & Torpier, G. (1976) Bull. Cancer 63, 123-134. J. (1975) Fenner, C., Traut, R.R., Mason, D.T. & Wikman-Coffelt, Analyt. Biochem. 2, 595-602. Tilney, L.G. & Detmers, P. (1975) J. cell. Biol. 66, 508-520. D.R., Huggins, J.W. & Carraway, K.L. (1976) Moore, P.B., Anderson, Biochem. Biophys. Res. Commun. 2, 288-294. Stone, K.R., Smith, R.E. & Johlik, W.K. (1974) Virology 58, 86-100. Isaka, T., Yoshida, M., Owada, M. & Toyoshima, K. (1975) Virology 65, 226-237.
584
Vol. 79, No. 2, 1977
INCREASE MEMBRANES
OF A HIGH
OF BHKZI
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MOLECULAR
CELLS
TRANSFORMED
A. Lage Davila* Viral Received
Oncology
October
WEIGHT
Unit,
PROTEIN
IN
BY HAMSTER
PLASMA
SARCOMA
VIRUS
and L. Montagnier
Institut
Pasteur,
Paris,
France
5,1977
SUMMARY - Electrophoretic analysis of plasma membrane fractions from '-3 cells non-transformed and transformed by the B34 strain of Hamster Sarcoma Virus showed a reproducible pattern of 18 main bands among which a band corresponding to a polypeptide of nominal molecular weight 177,000 daltons, is considerably increased in membranes of transformed cells. Labelling and solubilization studies suggest that it is an integral glycoprotein. INTRODUCTION The changes
that
occur
transformation
by oncogenic
recent
see (1).
review,
such as the loss cytoskeleton, which
in transformed which
proteins) density
BHK21.
are possible,
we chose
hydrophobic
viral
results
with
obtained of a large,
weight
of approximately were
the BHK21 cell
presumably 177,000
obtained
in
investigation We report
showing
in a transformed 100,000
systems of the
to
here
some
a significant with
the electrophoretic the
cell
observation
to be the main components
polypeptide
daltons
by optimalizing
of polypeptides
system,
using
of intramembranous
of this
(7 - 9).
integral,
the
bilayer
laboratory in three
are likely
fractures
known about
increase
a biochemical
a
or the endo-
the phospholipid
3-fold
(for
and microtubules
is
from our
transformation
which
(2),
cables
interpretations
to undertake
proteins
crease
MATERIALS
with
malignant
the exocytoskeleton,
Much less
studies
several
seen in freeze
the resolution
4).
associated
after
Although
affect
of actin
(3,
Previous
of the particles
results
changes
(6) had shown a 2-to
(IMP)
including
following documented
of the LETS protein
cells are
(5).
freeze-fracturing
integral
of these
or the diminution
proteins
(integral particle
Most
periphery
have been widely
such as the dissociation
occur
membrane
at the cell
viruses
300,000
in-
a molecular clone. system dalton
These for range.
AND METHODS
An asparagine-dependent subclone of BHK21/13 cells was used. It had the same growth properties as the untransformed population of BHKZI cells (IO). Following infection of C13/8 cells with Hamster *
Permanent
Copyright All rights
address:
0 1977 by Academic of reproduction in any
Instituto National de Oncologia y Radiobiologia, 29 y E, Vedado, La Habana, Cuba. Press, Inc. form reserved.
577 ISSN
0006-291X
BIOCHEMICAL
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sarcoma Virus (II) and cloning in agarose medium containing dextran sulphate (12), several transformed clones were isolated. One of these, HS5, was chosen for further study. It was found to be non-virus producing and highly malignant: less than 100 cells were sufficient to induce an invasive and metastasing tumor in the Syrian hamster, whereas the 50% tumorigenic dose of its untransformed counterpart was 3.104 cells (13). Conditions were established to obtain a purified plasma membrane fraction from both types of cells, beginning with Warren's Zn-fixation method (14). In each experiment, approximately 2.108 subconfluent cells were removed, either by trypsinisation or by scraping from large plastic plate, washed twice in saline and kept for 8 min at room temperature in I mM ZnC12. After centrifugation, the cells were allowed to swell in distilled water and were broken in a Dounce Homogenizer in medium containing 3 mM MgC12, 10 mM Na phosphate buffer, pH 8.0. Nuclei were removed by centrifugation and the ghosts in the supernatant were concentrated on a 65% (w/w) sucrose layer. A linear sucrose density gradient (1.23 - 1.15 g/cm3) was then formed over the ghost containing sucrose layer and centrifuged to equilibrium at 81,000 X g for 15 hours in a SW 27.1 Beckman rotor. Fractions were collected with an Isco fractionator with continuous W absorbance recording. The electrophoretic profile of membrane polypeptides was analyzed in polyacrylamide gels containing sodium dodecyl sulphate (SDS), using the discontinuous Tris-buffered system of Laemmli (15) with minor modifications. Separation gels were 7.5% polyacrylamide which were 0.57% crosslinked, containing 0.1% SDS.
RESULTS cation
The final
isopycnic
procedure
produced
centrated than
for
1.196
in
the first
Cl3/8
cells:
+ 0.005
obtained brane
fraction 0.75M
it
according
adsorbed
files,
types
18 major
molecular
weight
increased
quantities
was obtained in untreated cells. from
Cl318
177,000
material,
yielded
daltons
(Band
appeared
in
but
+ 0.005
g/cm3,
of the final
state
of large
they
described
could for
replicas in
transformed using experiments,
ghosts.
showed
the ghosts
band
cells. either
from both pro-
of nominal found
in
The same result trypsinised or band
(Band
in HS5 membranes
out
of non-
electrophoretic
migrating peak
mem-
be washed
removal
IO) was consistently
from
578
con-
HS5 cells
of
preparation
A prominent
as a double
were
for
respectively. controls
reproducible
a more rapidly
the purifi-
ghosts
denser density
Freeze-fracture
(Fig.]).
in membranes
10 independent In addition,
membranes,
experiments,
(16).
bands
Cell
was 1.186
microscopy
in an aggregated
of membranes
containing
density
to the conditions
ribosomes
peaks.
had a buoyant
ghost
in this
used
was somewhat
to be a homogeneous
present
intramembranous particles cell types (17). Both
ghosts
and electron
showed were
KCl,
specifically
which
16 and 9 independent
Some riboscmes with
of three
peak,
HS5 cell
contrast
centrifugation
a profile light
g/cm3 and the Cl318
from Phase
equilibrium
10) due to
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. I Electrophoretic patterns of the membrane proteins of C13/8 and H'S5 cells. Staining was with Coomasie Blue R 250. Direction of migration in the figure is downward. Gel A: membrane fraction of Cl318 cells. Gel B: membrane fraction of HS5 cells. Gel C contained standard proteins, from top to bottom: chicken muscle myosin, rabbit muscle phosphorylase B and bovine serum albumin. Mobilities of standard proteins were reproducible within + 4% and were linearly related to the logarithm of their molecular Geights.
the appearance 94,000
of a second
daltons. Three different
protein
criteria
having were
a nominal
used to quantitate 579
molecular
weight
of
the
increase
of
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
s 3.
, 2.
, 1.1
-0
60
90
Yolecrler
w6illL:b
Irll-31
250 Cl 6 I
B
*30
.2.0
.ltl
ac
0
580
BIOCHEMICAL
Vol. 79, No. 2, 1977
Band 10. Direct fold
increase.
sliced gels
UV scanning
(18)
Band
incubated
collagenase related
(Advance
polypeptides 10 could
to the conditions vesiculation
is also
for
components from
the membrane of actin
from human erythrocytes
ghosts
proteins
fairly
was
175 units
of
the same
that
collagen-
of the membrane
fraction.
preparation
by a
performed
according
and myosin
with
(19).
treatment
This
from Sarcoma
conco-
180 ascitic
(20).
Finally, HS5 cells were grown in culture 14 C-fucose in order to label their carbohydrate membranes
which
yielded
showing
M) treatment
the extraction
non-integral
of
Corporation) sample,
M) and KC1 (0.45
know to extract
ghosts
Biofacture
not be removed
used
from myosin
HS5 membranes
at 37°C in presence
are not major
EDTA (0.001
(Fig.ZB).
distinguished
as an untreated
dye from
of double-labelled
increase
of purified
two hours
profile
Band
cell
for
Form III
sequential
distribution
be electrophoretically A sample
of a three-
at 605 nm of eluted
of a two-fold
components.
electrophoretic
to the estimation
profile
and the radioactivity
10 could
previously
led
density
to the estimation
and collagen
mitant
(Fig.2A)
The optical
gels led
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
obtained
simple
corresponding and 90,000
from cells
electrophoretic to nominal
daltons
without
radioactivity molecular
(Fig.3).
to Band 10. When cells peak, as well electrophoretic
detached
initially
as the 200,000
peak peak,
gave a
with
of 200,000,
four
major
175,000,
in stained
detached
daltons
Fucose-labelled
trypsinisation pattern
weights
The second
were
medium containing chains.
gels
corresponded
by trypsinisation,
was not
found
peaks
115,000 this
in the
"C'fucose
pattern.
DISCUSSION These brane daltons
experiments
fucose-containing occurs
indicate protein
that
a significant
of apparent
in the virus-transformed
increase
molecular
cell
clone.
weight
This
of a mem177,000
protein
is
Quantitation of the electrophoretic patterns of Cl318 and Fig. 2 HS5 cell membrane proteins. A: Direct UV scanning of stained gels. Abcissa: mobility relative to bovine serum albumin, taken as 1.000. Ordinate: optical density at 280 run relative to that of band 9 (unvarying in both cell types) taken as an internal standard. Solid line: C13/8; dotted line: HS5. B: Double-labelling experiment: C13/8 and HS5 cells were grown for res48 hours in the presence of 14C and 3H labelled amino acids, pectively. Labelled membranes were isolated separately, then mixed and co-electrophoresed in the same gel. Direction of migration is from right to left. Ordinate: cpm of each gel slice as the percentage of total counts recovered. The
arrows
indicate
the
two
581
major
differences.
BIOCHEMICAL
Vol. 79, No. 2, 1977
30
II
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10
Sld”mmbw
0x( (WA ( Enlpnttnrr
I HI1
(I
14 Electrophoretic patterns of C fucose-labelled membrane from HS5 cells. After electrophoresis, the gel was stained and sliced and the radioactivity in each slice was determined. The diagram below the abcissa indicates the correspondence between slices and stained bands. Continuous line (m) corresponds to a membrane fraction purified from scraped, non-trypsinised cells. Protection against proteolysis was achieved by adding 0.2% diisopropyl-fluorophosphate to the collection and homogenisation media. Discontinuous line (A---A) corresponds to a membrane fraction purified from trypsinised cells. The arrow indicates the position of Band 10. Fig. 3 fractions
distinguishable
from myosin
association its
of this
enrichment
with
the
with
in a purified
total
cell
component
represents
proteins,
in the That
protein
and collagen
the plasma
plasma
homogenate.
components.
membrane
protein
is
labelled
is
indicated
preparation,
5% of the
case of the transformed
this
membrane
Densitometry
approximately
The specific by
compared
data
indicate
total
plasma
that
this
membrane
clone.
in
the presence
of radioactive
precursors
indicates that it is a product of cellular meta--in vitro bolism and is not a serum component which is adventitiously bound to the membrane. In addition, its labelling with fucose suggests that it
is
a glycoprotein.
significantly a small
portion
to be exposed Finally, protein,
Since
change
since
fucose
the migration
of the peptide
removal
upon
of the protein sequence
bound
is
not
removed
by trypsin,
582
band,
does not
apparently
to the sugar
at the cell surface. this protein has the characteristics it
trypsinisation chain
only seems
of an integral
EDTA and KC1 treatments.
BIOCHEMICAL
Vol. 79, No. 2, 1977
Although
we have no evidence
intramembranous
particles
quantitative
increase
transformed
clone
membranes
is
of the
protein/lipid
striking.
of membrane
a component
The increased cells
is
in
buoyant
also
of the
the parallel
and particles
the virus-
density
consistent
proteins
cell
of plasma
with
a higher
proteins certain
phenotype.
from
in
suggested
disorganization. transport it
in altered
is
it
could
for
in This
(21)
the change
and Isaka
17),
alter
could
some integral
their
the
the anchorage
which
or metabolites that
the increased
of transformed
which
may
transformed
of components
ultimately
result
proteins are
quantitative
insertion cells
are
critical increase
in
involved for
cell
may result
properties. a similar
clones
of BHKZI cells
rently
investigating
of virally
clone.
accompanying
if
Moreover, of ions
We found
(6,
alterations
conceivable
growth
optimal
daltons
by Stone
membrane
and the endocytoskeleton
in active
94,000
reported
the plasma
structural
division,
not
we nevertheless
the transformed
previously
For instance,
of the exo-
were
proteins,
systems.
As we have previously of integral
conditions weight
band of approximately
to those
in other
their
is
freeze-fractures,
the electrophoretic
an additional
explain
protein
the protein
of low molecular
may be similar (22)
seen in of both
transformed
Although
profile
this
ratio.
the resolution detected
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
177K protein
transformed
by hamster
sarcoma
the possibility
that
and chemically
increase
transformed
this
change
in virus.
two other We are cur-
is a general
feature
cells.
ACKNOWLEDGEMENTS One of us (A. L.D.) was Government (C.I.E.S.). We thank examination of plasma membrane B. Collandre, C. Axler-Blin and assistance.
supported by a fellowship of the French G. Torpier for the electron microscopy preparations and Mrs. J. Gruest, G. Jonkman for their skilful technical
REFERENCES I. 2. 3. 4. 5. 6. 7.
Nicolson, G.L. (1976) Biochim. Biophys. Acta 458, 1-72. Hynes, R.O. (1976) Biochim. Biophys. Acta 458, 73-107. Weber, K., Lazarides, E., Goldman, R.D., Vogel, A. & Pollack, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 2, 363-369. Pollack, R., Osborn, M. & Weber, K. (1975) Proc. Natl. Acad. Sci. USA 72-, 994-998. Singer, S.J. & Nicolson, G.L. (1972) Science 175, 720-731. Torpier, G., Montagnier, L., Biquard, J.M. & Gier, P. (1975) Proc. Natl. Acad. Sci. USA 72, 1695-1698. Branton, D. (1966) Proc. Natl. Acad. Sci. USA 55, 1048-1056.
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Vol. 79, No. 2, 1977
8. 9. IO.
Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Marchesi, V.T., Tillack, T.W., Jackson, R.L., Segrest, J.P. & Scott, R.E. (1972) Proc. Natl. Acad. Sci. USA 2, 1445-1449. Pinto da Silva, P. & Nicolson, G.L. (1974) Biochim. Biophys. Acta 363, 311-319. M. (1971) & "La L-asparaginase", MontagnG, L., Gruest, J. & Boccara, Colloque International CNRS n0197, pp. 159-164. Torpier, G., Gruest, J. & Montagnier, L. (1974) Exp. Cell Res. E, 437-442. Zavada, J. & MacPherson, I. (1970) Nature 225, 24-26. Montagnier, L. (1968) C. R. Acad. Sci. (Paz) D 267, 921-924. Torpier, G. 8 Montagnier, L. (1970) Int. .J. Cancer a, 529-535. 3, Warren, L., Glick, M.C. & Nass, N.K. (1966) J. cell Physiol. 269-287. Laemmli, U.K. (1970) Nature 227, 680-685. D.C. & Blobel, G. (1973) J. Cell Biol. 56, Aldeman, M.R., Sabatini, 206-229 * Montagnier, L. & Torpier, G. (1976) Bull. Cancer 63, 123-134. J. (1975) Fenner, C., Traut, R.R., Mason, D.T. & Wikman-Coffelt, Analyt. Biochem. 2, 595-602. Tilney, L.G. & Detmers, P. (1975) J. cell. Biol. 66, 508-520. D.R., Huggins, J.W. & Carraway, K.L. (1976) Moore, P.B., Anderson, Biochem. Biophys. Res. Commun. 2, 288-294. Stone, K.R., Smith, R.E. & Johlik, W.K. (1974) Virology 58, 86-100. Isaka, T., Yoshida, M., Owada, M. & Toyoshima, K. (1975) Virology 65, 226-237.
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