Vol. 64, No. 1, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ENHANCEMENT OF ANION PERMEABILITY IN LECITHIN VESICLES BY HYDROPHOBIC PROTEINS EXTRACTED FROM RED BLOOD CELL MEMBRANES* A. Rothstein, Research
Z.I.
Institute,
Received
Cabantchik"*,M
The Hospital
February
for
Sick
Balshin
and R. Juliano
Children,
Toronto,Ontario,
Canada
28,1975
SUMMARY: Triton X-100 extracts of membrane proteins from ghosts of normal and pronase treated cells enhance the anion permeability of lecithin vesicles. With proteins from cells pretreated with DIDS(4,4'-diisothiocyano-2,2'stilbene disulfonate), a specific inhibitor of anion transport, the anion On the basis that the Triton X-100 extracts permeability is not enhanced. are considerably enriched in a protein component of 95,000 molecular weight (or a 65,000 molecular weight segment in the case of pronase treated cells), and that DIDS is bound almost exclusively to the same proteins, it is suggested that the pronase resistant,65,000 molecular weight segment of the 95,000 molecular weight protein is directly involved in anion transport. It
has been proposed
ane,
a protein
from
its
tion
(1,2).
of apparent
position
4,4'-diisothiocyano
to proteins 65,000
range
molecular
weight
ment used had no effect molecular
weight
The band extractable
Triton the role
X-100,
is
segment
relationship
largely
localized
of this
protein.
contains
an anion
segment
3 protein
is
with
detergents
hydrophobically
the extraction
of band 3 proteins
(6,7). is
in anion
o 19 75 b-v Academic Press, Inc. of reproduction in any f&-m resewed.
with
it
the pronase that
site
(5).
associated
with
membrane
MA 4665. Institute
144
attempts
of Life
To further were
(95%)
resistant
transport
transport,
(2).
exclusively
was suggested
specific(6).
covalently
of inhibition
in a pronase Because
permea-
One comp-
binds
almost
3
disulfonic
In the case of the non-ionic
relatively
*The study was supported by MRC Grant **Present address: Biophysics Program, University, Jerusalem, Israel.
Cqyright AN rights
binds
band
in anion
(DIDS),
membr-
called
to the degree it
permeation,
a role
cell
transport(2,4).
disulfonate
on anion
(usually
from studies of anion
of concentrations,
3 and it
plays
primarily
inhibitors
in a linear
of 95,000 (3)),
2,2'-stilbene
(l),
in band
gels
derived
and specific
In the inhibitory
is
is
of the human red blood
weight
on SDS acrylamide
potent
to the membrane
a component
molecular
The evidence
stilbenes, ound,
that
treatthe 65,000
lipid
detergents establish
made to re-
Sciences,
and
Hebrew
BIOCHEMICAL
Vol. 64, No. 1,1975
constitute mine
Triton
X-100
the effects
extracted
AND BIOPHYSICAL
material
of such reconstitution
procedures
were
carried
inhibitory
concentrations
out with
in
and with
lecithin
on anion
extracts
from
extracts
RESEARCH COMMUNICATIONS
vesicles
permeability. cells
from
Parallel
pretreated
cells
and to deter-
with
pretreated
DIDS at
with
pronase. MATERIALS AND METHODS. Red blood cells obtained from fresh or recently outdated blood were washed several times with Hepes buffer saline (Hepes 2OmM, were pretreated for NaCl 14OmM, pH 7.4) at O'C. Aliquots o the suspension for 10 minutes with 0.1 10 minutes with 10 @I DIDS, 20 pit of { 5 HIDIDS and/or mg/ml of pronase at 370G as previously described(2). The method of detergent extraction was generally similar to that of Yu et al(6). One volume of ghosts in dilute Hepes buffered saline (Hepes lOmM, NaCl 2OmM) was treated with 3 volumes of Triton X-100 (Sigma, 1.0% w/v in dilute Hepes buffered saline,pH 7.4 at OOC) for 10 to 20 minutes followed by centrifugation at 30,000 x g for 10 minutes. Aliquots from the supematant and resuspended pellet were analyzed for 13H$IDS, protein content, and protein distribution by SDS acrylamide gel electrophoresis (3). The Triton X-100 extracted material from normal, DIDS-treated, and (Na SO4 51&l, pronase treated cells was placed in a buffered isotope solution Na235S04 lOOnCi, KC1 lOmM, EDTA 0.01 mM, Hepes 5mM, pH 7.0). One vo f ume was mixed with one volume of toluene containing lmg/ml of egg lecithin (Applied Sciences) to form a suspension(8). After low speed centrifugation (1000 rpm for 5 minutes), the clear aqueous phase was removed, N2 was bubbled through the {3HjDIDS-labelled material to remove traces of toluene and the sample was concentrated three to five fold with a Diaflo UM 20 E filter to a final protein concentration of 1.5 to 2.0 mg/ml. One ml was added to 200 g of pure egg or bovine brain lecithin (Applied Sciences) previously dried under vacuum and N2 and shaken vigorously under an N2 atmosphere. The protein-phospholipid dispersion was sonicated for 20 to 30 minutes at room temperature under N2 in a 1OOW bath sonicator (Heat Systems, Ultrasonics Inc.), to form vesicles. by a method reported previously (9). The The fluxes of 35S04- were determined external Na235S04 was removed from the vesicles by gel filtration on a Sephadex G-50 coarse column eluted with isotope free buffer. The 35~50~~ containing vesicles were placed in dialysis bags (0.33 in. flat dry diamzter). The flux of 35S04- into the bathing solution containing isotope free buffer was measured. Acrylamide gel electrophoresis was performed in 1% SDS by the method of Fairbanks et al(3). The 13H}DIDS distribution in gels was obtained by fractionation with a Savant gel extruder, solubilization overnight in a 10% protosol based solvent and counting in a Packard Liquid Scintillation Counter (2). Protein was estimated by a modified microbiuret method(l) that tolerates the presence of 1% Triton X-100. The detergent was measured chemically (10). RESULTS AND DISCUSSION. Triton and for
X-100
acid-Schiff
periodic
somewhat
glycoproteins.
ed in band ic
is
As demonstrated
3 (stained
specific
for
band
Thus in figure by
Coomassie
reagent).However,
acid-Schiff
previously
stained
1, the blue)
in the bands
3(95,000
were 145
extract
(6,7),
molecular
weight
was considerably
and glycoproteins case of pronase found
the extraction
and virtually
(stained treated
cells,
the only
by protein) enrich-
by periodno band
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PRONASE TREATED CELLS - . ..-_
CONTROL CELLS
2500,
3000
-GHOST
ii ~2000
2400
-EXTRACT
- GHOST
1
-EXTRACT
1
: \
g
1500,
s
1000
4
600
f m -
5oo
0.2
0.4
0.6
0.8
1.0 OIlGIN SDS FRONT
MOBILITY Figure
1.
Extraction of 13H}DIDS treated cells by Triton
labelled X-100
02 0:s SDS
0.2
FRONT
MOBILITY
proteins
from normal
and pronase
The upper sections are photographs of sodium dodecyl sulfate acrylamide gels of proteins derived from ghosts, or extracted by Triton X-100, stained for protein with Coomassie blue (CB) or for carbohydrate by periodic acid Schiff reagent (PAS). The lower sections are counts of 13H)~IDS in slices from gels prepared in parallel. In normal cells the counts are almost all at a position in the gel corresponding to band 3 (95,000 molecular weight) and in pronase treated cells, to band 3' (65,000 molecular weight).
stained
by Coomassie
molecular
weight
blue
(band
in the Triton
(band
3, and 85% is recovered 3')
labelled was found cells
after
pronase
material
is
in a single
to band 3', Quantitative
extract
was that
at 65,000
3').
Over 95% of C3H}DIDS bound in band
X-100
in the 65,000
treatment
also peak
and most
covalently
(2,5).
illustrated
information
molecular
The extraction in figure
corresponding of it
to the cell
1.
surface
weight
of the {3H)DIDS
The label
on the
146
in Triton
extraction
under
located
segment
in
to band 3, or in pronase
was extracted
is
the ghosts treated
X-100. the particular
BIOCHEMICAL
Vol. 64, No. 1,1975
EXTRACTION
TABLE 1
AND BIOPHYSICAL
OF 13H}~r~S-LAlrEL~~~ BY TRITON X-100
RESEARCH COMMUNICATIONS
GHOST PROTEIN
CONTROL CELLS
Protein
PRONASE TREATED CELLS
Radioactivity x 10 -3
Protein
Radioactivity
w/ml
cpm/ml
1.98(100)
1.299(100)
mglml
cpm/ml
Suspension
1.92(100)
1.020(100)
Extract
0.64(33)
867(85)
0.67(33)
977(77)
Pellet
1.30(68)
141(14)
1.28(65)
259 (20)
Values
in parentheses
conditions cells,
is
given
33% of the
i3H)DIDS
labelled
the labelled
30%(3).
It
ghost
protein
blue
protein
be calculated
that
in the Triton
X-100
protein
accounts
using
against
not
but
treated
the extraction
less
reported 3 must
Band 3 to be about
constitute
determined.
and by densitometry
known amounts
than
about
In the case of pronase
was more directly
of albumin
Assuming
behave
of Thus
enriched.
extract.
each other.
and albumin for
band
out by biuret gels
protein
protein,
the extract
carried
acrylamide
weight
the former
in
or pronase
was considerably ghost
that
the same with
85% of the
total
of to
the respect
to
extracted
protein. The Triton pronase
treated
cells
X-100 were
extracts
of ghosts
reconstituted
from normal,
in lecithin
147
-3
fraction.
85% and 77% respectively.
total
the two procedures
molecular
normal
was solubilized,
3 and 3')
were
stained
the particular
For either
of the
the total
in
was much higher,
(bands
determinations
standardize
staining,
total
fraction
total
cell,
Coomassie
65,000
1.
can therefore
85% of the
Parallel
Table
material a large
the % yield
in
material
involves
treated
denote
x 10
DIDS-treated
vesicles
by the
and
BIOCHEMICAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10 8 6 4 2 i TIME [hours)
Figure
2.
Anion Triton
permeability of lecithin vesicles X-100 extracted fractions.
constituted
with
1.2 to 2.0 mg of total protein derived from normal and DIDS-treated cells were reconstituted in lecithin vesicles (5OOug) as described in Methods. Control 2 contains pure phospholipids whereas Control 1 contains in addition 0.03% Triton X-100, the same concentration as that found in the protein reconstituted systems.
procedure
described
mixtures
reduced
the latter
in the Methods. the Triton
concentration
lecithin
vesicles,
reconstitution
concentration has only
the amount was determined
was added
to control
addition,
in each experiment
detergent
was pretreated
the anion
permeation
cells
were
the vesicles amount
carried for
fluxes. substantially
system
an aliquot
over,
non-specific
as illustrated
effect
on 35so--h over
of cells
The proteins
effects
fractions
patterns,
protein
served
148
In
with block
and DIDS-treated sulfate
did
not
fluxes alter
or the
of the
amount
of
as an internal
and of detergent
by the representative
of detergent
of sonication.
and the
increase
from
and irreversibly
gel
of proteins
Although
by protein
to be extracted
the DIDS treatment
the DIDS-treated
fluxes
from normal
in parallel,
0.03%.
sane amount
at the time
the acrylamide
The 95K- and 65K-rich
0.8% to about
DIDS to specifically (2).
toluene-lecithin
from
carried
(no protein)
Because
extracted,
with
in each case and the
reconstituted
was measured.
of protein
detergent control
with
a small
of detergent
vesicles
extracted,
Two extractions
the 35SO;experiment
on anion fluxes of figure
2.
BIOCHEMICAL
Vol. 64, No. 1,1975
The extent
of enhancement
different
experiments
amount
of protein
no increase (control
over l),
Fractions
controls
containing
to vesicles cells
immediately
50%.
interior
of pure
fluxes.
Firstly,
pronase
treatment)
anion
the
fluxes,
labelled
with
weight
is
that
located
amounts
without could
striking,
any effects
extracted the Triton
X-100
Some of the in the
on the sulfate
over
these
for
resulting
other
membrane
149
to enhance
the 95,000 1.
are
bound
DIDS
identifiable (5).
by pronase
or on DIDS inhibition observed are also are
that
has been
with
sialoglycoprotein
components
the
molecular
It
component
Some lipids
anion from
components
the reconstitution
protein
in the extracts.
cells
eliminated
cells.
weight
95% of the membrane
The only
permeability
treated
segment
in figure
however,
account
but
case
averaged
the enhanced
particular
4%) is the major is,
for
of DIDS for
range
on anion
(6),
to those
(2).
than
from pronase
treated
the
molecular
from DIDS-treated
inhibitory
therefore,
the DIDS
sites.
85% of the protein
as illustrated
protein
95,000
the 65,000
over
The specificity
of DIDS binding
not,
for
DIDS.
of DIDS (less
from
transport
responsible
(or
the effect
in that
component
protein
of protein
in the
but
or may be located
to the
component
limits
is
anion
(control
In this
variable
presumably
X-100
no Triton
step.
DIDS had no effect
strongly
accounts
failure
protein
reported
results
caused
vesicles.
point
this
cells
and pronase
and somewhat
vesicles.
3) as the membrane
and the
by adding
filtration
seven
of Triton
with
normal
in the vesicles
lecithin
The results
Secondly,
less
inward
of multilamellar
permeability
(band
the gel
inhibition
controls
from
in
DIDS-treated
be demonstrated
of some DIDS susceptible
may be oriented
fold)
preparation from
than
proteins
or after
The partial
to ten
the same concentration
also
was considerably
inaccessibility sites
with
before
three
obtained
higher
could
reconstituted
the inhibition about
somewhat effect
(from
on the particular
added.
The inhibitory
2).
was variable
depending
but were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not
This
treatment (5). from
It proteins
extracted significantly
by
BIOCHEMICAL
Vol. 64, No. 1,1975
labelled cannot
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
by DIDS under therefore
the
be directly
conditions involved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the present in the permeation
experiments
(Z),
and
function.
Cabantchik, Z.I., and Rothstein, A. (1972) .J.Membrane Biol, 10,311-330. Cabantchik, Z.I., and Rothstein, A. (1974) J.Membrane Biol, 15,207-226. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Biochemistry 10,2606-2617. Knauf, P., and Rothstein, A. (1971) J.Gen.Physiol, 58, 190-210. Cabantchik, Z.I., and Rothstein,A. (1974) J.Membrane Biol, 15,227-248. Yu, J., Fishman, D.A., and Steck, T.L. (1973) J.Supramol.Struct, 1,233-248. Rothstein, A., and Cabantchik, Z.I. (1974) in Comparative Biochemistry and Physiology of Transport, ed. Bloch, K., Bolis, L., and Luria, S.E. (North Holland Publ. Co., Amsterdam) in press. Gulik-Krizwicki, T. (1974) in Comparative Biochemistry and Physiology of Transport, ed. Bloch, K., Bolis, L., and Luria, S.E. (North Holland Publ. Co., Amsterdam) in press. Papahadjopoulos, D., and Watkins, J.C. (1967) Biochim. Biophys. Acta, 135, 639-652. Garewal, H.S. (1973) Anal. Biochem, 54,319-324.
150
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ENHANCEMENT OF ANION PERMEABILITY IN LECITHIN VESICLES BY HYDROPHOBIC PROTEINS EXTRACTED FROM RED BLOOD CELL MEMBRANES* A. Rothstein, Research
Z.I.
Institute,
Received
Cabantchik"*,M
The Hospital
February
for
Sick
Balshin
and R. Juliano
Children,
Toronto,Ontario,
Canada
28,1975
SUMMARY: Triton X-100 extracts of membrane proteins from ghosts of normal and pronase treated cells enhance the anion permeability of lecithin vesicles. With proteins from cells pretreated with DIDS(4,4'-diisothiocyano-2,2'stilbene disulfonate), a specific inhibitor of anion transport, the anion On the basis that the Triton X-100 extracts permeability is not enhanced. are considerably enriched in a protein component of 95,000 molecular weight (or a 65,000 molecular weight segment in the case of pronase treated cells), and that DIDS is bound almost exclusively to the same proteins, it is suggested that the pronase resistant,65,000 molecular weight segment of the 95,000 molecular weight protein is directly involved in anion transport. It
has been proposed
ane,
a protein
from
its
tion
(1,2).
of apparent
position
4,4'-diisothiocyano
to proteins 65,000
range
molecular
weight
ment used had no effect molecular
weight
The band extractable
Triton the role
X-100,
is
segment
relationship
largely
localized
of this
protein.
contains
an anion
segment
3 protein
is
with
detergents
hydrophobically
the extraction
of band 3 proteins
(6,7). is
in anion
o 19 75 b-v Academic Press, Inc. of reproduction in any f&-m resewed.
with
it
the pronase that
site
(5).
associated
with
membrane
MA 4665. Institute
144
attempts
of Life
To further were
(95%)
resistant
transport
transport,
(2).
exclusively
was suggested
specific(6).
covalently
of inhibition
in a pronase Because
permea-
One comp-
binds
almost
3
disulfonic
In the case of the non-ionic
relatively
*The study was supported by MRC Grant **Present address: Biophysics Program, University, Jerusalem, Israel.
Cqyright AN rights
binds
band
in anion
(DIDS),
membr-
called
to the degree it
permeation,
a role
cell
transport(2,4).
disulfonate
on anion
(usually
from studies of anion
of concentrations,
3 and it
plays
primarily
inhibitors
in a linear
of 95,000 (3)),
2,2'-stilbene
(l),
in band
gels
derived
and specific
In the inhibitory
is
is
of the human red blood
weight
on SDS acrylamide
potent
to the membrane
a component
molecular
The evidence
stilbenes, ound,
that
treatthe 65,000
lipid
detergents establish
made to re-
Sciences,
and
Hebrew
BIOCHEMICAL
Vol. 64, No. 1,1975
constitute mine
Triton
X-100
the effects
extracted
AND BIOPHYSICAL
material
of such reconstitution
procedures
were
carried
inhibitory
concentrations
out with
in
and with
lecithin
on anion
extracts
from
extracts
RESEARCH COMMUNICATIONS
vesicles
permeability. cells
from
Parallel
pretreated
cells
and to deter-
with
pretreated
DIDS at
with
pronase. MATERIALS AND METHODS. Red blood cells obtained from fresh or recently outdated blood were washed several times with Hepes buffer saline (Hepes 2OmM, were pretreated for NaCl 14OmM, pH 7.4) at O'C. Aliquots o the suspension for 10 minutes with 0.1 10 minutes with 10 @I DIDS, 20 pit of { 5 HIDIDS and/or mg/ml of pronase at 370G as previously described(2). The method of detergent extraction was generally similar to that of Yu et al(6). One volume of ghosts in dilute Hepes buffered saline (Hepes lOmM, NaCl 2OmM) was treated with 3 volumes of Triton X-100 (Sigma, 1.0% w/v in dilute Hepes buffered saline,pH 7.4 at OOC) for 10 to 20 minutes followed by centrifugation at 30,000 x g for 10 minutes. Aliquots from the supematant and resuspended pellet were analyzed for 13H$IDS, protein content, and protein distribution by SDS acrylamide gel electrophoresis (3). The Triton X-100 extracted material from normal, DIDS-treated, and (Na SO4 51&l, pronase treated cells was placed in a buffered isotope solution Na235S04 lOOnCi, KC1 lOmM, EDTA 0.01 mM, Hepes 5mM, pH 7.0). One vo f ume was mixed with one volume of toluene containing lmg/ml of egg lecithin (Applied Sciences) to form a suspension(8). After low speed centrifugation (1000 rpm for 5 minutes), the clear aqueous phase was removed, N2 was bubbled through the {3HjDIDS-labelled material to remove traces of toluene and the sample was concentrated three to five fold with a Diaflo UM 20 E filter to a final protein concentration of 1.5 to 2.0 mg/ml. One ml was added to 200 g of pure egg or bovine brain lecithin (Applied Sciences) previously dried under vacuum and N2 and shaken vigorously under an N2 atmosphere. The protein-phospholipid dispersion was sonicated for 20 to 30 minutes at room temperature under N2 in a 1OOW bath sonicator (Heat Systems, Ultrasonics Inc.), to form vesicles. by a method reported previously (9). The The fluxes of 35S04- were determined external Na235S04 was removed from the vesicles by gel filtration on a Sephadex G-50 coarse column eluted with isotope free buffer. The 35~50~~ containing vesicles were placed in dialysis bags (0.33 in. flat dry diamzter). The flux of 35S04- into the bathing solution containing isotope free buffer was measured. Acrylamide gel electrophoresis was performed in 1% SDS by the method of Fairbanks et al(3). The 13H}DIDS distribution in gels was obtained by fractionation with a Savant gel extruder, solubilization overnight in a 10% protosol based solvent and counting in a Packard Liquid Scintillation Counter (2). Protein was estimated by a modified microbiuret method(l) that tolerates the presence of 1% Triton X-100. The detergent was measured chemically (10). RESULTS AND DISCUSSION. Triton and for
X-100
acid-Schiff
periodic
somewhat
glycoproteins.
ed in band ic
is
As demonstrated
3 (stained
specific
for
band
Thus in figure by
Coomassie
reagent).However,
acid-Schiff
previously
stained
1, the blue)
in the bands
3(95,000
were 145
extract
(6,7),
molecular
weight
was considerably
and glycoproteins case of pronase found
the extraction
and virtually
(stained treated
cells,
the only
by protein) enrich-
by periodno band
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PRONASE TREATED CELLS - . ..-_
CONTROL CELLS
2500,
3000
-GHOST
ii ~2000
2400
-EXTRACT
- GHOST
1
-EXTRACT
1
: \
g
1500,
s
1000
4
600
f m -
5oo
0.2
0.4
0.6
0.8
1.0 OIlGIN SDS FRONT
MOBILITY Figure
1.
Extraction of 13H}DIDS treated cells by Triton
labelled X-100
02 0:s SDS
0.2
FRONT
MOBILITY
proteins
from normal
and pronase
The upper sections are photographs of sodium dodecyl sulfate acrylamide gels of proteins derived from ghosts, or extracted by Triton X-100, stained for protein with Coomassie blue (CB) or for carbohydrate by periodic acid Schiff reagent (PAS). The lower sections are counts of 13H)~IDS in slices from gels prepared in parallel. In normal cells the counts are almost all at a position in the gel corresponding to band 3 (95,000 molecular weight) and in pronase treated cells, to band 3' (65,000 molecular weight).
stained
by Coomassie
molecular
weight
blue
(band
in the Triton
(band
3, and 85% is recovered 3')
labelled was found cells
after
pronase
material
is
in a single
to band 3', Quantitative
extract
was that
at 65,000
3').
Over 95% of C3H}DIDS bound in band
X-100
in the 65,000
treatment
also peak
and most
covalently
(2,5).
illustrated
information
molecular
The extraction in figure
corresponding of it
to the cell
1.
surface
weight
of the {3H)DIDS
The label
on the
146
in Triton
extraction
under
located
segment
in
to band 3, or in pronase
was extracted
is
the ghosts treated
X-100. the particular
BIOCHEMICAL
Vol. 64, No. 1,1975
EXTRACTION
TABLE 1
AND BIOPHYSICAL
OF 13H}~r~S-LAlrEL~~~ BY TRITON X-100
RESEARCH COMMUNICATIONS
GHOST PROTEIN
CONTROL CELLS
Protein
PRONASE TREATED CELLS
Radioactivity x 10 -3
Protein
Radioactivity
w/ml
cpm/ml
1.98(100)
1.299(100)
mglml
cpm/ml
Suspension
1.92(100)
1.020(100)
Extract
0.64(33)
867(85)
0.67(33)
977(77)
Pellet
1.30(68)
141(14)
1.28(65)
259 (20)
Values
in parentheses
conditions cells,
is
given
33% of the
i3H)DIDS
labelled
the labelled
30%(3).
It
ghost
protein
blue
protein
be calculated
that
in the Triton
X-100
protein
accounts
using
against
not
but
treated
the extraction
less
reported 3 must
Band 3 to be about
constitute
determined.
and by densitometry
known amounts
than
about
In the case of pronase
was more directly
of albumin
Assuming
behave
of Thus
enriched.
extract.
each other.
and albumin for
band
out by biuret gels
protein
protein,
the extract
carried
acrylamide
weight
the former
in
or pronase
was considerably ghost
that
the same with
85% of the
total
of to
the respect
to
extracted
protein. The Triton pronase
treated
cells
X-100 were
extracts
of ghosts
reconstituted
from normal,
in lecithin
147
-3
fraction.
85% and 77% respectively.
total
the two procedures
molecular
normal
was solubilized,
3 and 3')
were
stained
the particular
For either
of the
the total
in
was much higher,
(bands
determinations
standardize
staining,
total
fraction
total
cell,
Coomassie
65,000
1.
can therefore
85% of the
Parallel
Table
material a large
the % yield
in
material
involves
treated
denote
x 10
DIDS-treated
vesicles
by the
and
BIOCHEMICAL
Vol. 64, No. 1, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10 8 6 4 2 i TIME [hours)
Figure
2.
Anion Triton
permeability of lecithin vesicles X-100 extracted fractions.
constituted
with
1.2 to 2.0 mg of total protein derived from normal and DIDS-treated cells were reconstituted in lecithin vesicles (5OOug) as described in Methods. Control 2 contains pure phospholipids whereas Control 1 contains in addition 0.03% Triton X-100, the same concentration as that found in the protein reconstituted systems.
procedure
described
mixtures
reduced
the latter
in the Methods. the Triton
concentration
lecithin
vesicles,
reconstitution
concentration has only
the amount was determined
was added
to control
addition,
in each experiment
detergent
was pretreated
the anion
permeation
cells
were
the vesicles amount
carried for
fluxes. substantially
system
an aliquot
over,
non-specific
as illustrated
effect
on 35so--h over
of cells
The proteins
effects
fractions
patterns,
protein
served
148
In
with block
and DIDS-treated sulfate
did
not
fluxes alter
or the
of the
amount
of
as an internal
and of detergent
by the representative
of detergent
of sonication.
and the
increase
from
and irreversibly
gel
of proteins
Although
by protein
to be extracted
the DIDS treatment
the DIDS-treated
fluxes
from normal
in parallel,
0.03%.
sane amount
at the time
the acrylamide
The 95K- and 65K-rich
0.8% to about
DIDS to specifically (2).
toluene-lecithin
from
carried
(no protein)
Because
extracted,
with
in each case and the
reconstituted
was measured.
of protein
detergent control
with
a small
of detergent
vesicles
extracted,
Two extractions
the 35SO;experiment
on anion fluxes of figure
2.
BIOCHEMICAL
Vol. 64, No. 1,1975
The extent
of enhancement
different
experiments
amount
of protein
no increase (control
over l),
Fractions
controls
containing
to vesicles cells
immediately
50%.
interior
of pure
fluxes.
Firstly,
pronase
treatment)
anion
the
fluxes,
labelled
with
weight
is
that
located
amounts
without could
striking,
any effects
extracted the Triton
X-100
Some of the in the
on the sulfate
over
these
for
resulting
other
membrane
149
to enhance
the 95,000 1.
are
bound
DIDS
identifiable (5).
by pronase
or on DIDS inhibition observed are also are
that
has been
with
sialoglycoprotein
components
the
molecular
It
component
Some lipids
anion from
components
the reconstitution
protein
in the extracts.
cells
eliminated
cells.
weight
95% of the membrane
The only
permeability
treated
segment
in figure
however,
account
but
case
averaged
the enhanced
particular
4%) is the major is,
for
of DIDS for
range
on anion
(6),
to those
(2).
than
from pronase
treated
the
molecular
from DIDS-treated
inhibitory
therefore,
the DIDS
sites.
85% of the protein
as illustrated
protein
95,000
the 65,000
over
The specificity
of DIDS binding
not,
for
DIDS.
of DIDS (less
from
transport
responsible
(or
the effect
in that
component
protein
of protein
in the
but
or may be located
to the
component
limits
is
anion
(control
In this
variable
presumably
X-100
no Triton
step.
DIDS had no effect
strongly
accounts
failure
protein
reported
results
caused
vesicles.
point
this
cells
and pronase
and somewhat
vesicles.
3) as the membrane
and the
by adding
filtration
seven
of Triton
with
normal
in the vesicles
lecithin
The results
Secondly,
less
inward
of multilamellar
permeability
(band
the gel
inhibition
controls
from
in
DIDS-treated
be demonstrated
of some DIDS susceptible
may be oriented
fold)
preparation from
than
proteins
or after
The partial
to ten
the same concentration
also
was considerably
inaccessibility sites
with
before
three
obtained
higher
could
reconstituted
the inhibition about
somewhat effect
(from
on the particular
added.
The inhibitory
2).
was variable
depending
but were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not
This
treatment (5). from
It proteins
extracted significantly
by
BIOCHEMICAL
Vol. 64, No. 1,1975
labelled cannot
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
by DIDS under therefore
the
be directly
conditions involved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the present in the permeation
experiments
(Z),
and
function.
Cabantchik, Z.I., and Rothstein, A. (1972) .J.Membrane Biol, 10,311-330. Cabantchik, Z.I., and Rothstein, A. (1974) J.Membrane Biol, 15,207-226. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Biochemistry 10,2606-2617. Knauf, P., and Rothstein, A. (1971) J.Gen.Physiol, 58, 190-210. Cabantchik, Z.I., and Rothstein,A. (1974) J.Membrane Biol, 15,227-248. Yu, J., Fishman, D.A., and Steck, T.L. (1973) J.Supramol.Struct, 1,233-248. Rothstein, A., and Cabantchik, Z.I. (1974) in Comparative Biochemistry and Physiology of Transport, ed. Bloch, K., Bolis, L., and Luria, S.E. (North Holland Publ. Co., Amsterdam) in press. Gulik-Krizwicki, T. (1974) in Comparative Biochemistry and Physiology of Transport, ed. Bloch, K., Bolis, L., and Luria, S.E. (North Holland Publ. Co., Amsterdam) in press. Papahadjopoulos, D., and Watkins, J.C. (1967) Biochim. Biophys. Acta, 135, 639-652. Garewal, H.S. (1973) Anal. Biochem, 54,319-324.
150
