Vol. 45, No. 4, 1971
BIOCHEMICAL
EXTERIOR
PROTEINS
ON THE
David
13,
ERYTHROCYTE
and Martin
of Biochemistry,
Research
September
HUMAN
R. Phillips
Laboratories
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hospital,
Morrison
St. Jude
Memphis,
MEMBRANE
Children’s
Tennessee
38101
1971
SUMMARY Membranes from pronase treated human erythrocytes were isolated and the membrane proteins se arated by disc gel electrophoresis in sodium dodecyl sulfate. A comparison of pronase treated ccl Ps and untreated cells show that pronase treatment of intact human erythrocytes results in the alteration of three membrane proteins with molecular weights of 90,000, 95,000, 105,000 and reduces their molecular size. All of the proteins with molecular weights of 90,000 and 105,000 were altered indicating that all proteins with these molecular weights must be exposed on the outside of the membrane. Protein fragments formed from proteolytic di stion remain with the membrane, and have an a parent molecular weight of 65,000. The residual ffragments are insensitive to further hydrolysis, an B appear to be portions of the hydrolyzed proteins which is buried within the membrane structure.
INTRODUCTION
Evidence membrane the
from
a number
is asymmetric.
exterior
surface.
neuraminidase
(l),
proteolytic
All
proteins.
From
data Most
sialic
most acid
carbohydrate
(2-4).
membrane,
membrane
investigators
example,
the
while
enzymes
erythrocyte
For
of
have of the
One
exception
is the acetylcholine
appears
reagents
which
react
evidence
that Most
membrane exposed
to be with
on
protein
the
convincing
with
to the
exterior
finding surface
suggesting
treatment
groups,
although
asymmetric
that
the
proteins
cell
not
distribution in
the
removed
in the
of functional
the inside
of the red cell. by
proteolytic
(8,9). have
membrane proteins
Chemical
also
provided
(10,ll). red
cell
class
of 90,000
are
lactoperoxidase
(12).
The
weight by
by
2,3-diphosphoglycerate
definitive,
of
on with
present
membrane
molecular iodinated
be
can be removed
in the erythrocvte
can be catalytically
1103
red
is exposed
activities
(5),
towards
which
of the
erythrocyte
distribution
ATPase
surface
to
can
an asymmetric
activity
human
of erythrocytes
of enzyme
(7) are oriented
assembled
the
determinants
including
regard
and
by
antigenic
esterase
functional
are asymmetrically
recent
removed
activities,
exterior
that
of the membrane
the localization
enzymes
the proteins
is the
of
enzyme
as the glycolytic
and
be
accumulated
(6) as well
enzymes
can
studies
suggested
of the carbohydrate
containing
phosphatase notable
has
in
the
BIOCHEMICAL
Vol. 45, No. 4, 1971
remaining
membrane
iodination
(13).
of the
cell,
proteins,
These
whiie
data
most
however, indicate
can only
that
membrane
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
only
proteins
be iodinated
when
a few membrane
are exposed
the
proteins
cell
was
lysed
are exposed
to the hemoglobin
prior
to
to the outside
containing
cell
interior
(13). Pronase amino
acids
is a non-specific (14).
erythrocyte
This
enzyme
membrane
examined
in order
protease has been
surface
(2).
to further
which
will
shown
those
Human cells were
erythrocytes
were
suspended
in 0.106
then
25 pgg/ml pronase periods were
of time, then
(Calbiochem
procedure
was repeated
membranes
were
sodium
a second
isolated
dodecyl
M phosphate
buffer,
time
and
modified
from
the
by pronase
are
of the erythrocyte
0.106
as
at 37OC
described
membrane.
electrophoresed
The
on 5%
&scribed
to a hematocrit constant
at 1,500
Xg
M phosphate
(13).
previously
with
buffer
to free the cells of pronase
as previously
sulfate
proteins
and centrifuged
of the
to their
carbohydrate
proteins
pH 7.4,
and incubated to 4’C,
in 10 volumes
completely
AND METHODS
of serum
cooled
almost
of the
on the outside
free
B grade)
the cells were
suspended
washed
much
membrane
the proteins
MATERIALS
proteins
to remove
In this report,
characterize
hydrolyze
(13).
of 20% containing shaking.
for
After
polyacrylamide
varying
I5 minutes.
and again
proteins gels by
The
centrifuged.
and the hydrolysis
membrane
The
cells This
products.
were
The
solubilized
standard
in
procedures
(13). RESULTS
The
effect
composition
of pronase
is shown
erythrocytes.
The
Identical
protein
60 minutes
Pronase membrane
of
B and
concentrations
is decreased.
Band
molecular
III,
weights
separate
components.
Concomitant
proteins
is an increase
of proteins
pronase
1 represent
alter the
at 37’C
for
the membrane
15 and molecular
molecular
weight
protein
and the
95,000,
decrease
a molecular
1104
weight
respectively, in
the
isolated
published 0.106
from
untreated (13,15).
M phosphate
proteins
from
molecular
buffer
of
between
90,000
removed.
become
identifiable molecular
IV. After
from After
is completely
Band
selected
composition respectively.
weights
for
the membrane.
weights
protein
90,000-105,000
of 65,000,
protein
previously
60 minutes,
with
105,000
with
does
remove
of proteins
the 90,000
with
in the
do not
on the membrane
proteins
to that
cells incubated
however,
C in Fig.
of
is similar
conditions
cells,
25 &g/ml
105,000
with
the
Gels
the
II,
these
with
incubation,
and
that
from
erythrocytes
the membrane
proteins
obtained
indicating
minutes
human
1. Gel A represents
were
treatment
treated
of intact
of separated
patterns
proteins.
erythrocytes
in Fig.
pattern
at 37OC,
treatment
AND DISCUSSION
15 and
Bands
I
as two weight
treatment
of
Vol. 45, No. 4, 1971
BIOCHEMICAL
AND BIOPHYSICAL
---B ?I,a_
RESEARCH COMMUNICATIONS
C
Figure 1 - Acrylamide gels of membrane proteins from normal and pronase treated erythrocytes. Stroma were isolated from untreated erythrocytes, gel A, and from er throcytes treated at a 20% hematocrit with 25 pg/ml pronase at 370C for 15 and 60 minutes, ge r s B and C respectively. The stroma were solubilized in 3% SDS, electrophoresed on 5% polyacrylamide gels containin 0.1% SDS and stained for protein with coomassie brilliant blue. Protein molecular weight bands Habeled correspond to molecular weights of 105,000, 95,000, and 90,000, and 65,000, numbers I, II, III and IV respectively.
the
erythrocytes
for
60
concentration
of
protein
concentration
of
the
minutes,
this
in Band
I, and
other
protein
pattern
is further
a further
molecular
accentuated
increase
weight
in
classes
the are
with
a decrease
intensity not
of
affected
Band by
in IV.
the
the The
pronase
treatment. These
protein
stained
gels shown
weight
regions,>
in protein reduced occur
which
alterations in Fig.
this time
2. No However,
coincides
with
by
I5
an additional
more
alterations
200,000.
in intensity after
are
easily
in protein
in the regions
the increase
minutes
of Bands
treatment,
incubation.
by
the
composition
in the amount
pronase
45 minutes
visualized
Band
densitometer appear
tracings
in the higher
I, II and III there of protein
in Band
however,
no further
I, however,
molecular
is a marked IV. Band changes
is completely
of the
decrease
II, is greatly in this
removed
band during
period. The
present
results
molecular
weights
of
molecular
weights
must
90,000
show and
be on the
that
pronase
105,000. outside
reduces This
the
means
of the membrane.
1105
molecular that
weight
all membrane The
95,000
protein
of all proteins
with
proteins
these
with
molecular
weight
BIOCHEMICAL
Vol. 45, No. 4, 1971
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
J L Figure 2 - Densitometer the gels and the numbers
class is only
partially
One
is digested
are
of these identical
different buried
with
proteins:
tracings of the ac lamide gels shown in Fig. 1. The to the protein mo 7 ecular weight classes in Fig. 1.
removed
by pronase
and
one is unaffected
different one
located
in the membrane Band
hydrolytic concentration
with
from
pronase
further
1 and
resulting time
membrane
present
pronase
types
that
these
or alternatively
in the stroma treatment.
treatment by
of two
It is possible
of the membrane
and
correspond
thus
they
of proteins. two
may
digestable
to
proteins
be entirely and the other
to the enzyme.
2 is not
of pronase
hydrolysis
the
on the outside
from
to be composed
by this enzyme.
in
and inaccessible
IV of Figs. product
orientation
and appears
letters
non
1106
untreated
erythrocytes
and is a
Since
this
protein
material
increases
that
this
peptide
fragment
is protected
and
this
it appears
protein
from
membrane
components,
that
lack
in
of
Vol. 45, No. 4, 1971
hydrolysis
is a function The
90,000
new
That
pronase
formed
molecular
this
susceptibility
of the membrane
peptide
to 105,000
lipid.
BIOCHEMICAL
65,000
of
the
fragment
concentration
organization,
in the present weight
peptide
region is not
and not
study
which
to
could
by
further
RESEARCH COMMUNICATIONS
of the protein represent
are buried
covered
fragment
used
AND BIOPHYSICAL
seems
hydrolysis.
intact
erythrocytes
if all the proteins
altered
by pronase
a portion
of proteins
in the membrane
protein
to incubate
structure.
and are covered
evident
Indeed,
due
to the
a twenty-fold
has no effect
in the by
lack
of
increase
on this peptide
of
molecular
weight. It is not
clear
on the membrane
surface.
Lactoperoxidase
labeling
90,000
molecular
of
the
polyacrylamide in this
disc
90,000
susceptible
are indeed
on the
probably protein
and
iodination
Since
this
are in essential
(12).
giving
becomes from
by carbohydrate
class
further
because
surface.
Thus,
lack
results
two
molecular
weight
weight
the
these
of exposed
tyrosines
proteins proteins
surface enzyme
proteins
5%
are
class of proteins
proteolytic
latter
in the by
different
on the membrane
when
components
as estimated
that
this molecular
proteins
solution
of the
proteins
90,000
that
protein
erythrocytes
indicates
These
other
the
the membrane
either
membrane
evidence
to
represent
of intact
evidence
(16).
The
exposed
of
Recent
of the membrane.
carbohydrate
iodination
class are labeled
to pronase
that
lactoperoxidase covered
weight
sruface
protein
weight
gel electrophoresis
molecular
also very
catalyzed
treatment
or more removes
are inaccessible or because
they
to are
or protein.
manuscript
agreement
was completed, with
the data
two
presented
publications
(17,18)
have appeared
whose
results
in this paper.
ACKNOWLEDGEMENTS
The This
work
National
authors was
Institutes
gratefully
supported of Health
acknowledge in part
by
the
grant
capable
number
technical
GM-15913,
assistance DE-02792
of Miss
Becky
Wailer.
and GM-39472
of the
and by ALSAC.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
E.H. Eylar, M.A. Maloof, O.V. Brody and J.O. Oncley, J. Biol. Chem., G.M.W. Cook and E.H. Eylar, Biochim. Biophys. Acta, 101:57 (1965). SKornfeld and R. Kornfeld, Proc. Nat. Acad. Sci., 63: 1439 (1969). R.J. Winzler, E.D. Harris, D.I. Pekas, C.A. Johnson and P. Weber, (1967). V.T. Marchesi and G.E. Palade, J. Cell Biol., 35:385 (1967). W. Schroter and M. Neuvians, J. Membrane Biol., 2:31 (1970). D.E. Green, G. Murer, H.O. Gultin, S.H. Richardson, B. Salmon, G.P. Arch Biochem. Biophys. 112:635 (1965). B.G. Firkin, R.W. Bed, G. Mitchell, Australian Annals of Med., 12: 26 F.Herz, E. Kaplan and J.H. Stevenson, Nature, 200:901 (1963). H.C. Berg, Biochim. Biophys. Acta, 183:65 (1969).
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
M.B. Bellhorn, 0.0. Blumenfeld and P.M. Gallor, Biochem. Biophys. Res. Commun., (1970). D.R. Phillips and M. Morrison, Biochem. Biophys. Res. Commun., 40:284 (1970). D.R. Phillips and M. Morrison, Biochemistry, 10: 1066 (1971). Nomoto, J,, Biochem. (Japan), 48:598,900 (1960). T. Lenard. Biochemistrv. 9: 1129 (1970). D.R. Philb s and M. Mbrrison, Fed. Prdc., 30, 1065, 1971. W.W. Ben cfer, H. Garan and A.C. Berg, J. Mol. Biol., 58:783 (1971). M.S. Bretscher, Nature, 231:229 (1971).
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