- Email: [email protected]
Cell-free translation of apocytochrome b5 messenger RNA and analysis of the peptide product
Vol.
94, No.
May
30,
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 606-611
1980
CELL-FREE TRANSLATION OF APOCYTOCHROME b5 MESSENGER RNA AND ANALYSIS OF THE PEPTIDE PRODUCT P.A.
Krieter
and T.K.
Shires
Department of Pharmacology The University of Iowa Iowa City, Iowa 52242 Received
April
3,198O
SUMMARY Using a reticulocyte lysate system, total rat liver poly A+ RNA was translated in vitro and the apocytochrome b5 product examined by immuno-chemical methods. Demonstrably specific antisera to cytochrome b5 was produced in sheep against whole purified apoprotein isolated from rat liver microsomes. The results show that apocytochrome b5 is translated as a protein that is not appreciably different in size from that isolated from membranes. INTRODUCTION Cytochrome
b5,
an integral
composed
of a hydrophilic
end that
anchors
the
protein
domain
apoprotein
Recent
membranes
studies
to get
mitochondrial sequence lytically
are
at or near
is an additional ER protein ent with
to their
(5),
from
have
-c reductase shown that
synthesized N-terminus
the protein.
The G-coat
studied
such as apocytochrome the presence
EXPERIMENTAL
in a precursor
example
which
protein
form having
b5 is also
N-terminal
cross (4)
removed
or
proteo-
stomatitus
to know if
made as a large
such
a hydrophobic
of vesicular We wanted
is
structure
secretory
may be subsequently
(6,7).
of a non-anchoring
are
ER proteins
two-domain
in general they
(ER),
on the carboxyl
Other
have a similar
whether
that
reticulum
segment (1).
proteins
destination,
the
well
and a hydrophobic to the membrane
as NADH- and NADPH-cytochrome (2,3).
of the endoplasmic
precursor
hydrophobic
virus
an consist-
sequence.
METHODS
Cytochrome b5 Purification of cytochrome b5 and production of antisera: was purified from rat liver microsomes according to 0~01s (8). Approximately 3 mg in Freund's complete adjuvent was injected into a sheep; a second Blood was collected one injection of 3 mg was administered 4 weeks later. 0006-291X/80/100606-06$01.00/0 Copyrighr i 1980 b-vAcademrc Press, Inc. All rights of reproduction in anv form reserved.
606
Vol.
94, No.
BIOCHEMICAL
2, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Immunoelectrophoresis was week after the booster and processed for antisera. performed according to Crowle (10) using an ethyelenediamineacetic acid Microsomes were solubilized by buffer system containing 1.5% Triton X-100. was collected stirring overnight with 1.5% Triton X-100 at 5'C; the supernatant Immunoelectrophoretic plates after centrifugation at 100,000 xg for 1 hour. The equivalence point of were incubated for 2 days at 4OC before analysis. the sheep antisera, determined by radial immunodiffusion with serial dilutions of antisera, was 10 pg of cytochrome b5 precipitated by 0.1 ml of sheep antisera. Isolation of liver mRNA and cell-free translation: Rat liver mRNA was isolated according to Naguchi --et al. (10). Reticulocyte lysates were prepared according to Hunt and Jackson (11) using phenylhydrazine to induce anemia. Lysates were treated with micrococcal nuclease (12). Twelve micrograms of rat liver polyA+ RNA was incubated in 4 ml of lysate with 25 UCi of (3H)-leucine for one hour at 30°C. After translation, the lysate was diluted and Triton X-100 and sodium deoxyto one ml with phosphate-buffered saline, After cholate were added to final concentrations of 1% and 0.5%, respectively. centrifugation at 10,000 xg for 10 minutes to clarify the sample, 0.1 ml of antisera was added to the supernatant. The mixture was incubated at room temperature for one-half hour, and then 6 up of carrier protein was added and stored for 2 days at 4'. The immunoprecipitate was then pelleted and washed once with PBS containing l? Triton X-ID0 and 0.5% deoxycholate. The pellet was resuspended in buffer and pelleted through 1.0 M sucrose at 10,000 x g for 10 minutes. The whole tube was then frozen. The tip of the frozen tube was cut off and the thawed contents solubilized in 90 ~1 of SDSsolubilization buffer (13). The protein was run on Laemmli gels containing 12.5% acrylamide (14). Gels were frozen, sliced into 2 mm discs and solubilized in scintillation vials containing !i ml of NCS:H2@ (9:l) overnight at 4oOc. Ten ml of toluene scintillation fluid was added and the vials counted. Total incorporation into protein was determined by TCA-precipitation of 5 111 lysate and collection on GF/C filters which were then dissolved in 1 ml of NCS:H20 (9:l) and counted in a toluene fluor. Analytical methods and materials: Protein determinations were done according to Lowry --et al. (15) using bovine serum albumin as the standard. Cytochrome b5 concentrations were measured according to Omura and Sato (16) using an Aminco DW-2 spectrophotometer. All glassware was acid-washed and solutions were autoclaved or prepared with autoclaved water. (3H)-leucine (140 Ci/mmole) , proteinase K and NCS were purchased from New England Nuclear or Boehringer Mannheim. RESULTS The detergent-solubilized gels
and had an apparent
has also It
confirmed
had a specific
reported
its
cytochrome
content
weight
molecular
purity
of 20-23
the NADH-cytochrome
membranes
by about
60%, whereas
Tryptic
(D. Peterson,
nmoles/mg a similar
of the b5 -antisera
inhibited
of 17,000.
and identity
by West and Lu who used
The specificity
b5 was homogeneous
protein,
technique
was demonstrated
-c reductase
activity
NADPH-cytochrome
607
digest
unpublished identical
isolation
on SDS-acrylamide
in
of isolated -c reductase
analysis results).
to that (17). 3 ways.
First,
microsomal activity
in the
it
Vol.
94, No.
2, 1980
Figure
1.
was unaffected
of antisera
were
somes and the reductase activity
was affected
microsomes
were
corresponding
Thirdly,
that
BIOPHYSICAL
RESEARCH
a constant
COMMUNICATIONS
sera.
cpm of
the
diffusion
reticulocyte per
a single
b 5 was obtained when preimmune
with
lysate
system
5 ~1 lysate
micro-
enzymatic
antigen (Fig.
sera
incorporated
from rat
1+X Triton
component
the purified
liver
1).
band No
was used.
containing
a single
identity
608
of liver
microsomal
b5-antisera
with
increasing
when detergent-solubilized
in 1% agarose
reacted
and showed
(3H)-leucine
concentration
Second,
cytochrome detected
experiments,
Neither
with
were
the antisera
In these
determined.
of purified
double
microsomes
In our hands 25-30,000
with
activity
bands
Ouchterlony
solubilized
mixed
18).
immunoelectrophoresed
b 5 antigen
100 confirmed
(viz,
by preimmune
to that
cytochrome
AND
Immunoelectrophoresis of triton-solubilized microsomes and purified cytochrome b5. Electrophoresis was performed as described in Methods. The Ig fraction was put into the trough and allowed to react for 2 days before photographing. Solubilized microsomes are on the right, purified protein on the left.
same membranes amounts
BIOCHEMICAL
in the
protein. approximately polyA+
RNA.
X-
Vol.
94, No.
BIOCHEMICAL
2, 1980
BSA
Ov
+
4
AND
Try’gen
BIOPHYSICAL
Cyt
4
RESEARCH
COMMUNICATIONS
bS Cyt 5;
BPB
4
1
4
l \
\
16 -
\
\
\
\
\
14 -
\
\
\
\
\
\
12 ?
\
\
\
\ ‘\
lo-
0
* E P 0
‘1
\
\
a-
\
\
6.
c
_-
--20
10
Slice
Figure
2.
were
with
no added
labeled
bromphenol
number
RNA incorporated
of bands
weights
from
15,000
to over
lysates
with
anti-cytochrome
on SDS-polyacrylamide This not with
band
had the
excess
amounts
peak represented As a further apoprotein,
were
detected
70,000.
After
a single
sera
approximately corroboration
the
1 ml DEAE-cellulose
complete column
weight
by autoradiography
0.052 of the
lysate under
or when
identity
mixture conditions
the
total
cytochrome immune sera
(Fig.
was preincubated in the
radioactivity
incorporated.
to bind
cytochrome was passed the
2)
b5 and was
The radioactivity
incubation used
products
was detected
of the translated after
molecular
of incubated
of the antigenic
bS.
of the
When proteins
with
immunoprecipitation
as authentic
cytochrome
cpm.
run on SDS-po.yacrylamide
band of radioactivity
was used
of purified
2,000
and subsequently
same molecular
seen when preimmune
approximately
b5 and presentation
gels
weight
blue.
( 35 S)-methionine
with
a large
gels,
Number
SDS-polyacrylamide gel electrophoresis of immunoprecipitate. Immunoprecipitated product using immune sera. o ---O *-* Immunoprecipitated product using preimmune sera. Molecular standards: bovine serum albumin (67,000), ovalbumin (44,000), trypsinogen (24,000), cytochrome bg (17,000), cytochrome 2
(13,400), Lysates
\
b5
through
hemoprotein
a
Vol.
94, No.
during
2, 1980
its
BIOCHEMICAL
purification
cyanate.
(8).
AND
The protein
Immunoprecipitation
was then
labelled
cytochrome
b5 was detected
weight
as the authentic
protein.
BIOPHYSICAL
RESEARCH
was then done using
on the
eluted
with
the eluted
SDS gels
COMMUNICATIONS
with
0.25
M thio-
material
and
the same molecular
DISCUSSION The results chrome
b
is not
5
processing
protein
though,
in cytochrome not
it
(4)
protein
not
product
lysate
protease
the N-terminal alter
that
b5 (20).
Also,
the membrane
in the reticulocyte appears
that
form,
apocytochrome
there
and released
is into
(22).
Since
be exposed
outside
was released
from
(19). weight
fractions
unlikely. proteins of the
on SDS gels.
Sequence
side for
chains
glycosylation
(21).
cytosol
synthesized that
where
the hydrophobic
the ribosome
appears
needed
b5 is not
the large
Endogenous
Glycosylation
the good possibility the
precursor.
of any carbohydrate
system
apocyto-
on newly-synthesized
molecular
the presence
system
currently
acts
methionine
the apparent
lysate
larger
by lysate
support
the membrane would
in the reticulocyte
does not
precusor
polysomes
it
also
present
Since
into
as a significantly
removes
could
larger
synthesized
known rabbit
is one that
are
that
of the translational
The only
data,
demonstrate
tail
ribosomal
it
it
would is
only
subunit
in significantly is made on free incorporate 30 residues until
then long,
the apo-
(23).
ACKNOWLEDGEMENTS This
study
was partially
supported
by NIH Grant
GM12675 and NS12121.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad. Sci. 68, 10421046. Mihara, K., Sato, R., Sakakibaro, R. and Wada, II. (1978) Biochemistry 17, 2829-2834. Gum, J.R. and Strobel, H.W. (1979) J. Biol. Chem. 254, 4177-4185. Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. Cote, C., Solioz, M. and Schatz, G. (1979) J. Biol. Chem. 254, 14371439. Morrison, T.G. and Lodish, H.F. (1975) J. Biol. Chem. 250, 6955-6962. Rothman, J.E. and Lodish, H.F. (1977) Nature 269, 775-780.
610
Vol.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
94, No.
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ozols, J. (1974) Biochemistry 13, 426-434. Crowle, A.J. Immunodiffusion, 2nd Ed. (1973) Acad. Press, N.Y. Naguchi, T., Diesterhaft, M. and Granner, D. (1978) J. Biol. Chem. 253, 1332-1335. Hunt, T. and Jackson, R.J. (1974) in Modern Trends in Human Leukemia, eds. Neth, R., Gallo, R.C., Spiegelman, S. and Stahlman, F. (J.F. Lehmans, Verlag, Munich), pp. 300-307. Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247-256, Palmiter, R.D., Oka, T. and Schimke, R.T. (1971) J. Biol. Chem. 246, 724-737. Laemmli, V.K. (1970) Nature 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, P.-J. (1951) J. Biol. Chem. 193, 265-275. Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378. West, S.B. and Lu, A.Y.H. (1977) Arch. Biochem. Biophys. 182, 369-378. Baron, J. (1976) Arch. Biochem. Biophys. 174, 226-238. Housman, D., Jacobs-Lorena, M., Rajbhandary, V.L. and Lodish, H.F. (1970) Nature 227, 913-918. Ozols, J., Gerard, C. and Nobrega, G. (1976) J. Biol. Chem. 251, 67676774. Czichi, V. and Lennarz, W.J. (1977) J. Biol. Chem. 252, 7901-7904. Strittmatter, P., Rogers, M.J. and Spatz, L. (1972) J. Biol. Chem. 247, 7188-7194. Blobel, G. and Sabatini, D.P. (1970) J. Cell. Biol. 45, 130-145.
611
94, No.
May
30,
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 606-611
1980
CELL-FREE TRANSLATION OF APOCYTOCHROME b5 MESSENGER RNA AND ANALYSIS OF THE PEPTIDE PRODUCT P.A.
Krieter
and T.K.
Shires
Department of Pharmacology The University of Iowa Iowa City, Iowa 52242 Received
April
3,198O
SUMMARY Using a reticulocyte lysate system, total rat liver poly A+ RNA was translated in vitro and the apocytochrome b5 product examined by immuno-chemical methods. Demonstrably specific antisera to cytochrome b5 was produced in sheep against whole purified apoprotein isolated from rat liver microsomes. The results show that apocytochrome b5 is translated as a protein that is not appreciably different in size from that isolated from membranes. INTRODUCTION Cytochrome
b5,
an integral
composed
of a hydrophilic
end that
anchors
the
protein
domain
apoprotein
Recent
membranes
studies
to get
mitochondrial sequence lytically
are
at or near
is an additional ER protein ent with
to their
(5),
from
have
-c reductase shown that
synthesized N-terminus
the protein.
The G-coat
studied
such as apocytochrome the presence
EXPERIMENTAL
in a precursor
example
which
protein
form having
b5 is also
N-terminal
cross (4)
removed
or
proteo-
stomatitus
to know if
made as a large
such
a hydrophobic
of vesicular We wanted
is
structure
secretory
may be subsequently
(6,7).
of a non-anchoring
are
ER proteins
two-domain
in general they
(ER),
on the carboxyl
Other
have a similar
whether
that
reticulum
segment (1).
proteins
destination,
the
well
and a hydrophobic to the membrane
as NADH- and NADPH-cytochrome (2,3).
of the endoplasmic
precursor
hydrophobic
virus
an consist-
sequence.
METHODS
Cytochrome b5 Purification of cytochrome b5 and production of antisera: was purified from rat liver microsomes according to 0~01s (8). Approximately 3 mg in Freund's complete adjuvent was injected into a sheep; a second Blood was collected one injection of 3 mg was administered 4 weeks later. 0006-291X/80/100606-06$01.00/0 Copyrighr i 1980 b-vAcademrc Press, Inc. All rights of reproduction in anv form reserved.
606
Vol.
94, No.
BIOCHEMICAL
2, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Immunoelectrophoresis was week after the booster and processed for antisera. performed according to Crowle (10) using an ethyelenediamineacetic acid Microsomes were solubilized by buffer system containing 1.5% Triton X-100. was collected stirring overnight with 1.5% Triton X-100 at 5'C; the supernatant Immunoelectrophoretic plates after centrifugation at 100,000 xg for 1 hour. The equivalence point of were incubated for 2 days at 4OC before analysis. the sheep antisera, determined by radial immunodiffusion with serial dilutions of antisera, was 10 pg of cytochrome b5 precipitated by 0.1 ml of sheep antisera. Isolation of liver mRNA and cell-free translation: Rat liver mRNA was isolated according to Naguchi --et al. (10). Reticulocyte lysates were prepared according to Hunt and Jackson (11) using phenylhydrazine to induce anemia. Lysates were treated with micrococcal nuclease (12). Twelve micrograms of rat liver polyA+ RNA was incubated in 4 ml of lysate with 25 UCi of (3H)-leucine for one hour at 30°C. After translation, the lysate was diluted and Triton X-100 and sodium deoxyto one ml with phosphate-buffered saline, After cholate were added to final concentrations of 1% and 0.5%, respectively. centrifugation at 10,000 xg for 10 minutes to clarify the sample, 0.1 ml of antisera was added to the supernatant. The mixture was incubated at room temperature for one-half hour, and then 6 up of carrier protein was added and stored for 2 days at 4'. The immunoprecipitate was then pelleted and washed once with PBS containing l? Triton X-ID0 and 0.5% deoxycholate. The pellet was resuspended in buffer and pelleted through 1.0 M sucrose at 10,000 x g for 10 minutes. The whole tube was then frozen. The tip of the frozen tube was cut off and the thawed contents solubilized in 90 ~1 of SDSsolubilization buffer (13). The protein was run on Laemmli gels containing 12.5% acrylamide (14). Gels were frozen, sliced into 2 mm discs and solubilized in scintillation vials containing !i ml of NCS:H2@ (9:l) overnight at 4oOc. Ten ml of toluene scintillation fluid was added and the vials counted. Total incorporation into protein was determined by TCA-precipitation of 5 111 lysate and collection on GF/C filters which were then dissolved in 1 ml of NCS:H20 (9:l) and counted in a toluene fluor. Analytical methods and materials: Protein determinations were done according to Lowry --et al. (15) using bovine serum albumin as the standard. Cytochrome b5 concentrations were measured according to Omura and Sato (16) using an Aminco DW-2 spectrophotometer. All glassware was acid-washed and solutions were autoclaved or prepared with autoclaved water. (3H)-leucine (140 Ci/mmole) , proteinase K and NCS were purchased from New England Nuclear or Boehringer Mannheim. RESULTS The detergent-solubilized gels
and had an apparent
has also It
confirmed
had a specific
reported
its
cytochrome
content
weight
molecular
purity
of 20-23
the NADH-cytochrome
membranes
by about
60%, whereas
Tryptic
(D. Peterson,
nmoles/mg a similar
of the b5 -antisera
inhibited
of 17,000.
and identity
by West and Lu who used
The specificity
b5 was homogeneous
protein,
technique
was demonstrated
-c reductase
activity
NADPH-cytochrome
607
digest
unpublished identical
isolation
on SDS-acrylamide
in
of isolated -c reductase
analysis results).
to that (17). 3 ways.
First,
microsomal activity
in the
it
Vol.
94, No.
2, 1980
Figure
1.
was unaffected
of antisera
were
somes and the reductase activity
was affected
microsomes
were
corresponding
Thirdly,
that
BIOPHYSICAL
RESEARCH
a constant
COMMUNICATIONS
sera.
cpm of
the
diffusion
reticulocyte per
a single
b 5 was obtained when preimmune
with
lysate
system
5 ~1 lysate
micro-
enzymatic
antigen (Fig.
sera
incorporated
from rat
1+X Triton
component
the purified
liver
1).
band No
was used.
containing
a single
identity
608
of liver
microsomal
b5-antisera
with
increasing
when detergent-solubilized
in 1% agarose
reacted
and showed
(3H)-leucine
concentration
Second,
cytochrome detected
experiments,
Neither
with
were
the antisera
In these
determined.
of purified
double
microsomes
In our hands 25-30,000
with
activity
bands
Ouchterlony
solubilized
mixed
18).
immunoelectrophoresed
b 5 antigen
100 confirmed
(viz,
by preimmune
to that
cytochrome
AND
Immunoelectrophoresis of triton-solubilized microsomes and purified cytochrome b5. Electrophoresis was performed as described in Methods. The Ig fraction was put into the trough and allowed to react for 2 days before photographing. Solubilized microsomes are on the right, purified protein on the left.
same membranes amounts
BIOCHEMICAL
in the
protein. approximately polyA+
RNA.
X-
Vol.
94, No.
BIOCHEMICAL
2, 1980
BSA
Ov
+
4
AND
Try’gen
BIOPHYSICAL
Cyt
4
RESEARCH
COMMUNICATIONS
bS Cyt 5;
BPB
4
1
4
l \
\
16 -
\
\
\
\
\
14 -
\
\
\
\
\
\
12 ?
\
\
\
\ ‘\
lo-
0
* E P 0
‘1
\
\
a-
\
\
6.
c
_-
--20
10
Slice
Figure
2.
were
with
no added
labeled
bromphenol
number
RNA incorporated
of bands
weights
from
15,000
to over
lysates
with
anti-cytochrome
on SDS-polyacrylamide This not with
band
had the
excess
amounts
peak represented As a further apoprotein,
were
detected
70,000.
After
a single
sera
approximately corroboration
the
1 ml DEAE-cellulose
complete column
weight
by autoradiography
0.052 of the
lysate under
or when
identity
mixture conditions
the
total
cytochrome immune sera
(Fig.
was preincubated in the
radioactivity
incorporated.
to bind
cytochrome was passed the
2)
b5 and was
The radioactivity
incubation used
products
was detected
of the translated after
molecular
of incubated
of the antigenic
bS.
of the
When proteins
with
immunoprecipitation
as authentic
cytochrome
cpm.
run on SDS-po.yacrylamide
band of radioactivity
was used
of purified
2,000
and subsequently
same molecular
seen when preimmune
approximately
b5 and presentation
gels
weight
blue.
( 35 S)-methionine
with
a large
gels,
Number
SDS-polyacrylamide gel electrophoresis of immunoprecipitate. Immunoprecipitated product using immune sera. o ---O *-* Immunoprecipitated product using preimmune sera. Molecular standards: bovine serum albumin (67,000), ovalbumin (44,000), trypsinogen (24,000), cytochrome bg (17,000), cytochrome 2
(13,400), Lysates
\
b5
through
hemoprotein
a
Vol.
94, No.
during
2, 1980
its
BIOCHEMICAL
purification
cyanate.
(8).
AND
The protein
Immunoprecipitation
was then
labelled
cytochrome
b5 was detected
weight
as the authentic
protein.
BIOPHYSICAL
RESEARCH
was then done using
on the
eluted
with
the eluted
SDS gels
COMMUNICATIONS
with
0.25
M thio-
material
and
the same molecular
DISCUSSION The results chrome
b
is not
5
processing
protein
though,
in cytochrome not
it
(4)
protein
not
product
lysate
protease
the N-terminal alter
that
b5 (20).
Also,
the membrane
in the reticulocyte appears
that
form,
apocytochrome
there
and released
is into
(22).
Since
be exposed
outside
was released
from
(19). weight
fractions
unlikely. proteins of the
on SDS gels.
Sequence
side for
chains
glycosylation
(21).
cytosol
synthesized that
where
the hydrophobic
the ribosome
appears
needed
b5 is not
the large
Endogenous
Glycosylation
the good possibility the
precursor.
of any carbohydrate
system
apocyto-
on newly-synthesized
molecular
the presence
system
currently
acts
methionine
the apparent
lysate
larger
by lysate
support
the membrane would
in the reticulocyte
does not
precusor
polysomes
it
also
present
Since
into
as a significantly
removes
could
larger
synthesized
known rabbit
is one that
are
that
of the translational
The only
data,
demonstrate
tail
ribosomal
it
it
would is
only
subunit
in significantly is made on free incorporate 30 residues until
then long,
the apo-
(23).
ACKNOWLEDGEMENTS This
study
was partially
supported
by NIH Grant
GM12675 and NS12121.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad. Sci. 68, 10421046. Mihara, K., Sato, R., Sakakibaro, R. and Wada, II. (1978) Biochemistry 17, 2829-2834. Gum, J.R. and Strobel, H.W. (1979) J. Biol. Chem. 254, 4177-4185. Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. Cote, C., Solioz, M. and Schatz, G. (1979) J. Biol. Chem. 254, 14371439. Morrison, T.G. and Lodish, H.F. (1975) J. Biol. Chem. 250, 6955-6962. Rothman, J.E. and Lodish, H.F. (1977) Nature 269, 775-780.
610
Vol.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
94, No.
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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