Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 874-885
October 30, 1978
BIOSYNTHESIS OF A UBIDUITIN-RELATED IN RAT BRAIN
AND IN
PITUITARY H.
Scherrer,
N.G.
Protein Clinical
HUFIAN AND MOUSE TUMORS
Seidah,
11. Lis
S.
and
and Pituitary Research
M.
Benjannet,
P.
Crine,
Chretien
Hormone Laboratory Institute of Montreal,
Affiliated the
PEPTIDE
to
HDtel-Dieu
de
Montreal
de
Montreal"
and the
Received
September
"Universite
1,1978
SUMMARY. A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH2 terminal amino acid a protein sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalamus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatograph this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems. INTRODUCTION Ubiquitin in
bovine
Requests Research
P.Q.,
thymus
is (1,2)
for reprints Institute Canada.
of
a polypeptide
of
during
isolation
should Montreal,
be
sent to 110 Pine
0006-291X/78/0844-0874$01.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
874
74
amino
of
the
acids, thymic
first polypeptide
identified hormone
Dr Michel Chretien, Clinical Avenue West, Montreal H2W lR7,
BIOCHEMICAL
Vol. 84, No. 4, 1978
thymopoietin.
It
animal
cells
higher
plants
human
and
in
cellular
bovine
receptor been
and
(1).
bovine
to
nuclear
products structural
the or
been
suggested
ref.
6-8).
for
in
ACTH
sequence
of
ubiquitin
peptides
was by
a similar rat
secreting
pituitary
pituitary
tumor
identity
isolated
of
further
established
graphic
properties.
found
of
to
a protein
tumors;
the
(AtT-20),
the peptide
on The
the
first second
with
one one
native
basis
of
biosynthetic
875
the bank
has
was was
purified
their
was
a
beta-
partial either
to
(10).
In
characterized in
two
mouse tumor.
ubiquitin
has and of
in
ACTH-
pituitary
electrophoretic
characterization
has
of
experimental
a human
it
This
search
and an
the
amino-terminal
been
striatum,
in
demonstrated
the
unrelated
of
see
studies
peptides:
data
as
review,
(9).
peptide and
involved
laboratory
with
has
likely
genome
biosynthetic
segment
hypothalamus
be
was
match
ubiquitin
most
(for
peptide
lympho-
component
ubiquitin
related
biosynthesized
brain,
this
and
then
in
a beta-adrenergic of
are
proteins
this
course
radiolabeled
or
The
the
via
eukaryotic
of in
ACTH
beta-LPH
of
intermedia
cyclase
peptides
the
distribution,
important
non-histone
could
biosynthesis
during
adenylate
the
chromosomal
vitro
an
sequence
two
of
wide
through
non-selective
acting
of
Ubiquitin
beta-endorphin, a pure
areas
that
these
(4).
pars
of
two
(5);
non-histone
first
sequence
report,
to
properties
discovery
lipotropin,
this
gene
of
amino-terminal
identical
its
induces
presumably
the
A-24
same
pituitary
fortuitous
(4),
be
activation
in
conserved
has
it
and
identical
highly
ubiquitin
of
bacteria
is
and
vitro
variety
yeasts,
been
structure
In
a large
which
have
that
tissues,
functional
The bovine
other
protein
of
its
unknown.
Recently
discovered
of
and
in
to
suggest
differentiation
in
ubiquitin,
appears
cells, still
precursors
detected
of
constancy
living
function,
cyte
in
the
(3)
RESEARCH COMMUNICATIONS
invertebrates,
sequence
species
all
lymphocyte
subsequently and
The
Both in
been
vertebrates (1).
evolution. likely
has
AND BIOPHYSICAL
been
chromato-
ubiquitin
Vol. 84, No. 4, 1978
could
provide
BIOCHEMICAL
a useful
of
its
biosynthesis,
in
the
field
MATERIALS
of
tool and
non
its
AND BIOPHYSICAL
to
study
relationship
histone-chromosomal
the
RESEARCH COMMUNICATIONS
kinetics to
the
and nuclear
the
regulation protein
A-24
proteins.
AND METHODS
Preparation of tissues and cells: Brains were removed from Sprague-Dawley male rats (Canadian Breeding Laboratory, St. Constant, Due.) (150-290 g) killed by decapitation. Hypothalamus or striatum were quickly dissected according to Glowinski and Iversen (11), cut into' small cubes (about l-2 mm square) and immediately Fmmersed in Krebs Ringer' buffer containing bicarbonate O.D25M, 0.2% glucose, 0.1% bovine serum Albumin (Fraction V, Sigma) (KRBGA)*, and 0.018% soybean trypsin inhibit07 (Sigma) previously gassed with 95% O2 / 5% C02, at room temperature. Pooled hypothalamus or striatum fragments were preincubated for 1 hour a,t 37O in fresh gassed buffer in a Dubnoff metabolic shaker with a gentle agitation. The AtT-20 tumor (12,13) obtained from Dr A.E. Bogden (Mason Research Institute, Worcester, Massachusetts) was periodically retransplanted in adrenalectomized LAFl mice. Three tumors (total wet weight 7.1 g) were dissected, washed with saline buffer, and cross-cut at 250 urn intervals using a McIlwain tissue chopper. An ACTH secreting human pituitary tumor following adrenalectomy in a patient having a Cushing's was excised by a transphenoidal approach; a fragment of the disease, Fragments of the tumor was collected in physiological saline at 4OC. mouse or human tumor were then immediately immersed in KRBGA buffer, incubated at 370C under 95% 02 / 5% CO2 in KRBGA containing collagenase (6 g/l) for 15 minutes followed by KRBGA containing DNase (8 mg/l) for After washing in fresh gassed KRBGA 15 minutes in the same conditions. buffer, the cells were isolated by sucking several times the fragments through a plastic tubing (3 mm inside diameter) attached to a disposable The plastic syringe, and filtered through a fine nylon net (100 mesh). isolated cells were harvested by low speed centrifugation and preincubatjeo before. Viability of the cells was for 1 hour at 37O as described determined with the trypan blue exclusion technique and found to be higher than 90%. Incorporation of labeled amino acids in vitro: After preincubrain fragments or cells harvested by low speed centrifugation wer bation, resuspended in fresh KRBGA buffer with trypsin inhibitor and a labeled amino acid. The incubation was carried out at 37O under 95% 02 / 5% CO2 The radioatmosphere in a Dubnoff metabolic shaker for 2 to 3 hours. 35S-methionine (1 to 1.5 mCi, 800active amino acids incorporated were 1100 Ci/mmol, Amersham), 3H-lysine (1 to 2 mCi, 60-80 Ci/mmol, New England Nuclear (NEN), and 3H-leucine (5 mCi, 115 Ci/mmol, NEN). Protein Extraction: After the incubation, tissue fragments or cells were diluted in cold KRBGA containins 10 mM of unlabeled amino acid and immediately centrifuged at low speed. -Brain fragments were homogenized using a TeflonR glass homogenizer in 1 N acetic acid containing 0.3 mg/ml phenylmethylsulfonyl fluoride, 0.3 mg/ml iodoacetamide, 5 x lD-4M bacitracin (Sigma), 0.5 mg/ml BSA and 10 mM of the corresponding unlabeled amino acid and centrifuges (15,000 rpm, 30 min, 4oC). Cell pellets were extracted in 5 N acetic acid containing the same enzymatic *
Krebs
Ringer
Buffer
Glucose
Albumin
876
Vol. 84,No.4,
BIOCHEMICAL
1978
inhibitors, except bacitracin. P2 or Sephadex G-25 columns
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The equilibrated
supernatants and
were eluted
desalted on with 1N acetic
Biogel acid.
Carboxymethyl cellulose chromatography: After desalting the fraction excluded from the gel was lyophylized, dissolved in 0.01 M NH40Ac buffer (pH 4.6) with 50 mg of sheep or human (for the human tumor extract) fraction 0 prepared as earlier described (14). The mixture was chromatographed on a carboxymethyl cellulose column (0.7 x 26 cm) at 4oC using the same NH40Ac gradients with a mixing flask of 100 ml (14). The protein content was determined by absorbance at 230 nm and an aliquot of 80 ul/tube was taken for determination of radioactivity by liquid scintillation counting. Disc electrophoresis: Polyacrylamide gel electrophoresis of carboxymethyl cellulose fractions were performed at pH 4.5 or pH 8.3 according to Reisfield et al. (15) and Davis et al. (16) respectively. Gels containing radioactive proteins were cut into 2 mm slices immediately after the electrophoresis with a Gilson Aliquogel fractionator; gel fragmentswere then digested by incubation overnight at 50°C in a mixture of 0.2 ml of 60% (vol/vol) perchloric acid and 0.4 ml of 30% (vol/vol) H2O2. Ubiquitin: Ubiquitin purified supplied by Dr G. Goldstein, was taken polyacrylamide gel electrophoresis and Sequencing the purified peptides nmoles of sperm whale The thiazolinone (17). directly in toluene-base Nuclear)/1 toluene).
from bovine thynus, kindly as a standard for comparison molecular sieving.
on
of labeled peptides: Automatic Edman degradation of was performed on a Beckman 890B sequencer using 150 apomyoglobin as carrier and 0.1 M quadrol buffer collected in butylchloride were counted scintillation mixture (4 g omnifluor (New England
RESULTS 1.
Rat
Fig. desalted of
3
133 at
H-leucine
pH 4.5
and
hypothalamus
and it
8.3;
as a single
2B) ; this logous
of
carboxymethylcellulose
is
seen
band
intermedia
(9).
with
us
to
establish
Rf
mobility peptide
The
that
contains
0.57
and
previously
availability the
identity
877
purified of
its
gel
tubes
with from bovine
mobilities
106-
electrophoresis
peptide
respectively
comparable isolated
of
to
a labeled 0.21
the
35 S-methionine
after
polyacrylamide
it
of
a 3 h incorporation
corresponding
by
was
after
obtained
fraction
characterized
electrophoretic biosynthetic
were
The
chromatography
homogenate
profiles
incorporation.
lyophilized
migrates
the
Comparable
was
allowed
1.A shows
supernatant
3H-lysine.
and
hypothalamus.
which (Fig.
that beef ubiquitin with
of
2A,
the
homo-
pituitary
pars later
those
of
this
Vol. 84, No. 4, 1978
BIOCHEMICAL
KU
0
Figure extracted tumor,
loo TURE 100 WmRER
Carbox methyl cellulose from (A r rat hypothalamus, (D) human pituitary tumor.
1:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
300
chromatography of (B) rat striatum, A concave gradient
878
labeled proteins (C) mouse AtT-PO was started at
BIOCHEMICAL
Vol. 84, No. 4, 1978
LB I
I 2
I 3
I1 4 5
AND BlOPHYSlCAL
I 6
I 7cm
(El
CP 400 k
I
RESEARCH COMMUNICATIONS
CCn 4+------
2
3
4
S
6
7m
IF)
350
1
300
250
300
200
200
150 loo
100 loo
SO
12
3
45
1
DISTANCE
Fp;
2
3
4
5
6
7cm
FROY ORISIN
Polyacrylamide gel electrophoresis at pH 4.5 (A, C, E) and at B, D, F) of the radioactive material obtained after the carboxymethyl cellulose chromatography of labeled proteins from hypothalamus (A,B), mouse AtT-20 tumor (C,D) and human pituitary tumor (E,F). The arrow shows the position of the tracking dye at the end of the migration. The positions of cold purified ubiquitin run as a reference standard on identical gels are shown in A (pH 4.5) and B (pH 8.3). Although not shown1 the same pattern was obtained with polyacrylamide gel electrophoresis at pH 4.5 of the corresponding rat striatal peptide.
Fig.1
‘r:
cont.'d.
tube 20 by the addition mixing chamber containing (180 for human tumor), increase the gradient. of 0.6 ml/min.
of
0.1 M NH40Ac buffer, pH 6.7 to a 100 ml pH 4.6. 0.01 M NH OAc buffer At tube 160 a 0.2 M NH40 A c buffer, pH 6.7 was used to Fractions of 0.8 ml were collected at a rate
879
BIOCHEMICAL
Vol. 84, No. 4, 1978
TIME
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF EXPOSURE
TO 0,
Fig. 3. Inactivation of the unidirectional oxygen. The unidirectional hydrogenase (in A- A) was from the washings of the protoplast hydrogenase (in Tris.Cl, pH 8, with (0 - 0) was partially purified through the Sephadex were described in Materials and Methods. The extensively
purified
or dichlorophenolindophenol
measured
activity
midpoint
redox
in
decreases potential
the heterocysts
acceptor
(10,
MV was added with
directional
difference
iron-sulfur
not
shown).
was not (10);
acceptor,
with
and the
a more negative
The H2-oxidizing
7120 was reported
and the bidirectional
the
pasteurianum This
(data
acceptor
to use 0
however,
activity
2 as the electron
significantly
in O2-sensitivity
in approximately30
Like
used
and the activity
and the bidirectional
CO) .
is
electron
enhanced
the tests
were
when Fd or
performed
heterocysts.
Significant
occurred
sole
when an electron
to the assay mixture
intact
H2ase can use Fd, MB, MV, benzyl
as the
of Anabaena
ll),
and the bidirectional hydrogenases by Tris.Cl, pH 8, with 0.58 M sucrose, preparation. The bidirectional or without (0 - 0) 0.58 M sucrose) G-100 column step (1). Other details
unidirectional
viologen
(mid
other is also
suggests protein,
H2ases
H ases were 2
too.
(12),
Fifty
the uni-
percent
after
inactivation
the unidirectional
the unidirectional
H2ase
monoxide
(98% inhibition
likely
a metalloenzyme,
the new H2ase is metal
between
to air.
by carbon
Direct
3).
respectively,
exposed
H2ases
inhibited that
(Fig.
and 5 min.,
classical
was found
analysis
of the enzyme.
1149
must
await
further
from C.
at 0.5 atm perhaps
an
purification
Vol. 84, No. 4, 1978
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I f”
WllH UBlOUlTlN IAB5ORBANCE AT 230nm) 100
0.2
-
- 0.1
10
20
30
40 50 FRACTION
60
‘70
60
90
100
NUMBER
Figure 4: Sephadex G-50 superfine chromatography (column 1 x 55 cm) in 0.1 N acetic acid of fraction 113-130 of CM-cellulose chromatography of incubated human pituitary tumor cell extract. Calibration with purified ubiquitin is shown. 0.5 ml fractions were collected.
3.
Pituitary
In
the
tumor
and
human
major
peak
of
of
the
and
found
to
ubiquitin
and
was 0.1
ubiquitin, gel
contain
to human performed N acetic was
electrophoresis
cellulose tumor
'H-lysine
(Fig.
the
with
pituitary
labeled
were a pure
proteins
labeled
2D).
Its
that
of
NH2 -terminal
sequence
fraction,
(Fig.
(Fig.
The
4).
2E,
identified 2F)
and
881
found
in
tumor,
peptide
with
with
respect of
superfine fraction
sequence
be
lC,
this
a region
fraction
the to
sane
lysine
step (1
x 55
48-65,
coeluting by (Fig.
was
Rf
as
residues
(Fig.
column
analysis
lD),
fractions
ubiquitin
ubiquitin
AtT-20
a comparable
purification
to
mouse
respectively
AtT-20
additional
G-50
and
(Fig.
portion
an
acid lyophilited
In
3H-lysine
a Sephadex
extracts
profile:
lyophilized.
on
of
was
elution
ZC,
tumor
chromatography
radiolabeled
cellulose
113-130
identical
With
carboxymethyl
carboxymethyl
120-140
is
tumors.
3D). was cm)
needed eluted
with
cold
polyacrylamide 3E).
Vol. 04, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION This
paper
related
peptide
tumors,
an experimental
Such
an
in
to
fit
brain
of
completely
Met
availability
of
the
biosynthetic
peptides
to
be
newly
produced
by
process 2)
is
protease
(pituitary the
fraction
apparent
dodecyl the
8,451
of
ubiquitin
sodium for
daltons
been acetic coelute
dodecyl peptides
found
by
with
standard
on
tography
remain
pituitary,
be
sodium
related
or
biosynthetic sheep
be
protein
known an
apparent
during
the
sulfate/urea
gel
and
of
in
these
at
cold
of
sodium
it
74 of
has
Sephadex
amino
been
4) as
for
acids on
reported weight
(Fig.
reasons
and
ubiquitin
molecular
on
carrier
(9)
the
4'C,
between
on
ubiquitin,
The
isolation
performed 3)
appear
were
disparity
chromatography
beta-endorphin.
that
they
daltons)
as
found
as
they
migration
and
(9).
establish
Thus
peptide
gel,
bio-
15 was
that
The
sequence
peptides
to
and
abnormal
G-50
of
Furthermore,
were
extract.
tumor fortui-
8,
8.3.
(3,500-4,OnO
to
(9)
Leu
possibility
A comparable
G-75
and
biosynthetic
the
secreting
peptides
us
extraction,
the
the
from
dodecyl to
to
pituitary
mobility
experiments the
two
course
related
allowed
polyacrylamide
Sephadex for
the
in
(2,9,10).
pH 4.5
electrophoresis
(18,19).
both
acid,
The
to
both
for
of
sulfate/urea
behavior
normal
weight
the
same electrophoretic
at
ubiquitin-
identified,
33 and
ubiquitin
all
added
calculated
seems
other
1)
29,
an
ACTH
been
ubiquitin
precursor
D) was
gel
first
27,
a larger
used
sulfate/urea
11,
The
were
molecular
a human
during
ubiquitin.
because
inhibitors
and
and
of
the
gels
of
unlikely
5,
bovine
have
degradation
and
striatum
intermedia
Lys
purified
biosynthesized
and
has
sequence
polyacrylamide
of
beta-endorphin
1,
known
the
on
pars
biosynthesis
tumor
peptide
beta-LPH,
the
vitro
mouse
pituitary
sequence:
ubiquitin
in
hypothalamus
AtT-20
beef
studies
partial
rat
the
biosynthesized
the
synthetic
Its
in
actively
tously,
reports
in
has 0.1
they this
gel
abnormal chroma-
clarified.
biosynthesis pituitary
ubiquitin in
tumors,
882
several
different mammalian
tissues: species:
brain,
N
BIQCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
vol. 84, No. 4, i 978
beef,
rat,
with
mouse
and
immunological
Hewly
man
1 to
of
the
experiments.
we
of
"ubiquitous"
confirmed
with
could
represent
a biosynthetic
cells
used
this
specific
for
an
synthetic
peptides
machinery
in
the
reported
to
significant
find
vitro
here
while
ubiquitin
was
pars
intermedia
(20).
Regarding could
provide
its
sequence that
of
which
and
amino
the
non
histone
origin
or
are
histone
of 2A:
help
that the
has 37
these
two
products
of
ubiquitin
component
of
been
recently
the
sequences a single (4)
a mutation would
protein
other
be A24,
883
biosynthetic in
in
a cleaved
its
as yet (4) is
unknown that
A-24
to
2A molecule simi-
evolutionary second
alternative
highly
rate
the
complex,
structural
the
and
identical
a histone
The
as
metabolite after
unable
bovine
of
a common
acceptance
which,
in
maturation
a large
of
expe-
preparation)
its
Such
view
the
we were
protein
(4).
or
bio-
of
to
gene
it
characterization
linked
share
in
sought
present
ubiquitin
nuclear
that
synthesize
discovered of
be
tissue
to
biosynthetic
residues of
the
hypothalamus,
elucidation
(21).
suggested with
the
to
Indeed,
kinetics
polypeptide
nucleosomes
ubiquitin thus
in
the
(1).
represents
suggest
impaired
were
cells
incubation
(manuscript
this
study
component
preferentially
structure
non
in
to
Both
itself,
histone
of
beta-endorphin
terminal
a non present
implies
histone
detectable.
It
the
is
been
readily
could
of
larity
has
of
it
various
enough
agreement
incubation
it;
that
viable
and
our
we
in of
remains
absence
striatum
to
the
cnnditions.
amounts
tool
for
testifying
attributable
rat
function.
contains
(5)
with
ubiquitin
and
cellular
be
of
However,
the
incubation
a useful
regulation,
basic
not
each
biosynthesis
are
Accordingly,
in
in
studies.
incubation
would
in
are
variety
looked
"marker",
vitro
peptide.
found
vitro
tissue
sequence
a large
proteins
in
other
in
been
labeled
its
in
systematically
of
fully
of
ubiquitin has
have
totality Its
riments
constancy
ubiquitin
whenever
10%
the
detection
biosynthesized
experiments
and
enzymatic
conserved low of
as the
that nuclear
cleavage,
of
Vol. 04,No.4,
would of
diffuse
active
logical
of
eukaryotes
the
vitro its
nucleus
AND BIOPHYSICAL
into
biosynthesis
in
particularly
the
of
biosynthetic
function
proteins, in
from in
standing
BIOCHEMICAL
1978
cytoplasm
ubiquitin
relationship
regard in
to the
the
of
contribute
protein
roles expression
The
(4).
could to
possible
control
RESEARCH COMMUNICATIONS
A24 of
non of
demonstration to
and histone genetic
its
the
under.
biochromosoma inforoatiotjl
(6-8).
ACKNOWLEDGMENTS. Work supported by a Program Grant from the Medical Research Council of Canada and bv the National Cancer Institute. We thank Dr G. Tolis and Dr G. Bertrand from the Royal Victoria Hospital-for having provided the human pituitary tumor fragment. We would also like to thank Mr R. Routhier for his help during the determinations of the as well as Mrs 0. Marcil for her secretarial assistance. sequence,
REFERENCES 1. Goldstein, 2. ii: 5. 6. 7. 8. 9.
10.
:i: 15.
18.
G., Scheid, M. Hammerling, U., Boyse, E.A., Schlesinger, D.H. and Niall, M.D. (1975), Proc. Nat. Acad. Sci. USA 72:11-15. Schlesinger, D.H., Goldstein, G. and Niall, H.D. (1975), Biochemistry 14:2214-2218. Schlesinger, D.H. and Goldstein, G. (1975), Nature 255:423-424. Hunt, L.T. and Dayhoff, M.O. (19771, Biochem. Biophys. Res. Commun. 74:650-654. Goldknopf, I.L. and Busch, H. (1977), Proc. Natl. Acad. Sci. USA 74:864-868. Elgin, S.C.R. and Weintraub, H. (1975), Ann. Rev. Biochem. 44~725-774. Stein, G.S., Spelsberg, T.C. and Kleinsmith, L.J. (1974), Science 183:817-824. Stein. G.S., Stein, J.L., Kleinsmith, L.J., Jansing, R.L., Park, W.D. and Thomson, J.A. (1977), Biochem. Sot. Symp. 42:137-163. Seidah, N.G., Crine, P., Benjannet, S., Scherrer, H. and Chr@tien, M. (1978), Biochem. Biophys. Res. Commun. 80:600-608. Dayhoff, M.O., Hunt, L.T., Baker, W.C. and Schwartz, R.M. (1977), Protein Sequence Data File, National Biomedical Research Foundation, Washington D.C. Glowinski, J. and Iversen, L.L. (1966), J. Neurochemistry 13:655-669. Furth, J., Gadsen, E.L. and Upton, A.C. (1953) Proc. Sot. Exp. Biol. Med. 84~253-254. Cohen, A.E. and Furth, J. (1959), Cancer Res. 19:72-78. Li, C.H., Barnafi, L., Chrctien, M. and Chung, D. (1965), Nature 208:1093-1094. Reisfield, R.A., Lewis, V.J. and Williams, D.E. (1962), Nature 195: 281-283. Davis, B.J. (1965), Ann. N.Y. Acad. Sci. 121:404-427. Brauer, A.W., Margolies, M.N. and Haber, E. (1975), Biochemistry 14:3029-3031. Weber, K. and Osborn, M. (1975), In: "The Proteins", Third Edition, Neurath, N., Hill, R.L., eds, Academic Press N.Y. 179-223.
884
Vol. 84, No. 4, 1978
19. 20. 21.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lehtovaara, P. (1978), Biochen. J. 169:251-253. Crine, P., Benjannet, S., Seidah, N.G., Lis, M. and (1977), Proc. Natl. Acad. Sci. USA 74:4276-4280. Goldknopf, I.L., French, M.F., Musso, R. and Busch, (1977), Proc. Natl. Acad. Sci. USA 74:5492-5495.
885
Chretien, H.
M.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 874-885
October 30, 1978
BIOSYNTHESIS OF A UBIDUITIN-RELATED IN RAT BRAIN
AND IN
PITUITARY H.
Scherrer,
N.G.
Protein Clinical
HUFIAN AND MOUSE TUMORS
Seidah,
11. Lis
S.
and
and Pituitary Research
M.
Benjannet,
P.
Crine,
Chretien
Hormone Laboratory Institute of Montreal,
Affiliated the
PEPTIDE
to
HDtel-Dieu
de
Montreal
de
Montreal"
and the
Received
September
"Universite
1,1978
SUMMARY. A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH2 terminal amino acid a protein sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalamus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatograph this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems. INTRODUCTION Ubiquitin in
bovine
Requests Research
P.Q.,
thymus
is (1,2)
for reprints Institute Canada.
of
a polypeptide
of
during
isolation
should Montreal,
be
sent to 110 Pine
0006-291X/78/0844-0874$01.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
874
74
amino
of
the
acids, thymic
first polypeptide
identified hormone
Dr Michel Chretien, Clinical Avenue West, Montreal H2W lR7,
BIOCHEMICAL
Vol. 84, No. 4, 1978
thymopoietin.
It
animal
cells
higher
plants
human
and
in
cellular
bovine
receptor been
and
(1).
bovine
to
nuclear
products structural
the or
been
suggested
ref.
6-8).
for
in
ACTH
sequence
of
ubiquitin
peptides
was by
a similar rat
secreting
pituitary
pituitary
tumor
identity
isolated
of
further
established
graphic
properties.
found
of
to
a protein
tumors;
the
(AtT-20),
the peptide
on The
the
first second
with
one one
native
basis
of
biosynthetic
875
the bank
has
was was
purified
their
was
a
beta-
partial either
to
(10).
In
characterized in
two
mouse tumor.
ubiquitin
has and of
in
ACTH-
pituitary
electrophoretic
characterization
has
of
experimental
a human
it
This
search
and an
the
amino-terminal
been
striatum,
in
demonstrated
the
unrelated
of
see
studies
peptides:
data
as
review,
(9).
peptide and
involved
laboratory
with
has
likely
genome
biosynthetic
segment
hypothalamus
be
was
match
ubiquitin
most
(for
peptide
lympho-
component
ubiquitin
related
biosynthesized
brain,
this
and
then
in
a beta-adrenergic of
are
proteins
this
course
radiolabeled
or
The
the
via
eukaryotic
of in
ACTH
beta-LPH
of
intermedia
cyclase
peptides
the
distribution,
important
non-histone
could
biosynthesis
during
adenylate
the
chromosomal
vitro
an
sequence
two
of
wide
through
non-selective
acting
of
Ubiquitin
beta-endorphin, a pure
areas
that
these
(4).
pars
of
two
(5);
non-histone
first
sequence
report,
to
properties
discovery
lipotropin,
this
gene
of
amino-terminal
identical
its
induces
presumably
the
A-24
same
pituitary
fortuitous
(4),
be
activation
in
conserved
has
it
and
identical
highly
ubiquitin
of
bacteria
is
and
vitro
variety
yeasts,
been
structure
In
a large
which
have
that
tissues,
functional
The bovine
other
protein
of
its
unknown.
Recently
discovered
of
and
in
to
suggest
differentiation
in
ubiquitin,
appears
cells, still
precursors
detected
of
constancy
living
function,
cyte
in
the
(3)
RESEARCH COMMUNICATIONS
invertebrates,
sequence
species
all
lymphocyte
subsequently and
The
Both in
been
vertebrates (1).
evolution. likely
has
AND BIOPHYSICAL
been
chromato-
ubiquitin
Vol. 84, No. 4, 1978
could
provide
BIOCHEMICAL
a useful
of
its
biosynthesis,
in
the
field
MATERIALS
of
tool and
non
its
AND BIOPHYSICAL
to
study
relationship
histone-chromosomal
the
RESEARCH COMMUNICATIONS
kinetics to
the
and nuclear
the
regulation protein
A-24
proteins.
AND METHODS
Preparation of tissues and cells: Brains were removed from Sprague-Dawley male rats (Canadian Breeding Laboratory, St. Constant, Due.) (150-290 g) killed by decapitation. Hypothalamus or striatum were quickly dissected according to Glowinski and Iversen (11), cut into' small cubes (about l-2 mm square) and immediately Fmmersed in Krebs Ringer' buffer containing bicarbonate O.D25M, 0.2% glucose, 0.1% bovine serum Albumin (Fraction V, Sigma) (KRBGA)*, and 0.018% soybean trypsin inhibit07 (Sigma) previously gassed with 95% O2 / 5% C02, at room temperature. Pooled hypothalamus or striatum fragments were preincubated for 1 hour a,t 37O in fresh gassed buffer in a Dubnoff metabolic shaker with a gentle agitation. The AtT-20 tumor (12,13) obtained from Dr A.E. Bogden (Mason Research Institute, Worcester, Massachusetts) was periodically retransplanted in adrenalectomized LAFl mice. Three tumors (total wet weight 7.1 g) were dissected, washed with saline buffer, and cross-cut at 250 urn intervals using a McIlwain tissue chopper. An ACTH secreting human pituitary tumor following adrenalectomy in a patient having a Cushing's was excised by a transphenoidal approach; a fragment of the disease, Fragments of the tumor was collected in physiological saline at 4OC. mouse or human tumor were then immediately immersed in KRBGA buffer, incubated at 370C under 95% 02 / 5% CO2 in KRBGA containing collagenase (6 g/l) for 15 minutes followed by KRBGA containing DNase (8 mg/l) for After washing in fresh gassed KRBGA 15 minutes in the same conditions. buffer, the cells were isolated by sucking several times the fragments through a plastic tubing (3 mm inside diameter) attached to a disposable The plastic syringe, and filtered through a fine nylon net (100 mesh). isolated cells were harvested by low speed centrifugation and preincubatjeo before. Viability of the cells was for 1 hour at 37O as described determined with the trypan blue exclusion technique and found to be higher than 90%. Incorporation of labeled amino acids in vitro: After preincubrain fragments or cells harvested by low speed centrifugation wer bation, resuspended in fresh KRBGA buffer with trypsin inhibitor and a labeled amino acid. The incubation was carried out at 37O under 95% 02 / 5% CO2 The radioatmosphere in a Dubnoff metabolic shaker for 2 to 3 hours. 35S-methionine (1 to 1.5 mCi, 800active amino acids incorporated were 1100 Ci/mmol, Amersham), 3H-lysine (1 to 2 mCi, 60-80 Ci/mmol, New England Nuclear (NEN), and 3H-leucine (5 mCi, 115 Ci/mmol, NEN). Protein Extraction: After the incubation, tissue fragments or cells were diluted in cold KRBGA containins 10 mM of unlabeled amino acid and immediately centrifuged at low speed. -Brain fragments were homogenized using a TeflonR glass homogenizer in 1 N acetic acid containing 0.3 mg/ml phenylmethylsulfonyl fluoride, 0.3 mg/ml iodoacetamide, 5 x lD-4M bacitracin (Sigma), 0.5 mg/ml BSA and 10 mM of the corresponding unlabeled amino acid and centrifuges (15,000 rpm, 30 min, 4oC). Cell pellets were extracted in 5 N acetic acid containing the same enzymatic *
Krebs
Ringer
Buffer
Glucose
Albumin
876
Vol. 84,No.4,
BIOCHEMICAL
1978
inhibitors, except bacitracin. P2 or Sephadex G-25 columns
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The equilibrated
supernatants and
were eluted
desalted on with 1N acetic
Biogel acid.
Carboxymethyl cellulose chromatography: After desalting the fraction excluded from the gel was lyophylized, dissolved in 0.01 M NH40Ac buffer (pH 4.6) with 50 mg of sheep or human (for the human tumor extract) fraction 0 prepared as earlier described (14). The mixture was chromatographed on a carboxymethyl cellulose column (0.7 x 26 cm) at 4oC using the same NH40Ac gradients with a mixing flask of 100 ml (14). The protein content was determined by absorbance at 230 nm and an aliquot of 80 ul/tube was taken for determination of radioactivity by liquid scintillation counting. Disc electrophoresis: Polyacrylamide gel electrophoresis of carboxymethyl cellulose fractions were performed at pH 4.5 or pH 8.3 according to Reisfield et al. (15) and Davis et al. (16) respectively. Gels containing radioactive proteins were cut into 2 mm slices immediately after the electrophoresis with a Gilson Aliquogel fractionator; gel fragmentswere then digested by incubation overnight at 50°C in a mixture of 0.2 ml of 60% (vol/vol) perchloric acid and 0.4 ml of 30% (vol/vol) H2O2. Ubiquitin: Ubiquitin purified supplied by Dr G. Goldstein, was taken polyacrylamide gel electrophoresis and Sequencing the purified peptides nmoles of sperm whale The thiazolinone (17). directly in toluene-base Nuclear)/1 toluene).
from bovine thynus, kindly as a standard for comparison molecular sieving.
on
of labeled peptides: Automatic Edman degradation of was performed on a Beckman 890B sequencer using 150 apomyoglobin as carrier and 0.1 M quadrol buffer collected in butylchloride were counted scintillation mixture (4 g omnifluor (New England
RESULTS 1.
Rat
Fig. desalted of
3
133 at
H-leucine
pH 4.5
and
hypothalamus
and it
8.3;
as a single
2B) ; this logous
of
carboxymethylcellulose
is
seen
band
intermedia
(9).
with
us
to
establish
Rf
mobility peptide
The
that
contains
0.57
and
previously
availability the
identity
877
purified of
its
gel
tubes
with from bovine
mobilities
106-
electrophoresis
peptide
respectively
comparable isolated
of
to
a labeled 0.21
the
35 S-methionine
after
polyacrylamide
it
of
a 3 h incorporation
corresponding
by
was
after
obtained
fraction
characterized
electrophoretic biosynthetic
were
The
chromatography
homogenate
profiles
incorporation.
lyophilized
migrates
the
Comparable
was
allowed
1.A shows
supernatant
3H-lysine.
and
hypothalamus.
which (Fig.
that beef ubiquitin with
of
2A,
the
homo-
pituitary
pars later
those
of
this
Vol. 84, No. 4, 1978
BIOCHEMICAL
KU
0
Figure extracted tumor,
loo TURE 100 WmRER
Carbox methyl cellulose from (A r rat hypothalamus, (D) human pituitary tumor.
1:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
300
chromatography of (B) rat striatum, A concave gradient
878
labeled proteins (C) mouse AtT-PO was started at
BIOCHEMICAL
Vol. 84, No. 4, 1978
LB I
I 2
I 3
I1 4 5
AND BlOPHYSlCAL
I 6
I 7cm
(El
CP 400 k
I
RESEARCH COMMUNICATIONS
CCn 4+------
2
3
4
S
6
7m
IF)
350
1
300
250
300
200
200
150 loo
100 loo
SO
12
3
45
1
DISTANCE
Fp;
2
3
4
5
6
7cm
FROY ORISIN
Polyacrylamide gel electrophoresis at pH 4.5 (A, C, E) and at B, D, F) of the radioactive material obtained after the carboxymethyl cellulose chromatography of labeled proteins from hypothalamus (A,B), mouse AtT-20 tumor (C,D) and human pituitary tumor (E,F). The arrow shows the position of the tracking dye at the end of the migration. The positions of cold purified ubiquitin run as a reference standard on identical gels are shown in A (pH 4.5) and B (pH 8.3). Although not shown1 the same pattern was obtained with polyacrylamide gel electrophoresis at pH 4.5 of the corresponding rat striatal peptide.
Fig.1
‘r:
cont.'d.
tube 20 by the addition mixing chamber containing (180 for human tumor), increase the gradient. of 0.6 ml/min.
of
0.1 M NH40Ac buffer, pH 6.7 to a 100 ml pH 4.6. 0.01 M NH OAc buffer At tube 160 a 0.2 M NH40 A c buffer, pH 6.7 was used to Fractions of 0.8 ml were collected at a rate
879
BIOCHEMICAL
Vol. 84, No. 4, 1978
TIME
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF EXPOSURE
TO 0,
Fig. 3. Inactivation of the unidirectional oxygen. The unidirectional hydrogenase (in A- A) was from the washings of the protoplast hydrogenase (in Tris.Cl, pH 8, with (0 - 0) was partially purified through the Sephadex were described in Materials and Methods. The extensively
purified
or dichlorophenolindophenol
measured
activity
midpoint
redox
in
decreases potential
the heterocysts
acceptor
(10,
MV was added with
directional
difference
iron-sulfur
not
shown).
was not (10);
acceptor,
with
and the
a more negative
The H2-oxidizing
7120 was reported
and the bidirectional
the
pasteurianum This
(data
acceptor
to use 0
however,
activity
2 as the electron
significantly
in O2-sensitivity
in approximately30
Like
used
and the activity
and the bidirectional
CO) .
is
electron
enhanced
the tests
were
when Fd or
performed
heterocysts.
Significant
occurred
sole
when an electron
to the assay mixture
intact
H2ase can use Fd, MB, MV, benzyl
as the
of Anabaena
ll),
and the bidirectional hydrogenases by Tris.Cl, pH 8, with 0.58 M sucrose, preparation. The bidirectional or without (0 - 0) 0.58 M sucrose) G-100 column step (1). Other details
unidirectional
viologen
(mid
other is also
suggests protein,
H2ases
H ases were 2
too.
(12),
Fifty
the uni-
percent
after
inactivation
the unidirectional
the unidirectional
H2ase
monoxide
(98% inhibition
likely
a metalloenzyme,
the new H2ase is metal
between
to air.
by carbon
Direct
3).
respectively,
exposed
H2ases
inhibited that
(Fig.
and 5 min.,
classical
was found
analysis
of the enzyme.
1149
must
await
further
from C.
at 0.5 atm perhaps
an
purification
Vol. 84, No. 4, 1978
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I f”
WllH UBlOUlTlN IAB5ORBANCE AT 230nm) 100
0.2
-
- 0.1
10
20
30
40 50 FRACTION
60
‘70
60
90
100
NUMBER
Figure 4: Sephadex G-50 superfine chromatography (column 1 x 55 cm) in 0.1 N acetic acid of fraction 113-130 of CM-cellulose chromatography of incubated human pituitary tumor cell extract. Calibration with purified ubiquitin is shown. 0.5 ml fractions were collected.
3.
Pituitary
In
the
tumor
and
human
major
peak
of
of
the
and
found
to
ubiquitin
and
was 0.1
ubiquitin, gel
contain
to human performed N acetic was
electrophoresis
cellulose tumor
'H-lysine
(Fig.
the
with
pituitary
labeled
were a pure
proteins
labeled
2D).
Its
that
of
NH2 -terminal
sequence
fraction,
(Fig.
(Fig.
The
4).
2E,
identified 2F)
and
881
found
in
tumor,
peptide
with
with
respect of
superfine fraction
sequence
be
lC,
this
a region
fraction
the to
sane
lysine
step (1
x 55
48-65,
coeluting by (Fig.
was
Rf
as
residues
(Fig.
column
analysis
lD),
fractions
ubiquitin
ubiquitin
AtT-20
a comparable
purification
to
mouse
respectively
AtT-20
additional
G-50
and
(Fig.
portion
an
acid lyophilited
In
3H-lysine
a Sephadex
extracts
profile:
lyophilized.
on
of
was
elution
ZC,
tumor
chromatography
radiolabeled
cellulose
113-130
identical
With
carboxymethyl
carboxymethyl
120-140
is
tumors.
3D). was cm)
needed eluted
with
cold
polyacrylamide 3E).
Vol. 04, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION This
paper
related
peptide
tumors,
an experimental
Such
an
in
to
fit
brain
of
completely
Met
availability
of
the
biosynthetic
peptides
to
be
newly
produced
by
process 2)
is
protease
(pituitary the
fraction
apparent
dodecyl the
8,451
of
ubiquitin
sodium for
daltons
been acetic coelute
dodecyl peptides
found
by
with
standard
on
tography
remain
pituitary,
be
sodium
related
or
biosynthetic sheep
be
protein
known an
apparent
during
the
sulfate/urea
gel
and
of
in
these
at
cold
of
sodium
it
74 of
has
Sephadex
amino
been
4) as
for
acids on
reported weight
(Fig.
reasons
and
ubiquitin
molecular
on
carrier
(9)
the
4'C,
between
on
ubiquitin,
The
isolation
performed 3)
appear
were
disparity
chromatography
beta-endorphin.
that
they
daltons)
as
found
as
they
migration
and
(9).
establish
Thus
peptide
gel,
bio-
15 was
that
The
sequence
peptides
to
and
abnormal
G-50
of
Furthermore,
were
extract.
tumor fortui-
8,
8.3.
(3,500-4,OnO
to
(9)
Leu
possibility
A comparable
G-75
and
biosynthetic
the
secreting
peptides
us
extraction,
the
the
from
dodecyl to
to
pituitary
mobility
experiments the
two
course
related
allowed
polyacrylamide
Sephadex for
the
in
(2,9,10).
pH 4.5
electrophoresis
(18,19).
both
acid,
The
to
both
for
of
sulfate/urea
behavior
normal
weight
the
same electrophoretic
at
ubiquitin-
identified,
33 and
ubiquitin
all
added
calculated
seems
other
1)
29,
an
ACTH
been
ubiquitin
precursor
D) was
gel
first
27,
a larger
used
sulfate/urea
11,
The
were
molecular
a human
during
ubiquitin.
because
inhibitors
and
and
of
the
gels
of
unlikely
5,
bovine
have
degradation
and
striatum
intermedia
Lys
purified
biosynthesized
and
has
sequence
polyacrylamide
of
beta-endorphin
1,
known
the
on
pars
biosynthesis
tumor
peptide
beta-LPH,
the
vitro
mouse
pituitary
sequence:
ubiquitin
in
hypothalamus
AtT-20
beef
studies
partial
rat
the
biosynthesized
the
synthetic
Its
in
actively
tously,
reports
in
has 0.1
they this
gel
abnormal chroma-
clarified.
biosynthesis pituitary
ubiquitin in
tumors,
882
several
different mammalian
tissues: species:
brain,
N
BIQCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
vol. 84, No. 4, i 978
beef,
rat,
with
mouse
and
immunological
Hewly
man
1 to
of
the
experiments.
we
of
"ubiquitous"
confirmed
with
could
represent
a biosynthetic
cells
used
this
specific
for
an
synthetic
peptides
machinery
in
the
reported
to
significant
find
vitro
here
while
ubiquitin
was
pars
intermedia
(20).
Regarding could
provide
its
sequence that
of
which
and
amino
the
non
histone
origin
or
are
histone
of 2A:
help
that the
has 37
these
two
products
of
ubiquitin
component
of
been
recently
the
sequences a single (4)
a mutation would
protein
other
be A24,
883
biosynthetic in
in
a cleaved
its
as yet (4) is
unknown that
A-24
to
2A molecule simi-
evolutionary second
alternative
highly
rate
the
complex,
structural
the
and
identical
a histone
The
as
metabolite after
unable
bovine
of
a common
acceptance
which,
in
maturation
a large
of
expe-
preparation)
its
Such
view
the
we were
protein
(4).
or
bio-
of
to
gene
it
characterization
linked
share
in
sought
present
ubiquitin
nuclear
that
synthesize
discovered of
be
tissue
to
biosynthetic
residues of
the
hypothalamus,
elucidation
(21).
suggested with
the
to
Indeed,
kinetics
polypeptide
nucleosomes
ubiquitin thus
in
the
(1).
represents
suggest
impaired
were
cells
incubation
(manuscript
this
study
component
preferentially
structure
non
in
to
Both
itself,
histone
of
beta-endorphin
terminal
a non present
implies
histone
detectable.
It
the
is
been
readily
could
of
larity
has
of
it
various
enough
agreement
incubation
it;
that
viable
and
our
we
in of
remains
absence
striatum
to
the
cnnditions.
amounts
tool
for
testifying
attributable
rat
function.
contains
(5)
with
ubiquitin
and
cellular
be
of
However,
the
incubation
a useful
regulation,
basic
not
each
biosynthesis
are
Accordingly,
in
in
studies.
incubation
would
in
are
variety
looked
"marker",
vitro
peptide.
found
vitro
tissue
sequence
a large
proteins
in
other
in
been
labeled
its
in
systematically
of
fully
of
ubiquitin has
have
totality Its
riments
constancy
ubiquitin
whenever
10%
the
detection
biosynthesized
experiments
and
enzymatic
conserved low of
as the
that nuclear
cleavage,
of
Vol. 04,No.4,
would of
diffuse
active
logical
of
eukaryotes
the
vitro its
nucleus
AND BIOPHYSICAL
into
biosynthesis
in
particularly
the
of
biosynthetic
function
proteins, in
from in
standing
BIOCHEMICAL
1978
cytoplasm
ubiquitin
relationship
regard in
to the
the
of
contribute
protein
roles expression
The
(4).
could to
possible
control
RESEARCH COMMUNICATIONS
A24 of
non of
demonstration to
and histone genetic
its
the
under.
biochromosoma inforoatiotjl
(6-8).
ACKNOWLEDGMENTS. Work supported by a Program Grant from the Medical Research Council of Canada and bv the National Cancer Institute. We thank Dr G. Tolis and Dr G. Bertrand from the Royal Victoria Hospital-for having provided the human pituitary tumor fragment. We would also like to thank Mr R. Routhier for his help during the determinations of the as well as Mrs 0. Marcil for her secretarial assistance. sequence,
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