Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOLOGICAL EVIDENCE FOR A PRECURSORPROTEIN OF SERUM ALBUMIN Jljrg
Urban and Gerhard
Schreiber
The Russell Grimwade School of Biochemistry, Parkville, Victoria, 3052, Australia. Received
April
8,
Melbourne
University,
1975
SUMMARY: Radioactive leucine was injected into the portal vein of rats followed after 15 seconds by a 180 fold excess of nonradioactive leucine. An albumin-like protein in the liver became highly labelled within 15 minutes after injection. After 150 minutes, the radioactivity in the albumin-like protein had decreased to one tenth. In the serum, radioactively labelled albumin started to appear after 15 minutes and increased thereafter up to 150 minutes after injection. Radioactivity in albumin within the liver remained constant at a low level. These results suggest that the albumin-like protein is a biological precursor protein of serum albumin, i.e. a proalbumin.
Albumin
isolated
from
immunoprecipitation
was
turnover
rate.
exchange
chromatography
amide gels
Further
addition suggesting
the existence
Isolation,
amino
sequence
of
purification
including
of
acid
from albumin
by an N-terminal
five
biological
amino acids In this
protein Copyright AN ri&ts
in liver
(8).
into
studies the
from
pentapeptide into
on
the
to liver of
(l-6). after
slices,
albumin
(7).
amino acid
liver
microsomes
protein
differed
extension, by
suggesting
removal
of the
(8). [
14 CI leucine
albumin
and total
The results
strongly
time course
albumin-like
778
continued
rat
albumin
and serum are described.
o 19 75 b.v Academic Press. /NC. of reproduction itI my form reserwd.
albumin
N-terminal
The albumin-like
conversion
pure
protein and
ion-
in polyacryl-
albumin
a precursor
by
of a high
repeated
C12CJleucine
from the N-terminus
paper,
incorporation
into
protein
recently
a protein
to yield
composition
an albumin-like
homogenates
electrophoreses
or excess
were reported
a possible
disc
pH is necessary
cycloheximide
hepatoma with
c14C1leucine
of
of
or
contaminated
and/or
of various
Incorporation
liver
protein,
of
BIOCHEMICAL
Vol. 64, No. 2, 1975
support
that
the hypothesis
proalbumin,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
a biological
i.e.
albumin-like
precursor
protein
protein
of
is
indeed
a
serum albumin.
MATERIALS AND METHODS Affinity
Chromatography:
cyanogen
bromide
per
purified
from
activated
Sepharose
plus
1 M
rat
100 g
on
albumin-Sepharose.
to
200 g
activated
of
serum (6) in
Sepharose
with
Albumin
(9).
was covalently
linked
Antibodies
(9).
rabbits
in
4B was activated
to
0.1 M sodium carbonate-bicarbonate,
sodium chloride
serum were raised
plus
Sepharose
(6) and isolated
One gram Sepharose
1 M sodium chloride
Minutes
to
of these
in
0.1 M
25 g
(50 mg) 20 g
of
pH 9,
albumin
from rat
by chromatography
antibodies sodium citrate,
was coupled pH 6.5,
(9).
after
injection
of
‘?-Leuclne
Fig. 1. Radioactivity in Protein and Non-Protein in Liver and Serum after Intraportal Injection of [14C)Leucine. Male buffalo rats starved overnight received 5 uCiL-[1-14C]leucine (59 Ci/mole) 00 g body weight followed 15 seconds later by 15 umoles Fef14C]leucine. At indicated times 2 ml of blood were taken from the caval vein and livers were clamp-frozen and homogenized in The number of animals for each time point is ice-cold buffer. Albumin given in parentheses. Standard deviations are indicated. was isolated to radiochemical purity (6) from the combined sera of each time group. The average liver weight of the starvedrats was 3.1 g per 100 g body weight. The average serum volume per 100 g body weight was assumed to be 3.3 ml (10).
779
Vol. 64, No. 2, 1975
Liver
homogenates
followed washes
of
washes
with
0.5
precipitation
the
precipitate
ether.
chromatography other
with The
binding on the
methods
treated with
with
5%
deoxycholate acid,
(1:2,
was
The extract
0.7%
trichloroacetic
ethanol/ether
precipitate
pH 7.7.
protein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were
by
M Tris/HCl,
and all
All
BIOCHEMICAL
then
v/v)
to anti-albumin
and
extracted
was diluted
two with
1:5 with
was separated
two
water
by affinity
anti-albumin-Sepharose.
were as described
previously
(6).
RESULTS AND DISCUSSION Radioactive followed
leucine after
was
15 seconds
injected
into
the
by a chase with
portal
vein
non-labelled
of
rats
leucine.
Number
Fraction
of Anti-Albumin Binding Fig. 2. DEAE-Cellulose Chromatography and 150 Minutes after Injection Protein from Livers 15 Minutes Protein binding to anti-albumin of [14C]Leucine (see Fig. 1). by chromatography on was separated from the liver homogenates anti-albumin-Sepharose 4B (see Materials and Methods). In the two specific radioactivity of ion-exchange chromatographies, the fractions 42 to 62 (= albumin albumin was constant throughout 280 nm (not shown in The curves of the absorbance at P-W. figure) and albumin concentration in the eluate ran parallel. at 19 ml/hr with Tris/HCl, pH 7.7, Column: 1 x 40 cm. Elution: 1) 300 ml 10 mM, 2) 40 ml 50 mu and in the following order: 250 ml of a linear gradient of 50 mM to 250 mM buffer. 3) Fractions of 2.1 ml were collected after 70 ml of the gradient had passed the column.
780
BIOCHEMICAL
Vol. 64, No. 2, 1975
)0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
40 FRACTION
80
NUMBER
Fig. 3. Co-Chromatography on DEAF-Cellulose of GlyValPheSerArgAlbumin (8) from Liver Microsomes and the Highly Labelled AlbuminLike Protein from Total Liver Homogenates. The non-labelled GlyValPheSerArg-albumin was isolated from microsomes as described (55,000 dpm) recently (8) and 1.3 mg were mixed with 0.5 mg highly labelled albumin-like protein eluted from DEAE-cellulose The highly labelled protein was isolated from prior to albumin. total liver homo enates 12 minutes after intracaval injection of 100 PCi L-Cl- 9 4C]leucine without using immunoprecipitation (6). Using anti-albumin, both proteins were measured in a single radial immunodiffusion technique (11) with rat serum albumin as a standard. Curves of absorbance at 280 nm (not shown in Figure), radioactivity and albumin coincided. Column: 1 x 24 cm. Elution: as described for Figure 2, except, 3) 160 ml of 50 mM to 300 mM buffer, collected in fractions of 1.3 ml.
The radioactivity minutes
after
further total
the
about
decreased
(Fig.
and
liver,
in
1).
the
within
of
radioactivity
only
2,600
of observation.
the
15 minute liver
dpm/mg
in
period
781
2).
labelled
to
2).
After
150
protein
had
contrast,
the
In
was No
and
highly
albumin-like value.
albumin
(Fig.
(Fig.
a
radioactivity
serum,
became
30
for
the whole
in
of
thereafter
15 minutes
15 minutes
the
about
after
protein
radioactivity
to a tenth
the
for
appeared
throughout
dpm/mg
constant
In
albumin
increased
protein
remained
an albumin-like
90,000
being
liver and
thereafter
minutes,
times,
injection
protein
specific
total
60 minutes
increased In
in
the
albumin-like
same at both
protein
Vol. 64, No. 2, 1975
can be found (Fig.
in
prior
AND BIOPHYSICAL
(6,8).
Co-chromatography
serum
3) suggests
eluted
BIOCHEMICAL
that
the highly
to albumin
(see Fig.
2) is
protein
different
from
extension
at
N-terminus
(8).
structures
and
kinetics
hypothesis
that
the
protein
to albumin,
albumin
during
of
or shortly
ACKNOWLEDGEMENT. excellent technical NHMRC Grant No. 162.
albumin
a proalbumin,
protein
with by
In
protein
after
albumin-like
identical
labelling
albumin-like i.e.
on DEAE-cellulose
labelled
described
the
RESEARCH COMMUNICATIONS
a
a recently pentapeptide
conclusion,
chemical
support
strongly
is
an in
vivo
which
is
the
precursor
converted
into
secretion.
We are very assistance.
grateful This
to work
Miss P. Heyma was supported
for by
REFERENCES 1.
Schreiber, G., (1969) Eur. J.
2.
Rotermund, Weigand,
K.
Rotermund, H.-M., Weigand, Biochem. 10, 355-361.
H.-M., Schreiber, (1970) Cancer
and
Lesch,
R.
U.,
and
G., Maeno, H., Weinssen, Res. -30, 2139-2146.
3.
Judah, J.D., 643-648.
4.
Schreiber, G. (1972) in Immunology (Kaufman, B., Energy Agency, Vienna.
5.
Geller, J. 127,
6.
Urban, J., Zimber, 58, 102-116.
7.
Judah, 655.
8.
Urban, J., Inglis, Biochem. Biophys.
9.
Methods in Cuatrecasas, P., and Anfinsen, C.B. (1971) in Enzymology (Colowick, S.P., and Kaplan, N.O., ed.) Vol. 22, Academic Press, New York and London. PP. 345-378,
1, II.
Nicholls,
G.M., Judah, 856-874.
J-D.,
10 . Schreiber,
Frosch,
and
K.,
U.
Mancini, G., Immunochemistry
J.D., P.,
M.R.
(1971)
J.
and Nicholls,
M.R.
(1971)
M.R. G.
(1974)
(1972)
Biochem.
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Biochem.
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649-
G.
(1974)
A.S., Edwards, K., and Schreiber, Res. Commun. 61, 494-501.
G., Urban, J., (1971) J. Biol.
123,
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and Schreiber,
and Nicholls,
Biochem.
Z&ringer, J., Reutter, Chem. 246, 4531-4538.
Carbonara, A.O., 2, 235-254.
782
and
Heremans,
W., J.F.
and (1965)