Vol. 90, No. 4, 1979 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
29, 1979
Pages 1393-1399
BIOSYNTHESIS
OF ELASTIN
BY AN ENDOTHELIAL
P.A.
W.H. Carries*,
Abraham*
CELL CULTURE
and V. Buonassisi?
*Department
of Pathology, University of California, School of Medicine Los Angeles, California 90024, and tDepartment of Biology, University of California, San Diego
Received
August
29, 1979 SUMMARY
An established endothelial cell culture line synthesizes and secretes into the medium a soluble protein of molecular weight approximately 75,000 that has other properties (e.g., hydroxyproline and valylproline dipeptide contents and solubility in propanol-butanol) identifying it as elastin. This protein is extractable from the washed cell layer. It is found in the medium in lower concentration together with characteristic lower molecular weight degradation products. No insoluble elastin could be detected. The development of endothelial
cells
characterization erties
is
including nectin
(l-4)
of endothelium
the
secretion
(11).
Jaffe fibers
of several
secretion
of elastin
possibility
that
by its
of soluble
(8)
Jones
of fetal
extracellular
elastic
in the matrix (6)
endothelium
may be the
distinctive
fiber
(6-10)
source
structure
MATERIALS
of aortic
microfibrils human
to detect
the
endothelium. of intimal
(12).
has been made by demonstrating
by a culture
and fibro-
of cultured
arterial
endothelial
prop-
matrix,
tissue
was unable
bovine
biological
discovered
collagen
demonstrated
microscopy
question
elastin
of the (6),
cultivation
to the
many recently
glycoprotein
by a culture
of this
Among its
However,
arterial
suggested
solution
synthesis
endothelium.
and continuous
substantially
components
(3),
by electron
vein
the isolation
(5).
and coworkers
umbilical
has been
for
has contributed
mucopolysaccharide
and elastin
to the
of methods
The
elastin
An approach the biocells.
AND METHODS
A cloned line (A5) from an established culture of rabbit aortic endothelial cells (13) was used. Cultures were grown to confluency in F-12 medium (Gibco), supplemented with 10% fetal calf serum, in 5% CO . The cultures dishes containing a total of 4-5 x lo7 cells were rinse 8 three times with protein-free F-12 medium, lacking proline and lysine, and supplemented Abbreviation:
SDS-sodium
dodecylsulfate. 0006-291X/79/201393-07$01.00/0 1393
Copyright @ I979 by Academic Press, Inc. All rights of reproduction in anyform reserved.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with 5@g/ml of ascorbic acid. The cultures were preincubated for twenty minutes in the same medium and lpCi/ml of both L-[5-3Hlproline (lS,OOOmCi/mmol) and L-Ill-14C]lysine (330 mCi/mmol) were added. After 4 hr labeling, protein synthesis was stopped by the addition of lOOpg/ml of cyclohexamide and the cell layer was rinsed three times with cold serumfree medium. The decanted pooled medium and the cell layer were heated to 100°C for 15 min in O.OlM phosphate buffer, pH 7.4, containing 5% SDS, 5mM EDTA and protease inhibitors (0.2mM phenylmethylsulfonyl fluoride, 5mM N-ethylmaleimide and 1mM p-aminobenzamidine). The centrifuged supernatants were dialyzed against 1% SDS in the same buffer and the inhibitors were added in this and all subsequent steps. Chromatography: The dialyzed medium and cell extract were chromatographed in 1% SDS on a column (2.5 x 80 cm) of Bio-Gel A-5m (6% cross-linked, 200400 mesh). Fractions of 6 ml per tube were collected. Aliquots of 200~1 were counted in a Beckman LS-250 scintillation counter. SDS gel electrophoresis: Electrophoresis was performed on 7.5% acrylamide gels in 0.2% SDS and 2M urea at pH 7.2 (14). The sample was heated at 50°C for 30 min. A current of 6mA per tube was applied for 6 hr. The gels were then sliced into lmm sections and digested with 0.5 ml of 30% H202 at 55°C for 4 hr. Catalase was added to destroy excess peroxide (15). [14C]lysinelabeled soluble elastin, prepared from aortas of copper-deficient pigs (15), was used as a standard. Propanol-butanol extraction of the cell extract was carried out by the method of Sandberg, et al (16). The cell extract was dialyzed exhaustively against O.lM ammonium formate at pH 4.5 and cooled to 4'C. The pH was adjusted to 5.2 and the ammonium formate concentration to 0.5M. Propanol (1.5 volumes) was added dropwise, followed by 2.5 volumes of butanol. The precipitate formed was removed by centrifugation and the supernatant was evaporated at 3O“C. The residue was extracted with 1% SDS, dialyzed against the buffer and chromatographed on the agarose column. Proline and hydroxyproline determination: Protein samples were hydrolyzed 6N HCl at 110°C and the hydrolysate was chromatographed on the long column the Beckman 120B amino acid analyzer. Fractions were collected at 2 minute intervals and the radioactivity of the fractions corresponding to proline and hydroxyproline was counted. Identification of valylproline dipeptide: Valylproline dipeptide was identified by the method of Hauschka and Gallop (17) with some modifications. The cell extract was hydrolyzed in 2N KOH and the hydrolysate was neutralized with perchloric acid. Chromatography was carried out on the long column of the amino acid analyzer using a two buffer system of 0.2M citrate, pH 3.28, at 31'C and 0.2M citrate, pH 4.25, at 55°C with a buffer change time of 160 min. Fractions were collected at 2 min intervals and their radioactivity was counted. Synthetic valylproline was used as the standard. Valylproline anhydride was prepared from the dipeptide by heating it with S-naphthol at 145°C for 3 hr according to the method of Lichtenstein (18). Preparation of insoluble elastin: The residue after extraction of the cells with 5% SDS solution was washed with water to remove the SDS and autoclaved in distilled water for 1 hr, four times. The residue was dried in a vacuum dessicator and hydrolyzed in 6N HCl at 110°C. The hydrolysate was evaporated and the residue was dissolved in water and its radioactivity was counted.
1394
in of
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
67,000
RESEARCH COMMUNICATIONS
45,000
25,000
50 m b 40 x E 30 Et ,'
20 10
10
20
30
40
50
FRACTION
1. Agarose (Bio-Gel labeled proteins of butanol soluble fraction
60
70
A-51x1) gel chromatography the cell layer (a), the of the cell extract (c).
“Fe[ H]
80
90
400
NUMBER
of the SDS-soluble, medium (b) and a propanol-
RESULTS The labeled for
most
of the
the proteins that
proteins
weight
volume
tion
55,000,
extract
and 25,000
other
cpm).
l,a),
at the void the first
molecular
volume.
of
as great
of the
the minor
cell
had ten
two corresponding
weights,
respectively.
extract
one eluting
at
times
The medium on gel
at positions
as
to a molecular
protein
eluting
in the peak
times
corresponding
weight
SDS accounted
The radioactivity
Gel filtration
(Fig.
molecular
three
by hot
cpm) was fifteen
one in the region
l,b),
was contained
the
filtra-
to the peaks
of
of approximately The bulk
corresponding
to the
lowest
of the molec-
weight. Gel filtration extract
solubilized SDS gel
peak of the
of the propanol-butanol
yielded
peak of the cell not
(Fig.
layer
recovered.
two peaks
The lower
and the
45,000
radioactivity
cell
x lo6
of the one eluted peaks
the cell
(32 x lo6
(2.2
yielded
75,000.
gave five cell
extract
and the major
of about
from
radioactivity
medium
column
radioactivity
ular
cell
of the dialyzed
the void
the
nondialyzable
of the
on the agarose
extracted
only
extract
a single (Fig.
by the alcohol electrophoresis
cell
extract
peak
l,a>.
soluble (Fig.
1,~)
The peak eluting
fraction
of the aqueous
corresponding
to the major
at the void
volume
was
mixture. of the
gave a single
labeled
protein
radioactive
1395
recovered band
(Fig.
from 2).
the major This
band
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
5
10
15 LENGTH
20 FROM
25 30 ORIGIN (mm)
35
40
Fig. 2. SDS-gel electrophoresis of the protein eluting at about 75,000 daltons on gel filtration (o-o-o) and of labeled soluble elastin (o---o---o) prepared from copper-deficient pig aorta by coacervation.
had identical from
mobility
to that
copper-deficient
band,
derived
from
the cell
pig
from
demonstrated
proline
the presence which
hydrolysates
hydroxylation
eluting
at the void
lower
showed
extraction
volume
weight
was absent
the cell
extract
16% hydroxylation of insoluble of the
peaks
cell
for
proline
weight
chromatography
and its
the
synthetic
anhydride,
standards
of hydroxyproline
and
peaks.
showed
The peak of
The presence after
prepared
molecular
exchange
valylproline,
the presence
analyzed
of the
molecular
hydroxylation.
also
of the major
percent
weight
high
aorta,
on ion
by using
unidentified
and the medium were
from
extract
identified
showed
extract
and the
A minor
elastin
of copper-deficient
of the dipeptide, were
and a few other
The protein
elastin
of the cell
The hydrolysate
Acid
by coacervation.
soluble
hydrolysate
diketopiperazine, 3).
aorta
soluble
extract.
The alkali
(Fig.
the
[14 C] labeled
of the
of the proline
the
highest
(Table
degree
at about
proteins
eluting
of proline elastin
75,000
at about
The peaks (Q~33%).
molecular
showed
only
75,000
weight about
5%
molecular
residues.
was sought
with
1).
cell
and the
of hydroxylation
of the medium
the medium
of the
and hydroxyproline
was calculated
eluting
layer
chromatograms
hot
1396
in the residue
SDS solution.
remaining
The residue
was
Vol. 90, No. 4, 1979
BIOCHEMICAL
HYPro
6
AND BIOPHYSICAL
Pro
VOI
1
1
1
RESEARCH COMMUNICATIONS
12 10
: I
08
0 u
06
2 0
04 02 0 40
00
160
720 TIME
Fig. 3. Ion exchange proteins of the cell the standard amino
were determined
200
240
200
320
IN MINUTES
chromatography layer labeled
of
the
with
acids, valylproline by the ninhydrin method.
2M KOH hydrolysate
of
the
[3H]proline. Elution positions of dipeptide and valylproline anhydride
Table
1
PERCENT HYDROXYLATION OF PROLINE RESIDUES IN SOLUBLE PROTEINS OF ENDOTHELIAL CELL CULTURE (Hypro j Pro + Hypro x 100) Proteins
Source
>125,000
hydrolyzed residue
Eluting Molecular
32.4
5.1
Culture medium
33.15
16.3
in 6M HCl before of the
SDS extract
and the autoclaved the low activity
residue no further
55,000
75,000
Cell extract
and after
at Approximate Weights
repeated
showed
a total
[14C]
showed
a total
activity
analysis
45,000
25,000
5.1
5.4
autoclaving. radioactiv
The insoluble .ty of 1.9 x lo4
of 9 x lo3
of the hydrolysates
cpm. Because
was attempted.
DISCUSSION The endothelial maintained in detail
cells
by continuous
used weekly
were
a subculture
transfers.
(19).
1397
Its
from properties
an established have been
line reviewed
w of
Vol. 90, No. 4, 1979
The results
BIOCHEMICAL
presented
prove
the biosynthesis
protein
of molecular
weight
soluble
elastin
copper-deficient
from
a propanol-butanol It
contains
and the (17).
These
are
in the cells.
high
of hydroxylation that
salt-
also
soluble
with
elastin
suggests
extracted
by hot
The three
protein
SDS
of the protein
of the proline
form. weight
(21)
by the culture.
the accumulation
(5%) hydroxylation
(16).
of elastin
of elastin
elastin
in
and coworkers
characteristic
synthesis
a presecretory
of a soluble
dalton is
of Sandberg
of the
same molecular
It
compatible
of the
medium low
with
of the
fold
in this higher
in the medium
percent reinforces
inference. The relatively
presence tion
extracts
of these
lower
ular
weight not
culture trasts wherein
molecular
along
with
were
identified
the endothelial protein
after
culture its
in the
prevailing label
(24).
secretion
is noteworthy
the medium protease
The relatively
to that
distribution
that
correspond
action
degradathe
of the cell
layer.
closely
on soluble
low percent
compared
of labeled steps
elastin
hydroxylation
of the higher
of hydroxyproline
insoluble
leading
in cultures
was rapidly
has not
It
suggests
in the extract
in
the
weights
molecresidues
chain.
amounts
situation
a [14C]lysine
crosslinks
that
a deficiency
the
found
fragments
peptide
of significant
not
(22).
indicates the
molecular
of neutral
weight
in the medium and the
secretion.
proteins
aorta
molecular
suggests
were
weight
of chick
uniform
of lower
as products
component
The lack
proteins
proteins
identified
in salt
of the elastin
in the medium after
weight
lower
those
radioactivity
labeled
elastin
molecular
The three with
low
of three
of the
lower
is
culture
(20).
anhydride)
radioactivity
in the
consistent
its
evidences
The relatively
is
aorta
tropoelastin
(and
culture
the 74,000
in a concentration
compelling
to that
swine
RESEARCH COMMUNICATIONS
by this
approximating
the
valylproline
The relatively
fraction
like
hydroxyproline
dipeptide
compared
closely
mixture
AND BIOPHYSICAL
in the
to crosslinkage.
This
con-
muscle
cells
of aortic
insolubilized
The nature been
elastin
may decrease
(23)
of the defect
established. the
1398
smooth
Rapid concentration
and lysine
derived
in crosslinkage degradation
of the
of the protein
in
Vol. 90, No. 4, 1979
available
for
retarded
thelial are
crosslinkage.
after
cells susceptible
lysyl
oxidation
in the primary may affect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Oxidation
due to deficient
Crosslinkage ferences
BIOCHEMICAL
of the
oxidase
may be inhibited
structure
of the
one or another
to experimental
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
lysyl
amino
or to an inhibitor
groups
of the
by an unknown
may be
enzyme.
mechanism.
elastin
(25)
synthesized
of these
steps.
These
Dif-
by endo-
possibilities
solution.
Acknowledgements: The assistance of Ziaeddin Patricia Colburn is gratefully acknowledged. U.S. Public Service research grants HL 12561 Heart, Lung and Blood Institute.
1.
free
Mohseni, Mary Lou Hart and This work was supported by and HL 17995 of the National
REFERENCES Jaffe, E.A., Nachman, R.L., Becker, C.G., and Minick, C.R. (1973) J. Clin. Invest. 52, 2745-2756. Lewis, L.J., Hoak, J.C., Maca, R.D., and Fry, G.L. (1973) Science 181, 453-454. Buonassisi, V. (1973) Exp. Cell Res. 76, 363-368. Gimbrone, M.A., Cotran, R.S., and Folkman, J. (1974) J. Cell Biol. 60, 673-684. Gimbrone, M.A. Jr. (1976). In: Progress in Hemostasis and Thrombosis, Ed. T.H. Spaet, pp. l-28, Grune and Stratton, Inc., New York. Jones, P.A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1882-1886. Howard, B.V., Macarak, E.J., Gunson, D., and Kefalides, N.A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2361-2364. Jaffe, E.A., Minick, C.R., Adelman, B., Becker, C.G., and Nachman, R. (1976) J. Exp. Med. 144, 209-225. Levene, C.I., and Heslop, J. (1977) J. Mol. Med. 2, 145-151. Barnes, M.J., Morton, L.F., and Levene, C.I. (1978) Biochem. Biophys. Res. Commun. 84, 646-653. Jaffe, E.A., and Mosher, D.F. (1978) J. Exp. Med. 147, 1779-1791. Hart, M.L., Beydler, S.A., and Carnes, W.H. (1978). In: Scanning Electron Microscopy II, Eds., R.P. Becker and 0. Johari, pp. 21-28, Scanning Electron Microscopy, Inc., AMF O'Hare, Illinois. Buonassisi, V., and Venter, J.C. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1612-1616. Furthmayr, H., and Timpl, R. (1971) Anal. Biochem. 41, 510-516. Smith, D.W., Abraham, P.A., and Carnes, W.H. (1975) Biochem. Biophys. Res. Commun. 66, 893-899. Sandberg, L.B., Zeikus, R.D., and Coltrain, I.M. (1971) Biochim. Biophys. Acta 236, 542-545. Hauschka, P.V., and Gallop, P.M. (1979) Anal. Biochem. 92, 61-66. Lichtenstein, N. (1938) 3. Am. Chem. Sot. 60, 560-563. Buonassisi, V., and Colburn, P. (1979) Adv. Microcirc. 9, 1-21. Smith, D.W., Brown, D.M., and Carnes, W.H. (1972) J. Biol. Chem. 247, 2427-2432. Uitto, J., Hoffmann, H.P., and Prockop, D.J. (1976) Arch. Biochem. Biophys. 173, 187-200. Mecham, R.P., and Foster, J.A. (1977) Biochem. J. 16, 3825-3830. Abraham, P.A., Smith, D.W., and Carnes, W.H. (1974) Biochem. Biophys. Res. Commun. 58, 597-604. Abraham, P.A., Smith, D.W., and Carnes, W.H. (1975) Biochem. Biophys. Res. Commun. 67, 723-727. Keith, D.A., Paz, M.A., and Gallop, P.M. (1979) Biochem. Biophys. Res. Commun. 87. 1214-1217.
1399
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
29, 1979
Pages 1393-1399
BIOSYNTHESIS
OF ELASTIN
BY AN ENDOTHELIAL
P.A.
W.H. Carries*,
Abraham*
CELL CULTURE
and V. Buonassisi?
*Department
of Pathology, University of California, School of Medicine Los Angeles, California 90024, and tDepartment of Biology, University of California, San Diego
Received
August
29, 1979 SUMMARY
An established endothelial cell culture line synthesizes and secretes into the medium a soluble protein of molecular weight approximately 75,000 that has other properties (e.g., hydroxyproline and valylproline dipeptide contents and solubility in propanol-butanol) identifying it as elastin. This protein is extractable from the washed cell layer. It is found in the medium in lower concentration together with characteristic lower molecular weight degradation products. No insoluble elastin could be detected. The development of endothelial
cells
characterization erties
is
including nectin
(l-4)
of endothelium
the
secretion
(11).
Jaffe fibers
of several
secretion
of elastin
possibility
that
by its
of soluble
(8)
Jones
of fetal
extracellular
elastic
in the matrix (6)
endothelium
may be the
distinctive
fiber
(6-10)
source
structure
MATERIALS
of aortic
microfibrils human
to detect
the
endothelium. of intimal
(12).
has been made by demonstrating
by a culture
and fibro-
of cultured
arterial
endothelial
prop-
matrix,
tissue
was unable
bovine
biological
discovered
collagen
demonstrated
microscopy
question
elastin
of the (6),
cultivation
to the
many recently
glycoprotein
by a culture
of this
Among its
However,
arterial
suggested
solution
synthesis
endothelium.
and continuous
substantially
components
(3),
by electron
vein
the isolation
(5).
and coworkers
umbilical
has been
for
has contributed
mucopolysaccharide
and elastin
to the
of methods
The
elastin
An approach the biocells.
AND METHODS
A cloned line (A5) from an established culture of rabbit aortic endothelial cells (13) was used. Cultures were grown to confluency in F-12 medium (Gibco), supplemented with 10% fetal calf serum, in 5% CO . The cultures dishes containing a total of 4-5 x lo7 cells were rinse 8 three times with protein-free F-12 medium, lacking proline and lysine, and supplemented Abbreviation:
SDS-sodium
dodecylsulfate. 0006-291X/79/201393-07$01.00/0 1393
Copyright @ I979 by Academic Press, Inc. All rights of reproduction in anyform reserved.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with 5@g/ml of ascorbic acid. The cultures were preincubated for twenty minutes in the same medium and lpCi/ml of both L-[5-3Hlproline (lS,OOOmCi/mmol) and L-Ill-14C]lysine (330 mCi/mmol) were added. After 4 hr labeling, protein synthesis was stopped by the addition of lOOpg/ml of cyclohexamide and the cell layer was rinsed three times with cold serumfree medium. The decanted pooled medium and the cell layer were heated to 100°C for 15 min in O.OlM phosphate buffer, pH 7.4, containing 5% SDS, 5mM EDTA and protease inhibitors (0.2mM phenylmethylsulfonyl fluoride, 5mM N-ethylmaleimide and 1mM p-aminobenzamidine). The centrifuged supernatants were dialyzed against 1% SDS in the same buffer and the inhibitors were added in this and all subsequent steps. Chromatography: The dialyzed medium and cell extract were chromatographed in 1% SDS on a column (2.5 x 80 cm) of Bio-Gel A-5m (6% cross-linked, 200400 mesh). Fractions of 6 ml per tube were collected. Aliquots of 200~1 were counted in a Beckman LS-250 scintillation counter. SDS gel electrophoresis: Electrophoresis was performed on 7.5% acrylamide gels in 0.2% SDS and 2M urea at pH 7.2 (14). The sample was heated at 50°C for 30 min. A current of 6mA per tube was applied for 6 hr. The gels were then sliced into lmm sections and digested with 0.5 ml of 30% H202 at 55°C for 4 hr. Catalase was added to destroy excess peroxide (15). [14C]lysinelabeled soluble elastin, prepared from aortas of copper-deficient pigs (15), was used as a standard. Propanol-butanol extraction of the cell extract was carried out by the method of Sandberg, et al (16). The cell extract was dialyzed exhaustively against O.lM ammonium formate at pH 4.5 and cooled to 4'C. The pH was adjusted to 5.2 and the ammonium formate concentration to 0.5M. Propanol (1.5 volumes) was added dropwise, followed by 2.5 volumes of butanol. The precipitate formed was removed by centrifugation and the supernatant was evaporated at 3O“C. The residue was extracted with 1% SDS, dialyzed against the buffer and chromatographed on the agarose column. Proline and hydroxyproline determination: Protein samples were hydrolyzed 6N HCl at 110°C and the hydrolysate was chromatographed on the long column the Beckman 120B amino acid analyzer. Fractions were collected at 2 minute intervals and the radioactivity of the fractions corresponding to proline and hydroxyproline was counted. Identification of valylproline dipeptide: Valylproline dipeptide was identified by the method of Hauschka and Gallop (17) with some modifications. The cell extract was hydrolyzed in 2N KOH and the hydrolysate was neutralized with perchloric acid. Chromatography was carried out on the long column of the amino acid analyzer using a two buffer system of 0.2M citrate, pH 3.28, at 31'C and 0.2M citrate, pH 4.25, at 55°C with a buffer change time of 160 min. Fractions were collected at 2 min intervals and their radioactivity was counted. Synthetic valylproline was used as the standard. Valylproline anhydride was prepared from the dipeptide by heating it with S-naphthol at 145°C for 3 hr according to the method of Lichtenstein (18). Preparation of insoluble elastin: The residue after extraction of the cells with 5% SDS solution was washed with water to remove the SDS and autoclaved in distilled water for 1 hr, four times. The residue was dried in a vacuum dessicator and hydrolyzed in 6N HCl at 110°C. The hydrolysate was evaporated and the residue was dissolved in water and its radioactivity was counted.
1394
in of
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
67,000
RESEARCH COMMUNICATIONS
45,000
25,000
50 m b 40 x E 30 Et ,'
20 10
10
20
30
40
50
FRACTION
1. Agarose (Bio-Gel labeled proteins of butanol soluble fraction
60
70
A-51x1) gel chromatography the cell layer (a), the of the cell extract (c).
“Fe[ H]
80
90
400
NUMBER
of the SDS-soluble, medium (b) and a propanol-
RESULTS The labeled for
most
of the
the proteins that
proteins
weight
volume
tion
55,000,
extract
and 25,000
other
cpm).
l,a),
at the void the first
molecular
volume.
of
as great
of the
the minor
cell
had ten
two corresponding
weights,
respectively.
extract
one eluting
at
times
The medium on gel
at positions
as
to a molecular
protein
eluting
in the peak
times
corresponding
weight
SDS accounted
The radioactivity
Gel filtration
(Fig.
molecular
three
by hot
cpm) was fifteen
one in the region
l,b),
was contained
the
filtra-
to the peaks
of
of approximately The bulk
corresponding
to the
lowest
of the molec-
weight. Gel filtration extract
solubilized SDS gel
peak of the
of the propanol-butanol
yielded
peak of the cell not
(Fig.
layer
recovered.
two peaks
The lower
and the
45,000
radioactivity
cell
x lo6
of the one eluted peaks
the cell
(32 x lo6
(2.2
yielded
75,000.
gave five cell
extract
and the major
of about
from
radioactivity
medium
column
radioactivity
ular
cell
of the dialyzed
the void
the
nondialyzable
of the
on the agarose
extracted
only
extract
a single (Fig.
by the alcohol electrophoresis
cell
extract
peak
l,a>.
soluble (Fig.
1,~)
The peak eluting
fraction
of the aqueous
corresponding
to the major
at the void
volume
was
mixture. of the
gave a single
labeled
protein
radioactive
1395
recovered band
(Fig.
from 2).
the major This
band
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
5
10
15 LENGTH
20 FROM
25 30 ORIGIN (mm)
35
40
Fig. 2. SDS-gel electrophoresis of the protein eluting at about 75,000 daltons on gel filtration (o-o-o) and of labeled soluble elastin (o---o---o) prepared from copper-deficient pig aorta by coacervation.
had identical from
mobility
to that
copper-deficient
band,
derived
from
the cell
pig
from
demonstrated
proline
the presence which
hydrolysates
hydroxylation
eluting
at the void
lower
showed
extraction
volume
weight
was absent
the cell
extract
16% hydroxylation of insoluble of the
peaks
cell
for
proline
weight
chromatography
and its
the
synthetic
anhydride,
standards
of hydroxyproline
and
peaks.
showed
The peak of
The presence after
prepared
molecular
exchange
valylproline,
the presence
analyzed
of the
molecular
hydroxylation.
also
of the major
percent
weight
high
aorta,
on ion
by using
unidentified
and the medium were
from
extract
identified
showed
extract
and the
A minor
elastin
of copper-deficient
of the dipeptide, were
and a few other
The protein
elastin
of the cell
The hydrolysate
Acid
by coacervation.
soluble
hydrolysate
diketopiperazine, 3).
aorta
soluble
extract.
The alkali
(Fig.
the
[14 C] labeled
of the
of the proline
the
highest
(Table
degree
at about
proteins
eluting
of proline elastin
75,000
at about
The peaks (Q~33%).
molecular
showed
only
75,000
weight about
5%
molecular
residues.
was sought
with
1).
cell
and the
of hydroxylation
of the medium
the medium
of the
and hydroxyproline
was calculated
eluting
layer
chromatograms
hot
1396
in the residue
SDS solution.
remaining
The residue
was
Vol. 90, No. 4, 1979
BIOCHEMICAL
HYPro
6
AND BIOPHYSICAL
Pro
VOI
1
1
1
RESEARCH COMMUNICATIONS
12 10
: I
08
0 u
06
2 0
04 02 0 40
00
160
720 TIME
Fig. 3. Ion exchange proteins of the cell the standard amino
were determined
200
240
200
320
IN MINUTES
chromatography layer labeled
of
the
with
acids, valylproline by the ninhydrin method.
2M KOH hydrolysate
of
the
[3H]proline. Elution positions of dipeptide and valylproline anhydride
Table
1
PERCENT HYDROXYLATION OF PROLINE RESIDUES IN SOLUBLE PROTEINS OF ENDOTHELIAL CELL CULTURE (Hypro j Pro + Hypro x 100) Proteins
Source
>125,000
hydrolyzed residue
Eluting Molecular
32.4
5.1
Culture medium
33.15
16.3
in 6M HCl before of the
SDS extract
and the autoclaved the low activity
residue no further
55,000
75,000
Cell extract
and after
at Approximate Weights
repeated
showed
a total
[14C]
showed
a total
activity
analysis
45,000
25,000
5.1
5.4
autoclaving. radioactiv
The insoluble .ty of 1.9 x lo4
of 9 x lo3
of the hydrolysates
cpm. Because
was attempted.
DISCUSSION The endothelial maintained in detail
cells
by continuous
used weekly
were
a subculture
transfers.
(19).
1397
Its
from properties
an established have been
line reviewed
w of
Vol. 90, No. 4, 1979
The results
BIOCHEMICAL
presented
prove
the biosynthesis
protein
of molecular
weight
soluble
elastin
copper-deficient
from
a propanol-butanol It
contains
and the (17).
These
are
in the cells.
high
of hydroxylation that
salt-
also
soluble
with
elastin
suggests
extracted
by hot
The three
protein
SDS
of the protein
of the proline
form. weight
(21)
by the culture.
the accumulation
(5%) hydroxylation
(16).
of elastin
of elastin
elastin
in
and coworkers
characteristic
synthesis
a presecretory
of a soluble
dalton is
of Sandberg
of the
same molecular
It
compatible
of the
medium low
with
of the
fold
in this higher
in the medium
percent reinforces
inference. The relatively
presence tion
extracts
of these
lower
ular
weight not
culture trasts wherein
molecular
along
with
were
identified
the endothelial protein
after
culture its
in the
prevailing label
(24).
secretion
is noteworthy
the medium protease
The relatively
to that
distribution
that
correspond
action
degradathe
of the cell
layer.
closely
on soluble
low percent
compared
of labeled steps
elastin
hydroxylation
of the higher
of hydroxyproline
insoluble
leading
in cultures
was rapidly
has not
It
suggests
in the extract
in
the
weights
molecresidues
chain.
amounts
situation
a [14C]lysine
crosslinks
that
a deficiency
the
found
fragments
peptide
of significant
not
(22).
indicates the
molecular
of neutral
weight
in the medium and the
secretion.
proteins
aorta
molecular
suggests
were
weight
of chick
uniform
of lower
as products
component
The lack
proteins
proteins
identified
in salt
of the elastin
in the medium after
weight
lower
those
radioactivity
labeled
elastin
molecular
The three with
low
of three
of the
lower
is
culture
(20).
anhydride)
radioactivity
in the
consistent
its
evidences
The relatively
is
aorta
tropoelastin
(and
culture
the 74,000
in a concentration
compelling
to that
swine
RESEARCH COMMUNICATIONS
by this
approximating
the
valylproline
The relatively
fraction
like
hydroxyproline
dipeptide
compared
closely
mixture
AND BIOPHYSICAL
in the
to crosslinkage.
This
con-
muscle
cells
of aortic
insolubilized
The nature been
elastin
may decrease
(23)
of the defect
established. the
1398
smooth
Rapid concentration
and lysine
derived
in crosslinkage degradation
of the
of the protein
in
Vol. 90, No. 4, 1979
available
for
retarded
thelial are
crosslinkage.
after
cells susceptible
lysyl
oxidation
in the primary may affect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Oxidation
due to deficient
Crosslinkage ferences
BIOCHEMICAL
of the
oxidase
may be inhibited
structure
of the
one or another
to experimental
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
lysyl
amino
or to an inhibitor
groups
of the
by an unknown
may be
enzyme.
mechanism.
elastin
(25)
synthesized
of these
steps.
These
Dif-
by endo-
possibilities
solution.
Acknowledgements: The assistance of Ziaeddin Patricia Colburn is gratefully acknowledged. U.S. Public Service research grants HL 12561 Heart, Lung and Blood Institute.
1.
free
Mohseni, Mary Lou Hart and This work was supported by and HL 17995 of the National
REFERENCES Jaffe, E.A., Nachman, R.L., Becker, C.G., and Minick, C.R. (1973) J. Clin. Invest. 52, 2745-2756. Lewis, L.J., Hoak, J.C., Maca, R.D., and Fry, G.L. (1973) Science 181, 453-454. Buonassisi, V. (1973) Exp. Cell Res. 76, 363-368. Gimbrone, M.A., Cotran, R.S., and Folkman, J. (1974) J. Cell Biol. 60, 673-684. Gimbrone, M.A. Jr. (1976). In: Progress in Hemostasis and Thrombosis, Ed. T.H. Spaet, pp. l-28, Grune and Stratton, Inc., New York. Jones, P.A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1882-1886. Howard, B.V., Macarak, E.J., Gunson, D., and Kefalides, N.A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2361-2364. Jaffe, E.A., Minick, C.R., Adelman, B., Becker, C.G., and Nachman, R. (1976) J. Exp. Med. 144, 209-225. Levene, C.I., and Heslop, J. (1977) J. Mol. Med. 2, 145-151. Barnes, M.J., Morton, L.F., and Levene, C.I. (1978) Biochem. Biophys. Res. Commun. 84, 646-653. Jaffe, E.A., and Mosher, D.F. (1978) J. Exp. Med. 147, 1779-1791. Hart, M.L., Beydler, S.A., and Carnes, W.H. (1978). In: Scanning Electron Microscopy II, Eds., R.P. Becker and 0. Johari, pp. 21-28, Scanning Electron Microscopy, Inc., AMF O'Hare, Illinois. Buonassisi, V., and Venter, J.C. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1612-1616. Furthmayr, H., and Timpl, R. (1971) Anal. Biochem. 41, 510-516. Smith, D.W., Abraham, P.A., and Carnes, W.H. (1975) Biochem. Biophys. Res. Commun. 66, 893-899. Sandberg, L.B., Zeikus, R.D., and Coltrain, I.M. (1971) Biochim. Biophys. Acta 236, 542-545. Hauschka, P.V., and Gallop, P.M. (1979) Anal. Biochem. 92, 61-66. Lichtenstein, N. (1938) 3. Am. Chem. Sot. 60, 560-563. Buonassisi, V., and Colburn, P. (1979) Adv. Microcirc. 9, 1-21. Smith, D.W., Brown, D.M., and Carnes, W.H. (1972) J. Biol. Chem. 247, 2427-2432. Uitto, J., Hoffmann, H.P., and Prockop, D.J. (1976) Arch. Biochem. Biophys. 173, 187-200. Mecham, R.P., and Foster, J.A. (1977) Biochem. J. 16, 3825-3830. Abraham, P.A., Smith, D.W., and Carnes, W.H. (1974) Biochem. Biophys. Res. Commun. 58, 597-604. Abraham, P.A., Smith, D.W., and Carnes, W.H. (1975) Biochem. Biophys. Res. Commun. 67, 723-727. Keith, D.A., Paz, M.A., and Gallop, P.M. (1979) Biochem. Biophys. Res. Commun. 87. 1214-1217.
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