Vol. 67, No. 3, 1975
BIOCHEMICAL
PURIFICATION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF BOVINE SOMATOMEDIN 192 3
J. P. Liberti
Department of Biochemistry, Medical College Virginia Cormaonwealth University, Richmond, Received
October
of Virginia Virginia 23298
8,1975
SUMMARY: A procedure for the purification of bovine somatomedin (SM 4, is presented. The purification scheme utilizes ultrafiltration through membranes of nominal mol. wt. cutoffs, molecular sieve chromatography and finally isoelectric focusing. Two peaks of SM activity, measured by the --in vitro stimulation of 35S-Na2SO4 and 3H-thymidine uptake by costal cartilage, were present after focusing; an acidic component having a p1 of 6.0 - 6.7 and a basic component having a p1 in the range of 7.8 - 8.3. The acidic component comprised 2% of the initial activity and was 120,000-fold purified: the basic component comprised 10% of the initial activity and was 350,000-fold purified relative to the starting material. These components are similar in molecular size and pI to SM-A and SM-C isolated from human plasma. INTRODUCTION: a family
increasingly
promoting
substances
activities, necessary
A number degrees
has become
of growth
insulin-like factors
It
for
from
SM-B isolated
by Hall
leagues
(4).
Both
as the
starting
include purification majority
groups
SM-A is
a Cohn II
of activity
bovine
in tissue
has been species.
appears
a neutral
fraction
similar
(1) is
isolated
to that
which
from
obtained
serum growth
to varying are SM-A and
outdated
human plasma
(p1 = 7),
SM-B is acidic
- 9.4).
in
have
by Van Wyk and col-
Other
and MSA from calf
presented
in plasma
culture.
Among these
peptide
(PI 8.6
exists
peptides,
and SM-C isolated
peptide
blood
there
the so-called
used a Cohn IV fraction
and SM-C is a basic from
with
substances
(3,5)
that These
of cells
serum of different and Uthne
of SM from
human plasma
common traits
promoting
material.
NSILA-S
(l-4).
the proliferation
of growth
of purity
(p1 4 - 5.2)
share
evident
this
substances serum
(2).
communication.
by Van Wyk --et al.
(4).
lsupported by NIH grant AM-16457. 2A portion of this work has been presented at the Endocrine Meetings, New York (7). 3Recipient of Research Career Development Award, K 04-AMOO125. 4Abbreviations: SM, Somatomedin; MSA, multiplication stimulating activity; NSILA-S, non-supressible insulin-like activity-soluble. Copyright All rights
o 19 75 b.v Academic Press, Inc. of reproductim bl any form resrrved.
1226
The The using
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: Bovine blood was collected at a local slaughter house and transported to our laboratory in plastic buckets. The blood was allowed to clot at room temperature. After 4-5 hours, the fluid surrounding the retracted clot was decanted and its volume measured. The procedures described below are for 4 - L portions. Four L of the supernatant were heated in a water bath. When the temperature reached 52', 20 ml of glacial acetic acid and 100 ml of 1 M Na acetate pH 5.5 were added. The mixture was maintained between 53' and 57" and it was stirred continuously. After 30 min., the mixture, which appeared dark brown and had a great quantity of coagulated material, was cooled in an ice water bath. The mixture was centrifuged for 15 min. at 5000 x g and 10" and the precipitate wasdiscarded. The supernatant of this step was ultrafiltered at room temperature through a 50,000 mol. wt. cutoff membrane (Amicon, Lexington, Mass.) with stirring and at 30 psi. The filtrate was concentrated by ultrafiltration through a 1000 mol. wt. cutoff membrane at 50 psi at room temperature. The 1000 mol. wt. retentate was chromatographed on a (3.18 x 61 cm) column of Sephadex G-50 medium. Eluates of peaks having biological activity were pooled, concentrated using a 1000 mol. wt. cutoff membrane and subjected to isoelectric focusing in the pH range of 3.5 to 10. Most of the ampholyte was removed from the focused fraction by chromatography on G-50 Sephadex followed by ultrafiltration through a 1000 mol. wt. cutoff membrane. The retentates were assayed for SM activity. ASSAY: Hypophysectomized, male rats (100-110 g) were obtained from ZivicMiller Labs, Allison Park, Pa., and were used 7-10 days post-operation. The procedures for the --in vitro uptake of various precursors (carrier-free 35S-Na2S04 or 3H-thymidine purchased from New England Nuclear Corp.) by costal cartilage segments have been described elsewhere (6,7). The reference standard (bovine serum) and each test preparation were assayed at 2 or 3 dosages. The test preparations were diluted with saline to be equivalent in volume to the initial serum. When 35S-Na2SO 4 uptake was determined, the incubation reaction was stopped by boiling, washed overnite with tap water, air dried and weighed. Cartilage segments were treated similarly for the 3H-thymidine uptake experiments except that after boiling, the cartilage was soaked for 4 h in 5% trichloacetic acid. The cartilage segments were digested with formic acid and counted in a Beckman scintillation spectrometer. Results are expressed as c/m/mg cartilage. SM units were obtained from a 4-or 6-point symmetrical comparison with the standard reference serum using classic bioassay procedures as the activity present in 1 ml of the reference (8). One SM unit is defined standard. Protein was determined by the method of Lowry with bovine serum albumin as the standard (9). RESULTS: bovine ever,
The purification serum was prepared
it
was found
the retracted activity
possessed
fitted
decreased
ca.
30% protein
of supernatewith to half
a 2 L reservoir. that
presented
prior
suitable
was comparable supernate
is
yielded
preparation.
to that a dark
The flow
of serum. brown
when the volume
How-
surrounding the SM
Centrifugation
fluid
(supernate-2) of supernate-1. in a stirred
was 1 ml min -1 initially
of the retentate 1227
Initially,
step.
Thus,
at room temperature rate
I.
(supernate-1)
and 45% of the SM activity was performed
in Table
to the acid/heat
of the supernate
an equally
of the supernate-l
Ultrafiltration cell
decantation
provided
treated
devised
by centrifugation
that
clot
of the acid/heat which
procedure
was reduced
and
by 70%.
Vol. 67, No. 3, 1975
BIOCHEMICAL
TABLE 1:
AND BIOPHYSICAL
PURIFICATION
RESEARCH COMMUNICATIONS
SCHEME FOR BOVINE SOMATOMEDIN
Bovine
blo
d supernate-l heat
and acid
treatment
-centrifugation
\
d
Pellet
SupernateI
XM-50
membrane
50,oooa Retentate
Filtrate UM-2 membrane 1, oooa
RetentatehFiltrate G-50 Sephadex Chromatography Pool
Active
fractions
Isoelectric focusing pH 3.5 - 10 a Approximate
The filtrate 35 S-Na2S04 50,000 wt. (18.5 Five their
mol.
cutoff
exhibited
wt.
filtrate
was widely Fractions
on a column collected,
to stimulate distributed D and E were
of membrane
of SM as measured
with
flow
rate
pooled
concentrated
was 1.8 ml min -1 . and eluted
as indicated
(Fig.
and 3H-thymidine
the greatest
amount
with
1228
The
wt.
a 1000 mol. The concentrate
with
0.02
M NaCl.
1) and assayed
uptake.
residing
a 1000 mol.
of
of 2.66.
to 19 ml using
of G-50 Sephadex
35 S-Na2S04
by the uptake
activity
L) was concentrated
The initial
were
cutoff
and had a specific (1.7
membrane.
ml fractions
wt.
1360 units
and 3H-thymidine
ml) was placed
ability
mol.
SM activity
in fractions cutoff
for
D and E.
membrane
and
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
Effluent
RESEARCH COMMUNICATIONS
(mls)
sieve chromatography of bovine somatomedin. Fig. 1: Molecular was applied to a Sephadex UPPER PANEL - 19 mls of the 1000 mol. wt. retentate G-50 column and eluted with 0.02 M NaCl. The flow rate was 1.2 ml. mine1 and Chromatography was done at 4' C. 5 ml fractions were collected. LOWER PANEL - Fractions were pooled as indicated and the biologic activity of each was determined as described in Methods.
focusing of Bovine Somatomedin. Fig. 2: Isoelectric UPPER PANEL - Isoelectric focusing between pH 3.5 and 10 was performed in a 110 ml LKB ampholine column with a 1% ampholyte solution. Fractions D and E Sucrose of G-50 (Fig. 1) were concentrated and placed in the light solution. The cathode (-) was 0.25 M gradients were formed with an LKB gradient mixer. Focusing was done NaOH in water and the anode (+) was 1.3 N H2H04 in sucrose. at 4" for 63 hr. at ca. 600 V. LOWER PANEL - SM activity was measured as described in Methods section.
1229
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE RECOVERY AND PURIFICATION
TABLE 2: Preparation
Protein (mg>
OF SOMATOMEDIN FROM 4 L of BOVINE SERUM
Units*
Recovery %
Unitslmg
1) Supernate-l
3.44
x 105
4000
(100)
0.0116
2) Supernate[Acid/heat treatment
1.03
x 105
1800
45
0.0175
1360
34
2.66
Purification
(1) 1.5
of O)l 3) 50,000 mol. wt. filtrate of (2)
511
4) Eluates of G-50 Sephadex fractions of (3)
2.26
920
23
407
35 x 103
0.057
80
2
1404
12 x 104
0.098
400
10
4082
35 x 104
5) Eluates of isoelectric-focused fractions of (4) A) pH 6.0 to 6.8 B) pH 7.9 8.3 * 1 unit
defined
subjected ity
to
as SM activity
to isoelectric
of SM activity
to 4.4
pH 7.9
fraction
A minor
(Fig.
2) show that
the major-
one in the pH range
peak of activity
SM activity native
and had a specific
serum.
The basic
DISCUSSION:
The purification
the appearance component
of the purification
in the pH range
SM activity
protein
in 2 regions:
and 8.3.
the original
acidic
The results
of the progress
The isoelectric
than
in 1 ml of Supernate-l
6.0
was noted
to 6.8
in
the
pH range.
A tabulation
initial
focusing.
was located
and one between 4.2
present
229
and represented
focused
times
procedure
in
a recovery
described exhibit
the 6.0
1230
7.9
in this SM activity.
2.
2% of the
to 8.3)
more pure
initial
in Table
was 120,000
to 6.8 pH range
of 2% of the
presented
contained
which
(pH range
and was 350,000
which
to 6.8
activity
component
of two components that
6.0
is
times
possessed
than
native
report
greater 10% of
serum.
results
in
The slightly contained SM activity.
57 ug of This
com-
Vol. 67, No. 3, 1975
BIOCHEMICAL
ponent
more pure
was 120,000-fold
protein
analysis.
350,000
times
of
the minute
The basic
and accounted
amounts
of SM in blood, Thus,
the 98 ug of SM (350,000
pure
The activities
Both basic
exhibited component
theirs noted
is
slightly
in the
activities
of Sephadex
the course uptake
which
indicates
at least
two forms:
one which
unbound
(free)
and the
has a mol.
'free'
the material this
is
treatment
is
reassociation
acetic
acid
molecules
used
similar
to that
promoting
small
percent
to substances
peak.
in the
wt.
with
fractions
of the
SM activity
having
mol.
wts.
these
forms
in blood
SM exists which
wt.
This
during
of > 50,000 (3,4)
which most
prevents
of the SM
(3-5)
of
SM from
larger
chromatography.
chromatography
resided
This
as well
size
as other
is growth
by radioimmunoassay,
the effluent is
is bound
the concentration
the majority in
in
Presumably,
acid
to dissociate
similar
the
Furthermore,
conditions,
after
of
laboratories
of 5- to 12000.
having
1231
although
species.
may be that
The
multiple
In other
the
of SM activity
has a mol.
as measured
of i 1500.
(4),
of 1% formic
the range
was noted
steps.
group
the larger
noted
No insulin,
cartilage
and the other
SM from human serum
(1,2). Sephadex
in
segments.
reassociation
of SM activity
a mol.
observed
It
4% of
to chromatography.
was too low
or to minimize
substances
be detected
Under
in our procedures
having
prior from
procedure.
the majority
in components
acid
of
by costal
that
(11).
level
were
form
in the presence
in a single
effectively
Nonetheless,
out
this
is recorded
formic
7000
SM activity
carried
during
The bound
of ca.
with
dissociates
chromatography
activity
treated
wt.
of SM
contains
suggests
has accumulated
(3,10,11).
purification
The distribution
- 9.4).
Because
of the purification
by Van Wyk's
Evidence
molecule
activity.
the purification
hormone.
to a transport
initial
material)
chromatography
is
on the Lowry
of 7000 and a blood
throughout
(PI 8.6
based
pH 7.9 and 8.3 was
of fold
purified
SM-C isolated
more basic
eluates
fold
wt.
and 3H-thymidine
resembles
between
10% of the
a mol.
in
RESEARCH COMMUNICATIONS
material
the number
determined
parallel
initial
focused for
assuming
of 35 S-Na2S04
the
component,
10 rig/ml,
stimulation
than
purified
can be misleading.
SM.
AND BIOPHYSICAL
of SM activity. which
could A
corresponded
to an activity
which
Vol. 67, No. 3, 1975
we had observed
BIOCHEMICAL
previously
we have no direct or oxidized It
is
(10)
evidence
interesting
when purifying
of its
glutathione,
nature,
serine,
to note
from human serum which
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SM by different
experiments
glutamate,
that
Pickart
stimulates
means.
indicate
it
or the nucleotide
and Thaler
(12)
is not
reduced
triphosphates.
isolated
DNA, RNA, and protein
Although
a tripeptide
synthesis
in rat
liver
cells. Both MSA and SM activities peptides tinct from
(13).
It
naturally
is
determined
in chick
SM-C was assayed These
peptides
possessed pete
exhibit
similar
binding
and insulin
sites
augment
point,
it
SM could
be MSA or NSILA-S.
biologic
activity
majority
of the activity
assigned
SM-C to our
which
is
e,
glucose
that
similar focuses
culture
insulin
by rat
because
to that in
which
has been
(2).
(16);
Lastly, (15). SM and SM com-
membranes
(17).
cartilage
we isolated
NSILA-S
(4).
and refer
we have employed
region,
rats
insulin
and chick
exists
was
and purified
of Van Wyk's
the basic
arising
the hormonal
SM-A activity
and liver
the material
However,
are dis-
which
MSA activity
uptake
cells
of 35S-Na2SC4
possible
whereas
poly-
compound
of hypophysectomized
responses;
fat
with
Thus,
cartilage
of isolated
uptake is
systems
in monolayer
and increased
dependent
Some of the confusion
(14),
fibroblasts
the costal
MSA activity
for
At this
using
of a single
the purification. assay
hormone
MSA, and the SM’s
or products
bioassay
cartilage
embryo
NSILA-S,
procedures.
during
by a chick
assessed
molecules
to the different
is measured
to be growth
whether
purification
can be attributed response
not known
occurring
the different
appear
to as
an assay
and because
we tentatively
of
the have
preparation.
ACKNOWLEDGEMENTS: The technical is gratefully acknowledged.
expertise
of Yolanda
P. Lyles
and Lynne
Banta
REFERENCES: C. H., and Froesch, E. R., J. Clin. Invest., (1968) 1. Jakob, A., Houri, 47: 2678. 2. Pierson, R. W., Jr. and Temin, H. M., J. Cell Physiol., (1972) 2: 319. suppl., (1973) 175. 3. Uthne, K., Acta. Endocrinol. L. E., Hintz, R. L., Clemmons, D. R., Voina, 4. Van Wyk, J. J., Underwood, and Weaver, R. P., Rec. Progr. Horm. Res., (1974) 30: 259. 5. Hall, K., Acta Endocrinol. (1972) 163. suppl.,
1232
S.J.,
Vol. 67, No. 3, 1975
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Liberti, J. P., Can. 3. Biochem., (1973) 51: 1113. Liberti, J. P., Endocrine Sot. Meetings, r Y., (1975) Abs. 456. Finney, D. J., in Statistical Method in Biological Assay, (1952) Hafner Publ. Co., N. Y. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem., (1951) 193: 265. Liberti, J. P., Biochem. Biophys. Res. Comm., (1970) 2, 356. Society Meetings, N. Y., Uthne, K., Uthne, T., and Ellis, S., Endocrine (1975) Abs. No. 131. Pickart, L. and Thaler, M. M., Nature, (1973) 243: 85. Cohen, K. L., Short, P. A., and Nissley, S. P., Endocrinol. (1975) 96: 193. li;;ll, K., Acta Endocrinol., (1970) 63: 338. Van Wyk, J. J., Hall, K. and WeaverFR. P., Biochim. Biophys. Acta., (1969) 192: 560. Smith, G. L., and Temin, H. H., J. Cell. Physiol., (1974) 84: 181. Hints, R. L., Clemmons, D. R., Underwood, L. E., and Van W$, J. J., Proc. Natl. Acad. Sci., (U.S.), (1972) -69: 2351.
1233
BIOCHEMICAL
PURIFICATION
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF BOVINE SOMATOMEDIN 192 3
J. P. Liberti
Department of Biochemistry, Medical College Virginia Cormaonwealth University, Richmond, Received
October
of Virginia Virginia 23298
8,1975
SUMMARY: A procedure for the purification of bovine somatomedin (SM 4, is presented. The purification scheme utilizes ultrafiltration through membranes of nominal mol. wt. cutoffs, molecular sieve chromatography and finally isoelectric focusing. Two peaks of SM activity, measured by the --in vitro stimulation of 35S-Na2SO4 and 3H-thymidine uptake by costal cartilage, were present after focusing; an acidic component having a p1 of 6.0 - 6.7 and a basic component having a p1 in the range of 7.8 - 8.3. The acidic component comprised 2% of the initial activity and was 120,000-fold purified: the basic component comprised 10% of the initial activity and was 350,000-fold purified relative to the starting material. These components are similar in molecular size and pI to SM-A and SM-C isolated from human plasma. INTRODUCTION: a family
increasingly
promoting
substances
activities, necessary
A number degrees
has become
of growth
insulin-like factors
It
for
from
SM-B isolated
by Hall
leagues
(4).
Both
as the
starting
include purification majority
groups
SM-A is
a Cohn II
of activity
bovine
in tissue
has been species.
appears
a neutral
fraction
similar
(1) is
isolated
to that
which
from
obtained
serum growth
to varying are SM-A and
outdated
human plasma
(p1 = 7),
SM-B is acidic
- 9.4).
in
have
by Van Wyk and col-
Other
and MSA from calf
presented
in plasma
culture.
Among these
peptide
(PI 8.6
exists
peptides,
and SM-C isolated
peptide
blood
there
the so-called
used a Cohn IV fraction
and SM-C is a basic from
with
substances
(3,5)
that These
of cells
serum of different and Uthne
of SM from
human plasma
common traits
promoting
material.
NSILA-S
(l-4).
the proliferation
of growth
of purity
(p1 4 - 5.2)
share
evident
this
substances serum
(2).
communication.
by Van Wyk --et al.
(4).
lsupported by NIH grant AM-16457. 2A portion of this work has been presented at the Endocrine Meetings, New York (7). 3Recipient of Research Career Development Award, K 04-AMOO125. 4Abbreviations: SM, Somatomedin; MSA, multiplication stimulating activity; NSILA-S, non-supressible insulin-like activity-soluble. Copyright All rights
o 19 75 b.v Academic Press, Inc. of reproductim bl any form resrrved.
1226
The The using
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: Bovine blood was collected at a local slaughter house and transported to our laboratory in plastic buckets. The blood was allowed to clot at room temperature. After 4-5 hours, the fluid surrounding the retracted clot was decanted and its volume measured. The procedures described below are for 4 - L portions. Four L of the supernatant were heated in a water bath. When the temperature reached 52', 20 ml of glacial acetic acid and 100 ml of 1 M Na acetate pH 5.5 were added. The mixture was maintained between 53' and 57" and it was stirred continuously. After 30 min., the mixture, which appeared dark brown and had a great quantity of coagulated material, was cooled in an ice water bath. The mixture was centrifuged for 15 min. at 5000 x g and 10" and the precipitate wasdiscarded. The supernatant of this step was ultrafiltered at room temperature through a 50,000 mol. wt. cutoff membrane (Amicon, Lexington, Mass.) with stirring and at 30 psi. The filtrate was concentrated by ultrafiltration through a 1000 mol. wt. cutoff membrane at 50 psi at room temperature. The 1000 mol. wt. retentate was chromatographed on a (3.18 x 61 cm) column of Sephadex G-50 medium. Eluates of peaks having biological activity were pooled, concentrated using a 1000 mol. wt. cutoff membrane and subjected to isoelectric focusing in the pH range of 3.5 to 10. Most of the ampholyte was removed from the focused fraction by chromatography on G-50 Sephadex followed by ultrafiltration through a 1000 mol. wt. cutoff membrane. The retentates were assayed for SM activity. ASSAY: Hypophysectomized, male rats (100-110 g) were obtained from ZivicMiller Labs, Allison Park, Pa., and were used 7-10 days post-operation. The procedures for the --in vitro uptake of various precursors (carrier-free 35S-Na2S04 or 3H-thymidine purchased from New England Nuclear Corp.) by costal cartilage segments have been described elsewhere (6,7). The reference standard (bovine serum) and each test preparation were assayed at 2 or 3 dosages. The test preparations were diluted with saline to be equivalent in volume to the initial serum. When 35S-Na2SO 4 uptake was determined, the incubation reaction was stopped by boiling, washed overnite with tap water, air dried and weighed. Cartilage segments were treated similarly for the 3H-thymidine uptake experiments except that after boiling, the cartilage was soaked for 4 h in 5% trichloacetic acid. The cartilage segments were digested with formic acid and counted in a Beckman scintillation spectrometer. Results are expressed as c/m/mg cartilage. SM units were obtained from a 4-or 6-point symmetrical comparison with the standard reference serum using classic bioassay procedures as the activity present in 1 ml of the reference (8). One SM unit is defined standard. Protein was determined by the method of Lowry with bovine serum albumin as the standard (9). RESULTS: bovine ever,
The purification serum was prepared
it
was found
the retracted activity
possessed
fitted
decreased
ca.
30% protein
of supernatewith to half
a 2 L reservoir. that
presented
prior
suitable
was comparable supernate
is
yielded
preparation.
to that a dark
The flow
of serum. brown
when the volume
How-
surrounding the SM
Centrifugation
fluid
(supernate-2) of supernate-1. in a stirred
was 1 ml min -1 initially
of the retentate 1227
Initially,
step.
Thus,
at room temperature rate
I.
(supernate-1)
and 45% of the SM activity was performed
in Table
to the acid/heat
of the supernate
an equally
of the supernate-l
Ultrafiltration cell
decantation
provided
treated
devised
by centrifugation
that
clot
of the acid/heat which
procedure
was reduced
and
by 70%.
Vol. 67, No. 3, 1975
BIOCHEMICAL
TABLE 1:
AND BIOPHYSICAL
PURIFICATION
RESEARCH COMMUNICATIONS
SCHEME FOR BOVINE SOMATOMEDIN
Bovine
blo
d supernate-l heat
and acid
treatment
-centrifugation
\
d
Pellet
SupernateI
XM-50
membrane
50,oooa Retentate
Filtrate UM-2 membrane 1, oooa
RetentatehFiltrate G-50 Sephadex Chromatography Pool
Active
fractions
Isoelectric focusing pH 3.5 - 10 a Approximate
The filtrate 35 S-Na2S04 50,000 wt. (18.5 Five their
mol.
cutoff
exhibited
wt.
filtrate
was widely Fractions
on a column collected,
to stimulate distributed D and E were
of membrane
of SM as measured
with
flow
rate
pooled
concentrated
was 1.8 ml min -1 . and eluted
as indicated
(Fig.
and 3H-thymidine
the greatest
amount
with
1228
The
wt.
a 1000 mol. The concentrate
with
0.02
M NaCl.
1) and assayed
uptake.
residing
a 1000 mol.
of
of 2.66.
to 19 ml using
of G-50 Sephadex
35 S-Na2S04
by the uptake
activity
L) was concentrated
The initial
were
cutoff
and had a specific (1.7
membrane.
ml fractions
wt.
1360 units
and 3H-thymidine
ml) was placed
ability
mol.
SM activity
in fractions cutoff
for
D and E.
membrane
and
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
Effluent
RESEARCH COMMUNICATIONS
(mls)
sieve chromatography of bovine somatomedin. Fig. 1: Molecular was applied to a Sephadex UPPER PANEL - 19 mls of the 1000 mol. wt. retentate G-50 column and eluted with 0.02 M NaCl. The flow rate was 1.2 ml. mine1 and Chromatography was done at 4' C. 5 ml fractions were collected. LOWER PANEL - Fractions were pooled as indicated and the biologic activity of each was determined as described in Methods.
focusing of Bovine Somatomedin. Fig. 2: Isoelectric UPPER PANEL - Isoelectric focusing between pH 3.5 and 10 was performed in a 110 ml LKB ampholine column with a 1% ampholyte solution. Fractions D and E Sucrose of G-50 (Fig. 1) were concentrated and placed in the light solution. The cathode (-) was 0.25 M gradients were formed with an LKB gradient mixer. Focusing was done NaOH in water and the anode (+) was 1.3 N H2H04 in sucrose. at 4" for 63 hr. at ca. 600 V. LOWER PANEL - SM activity was measured as described in Methods section.
1229
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE RECOVERY AND PURIFICATION
TABLE 2: Preparation
Protein (mg>
OF SOMATOMEDIN FROM 4 L of BOVINE SERUM
Units*
Recovery %
Unitslmg
1) Supernate-l
3.44
x 105
4000
(100)
0.0116
2) Supernate[Acid/heat treatment
1.03
x 105
1800
45
0.0175
1360
34
2.66
Purification
(1) 1.5
of O)l 3) 50,000 mol. wt. filtrate of (2)
511
4) Eluates of G-50 Sephadex fractions of (3)
2.26
920
23
407
35 x 103
0.057
80
2
1404
12 x 104
0.098
400
10
4082
35 x 104
5) Eluates of isoelectric-focused fractions of (4) A) pH 6.0 to 6.8 B) pH 7.9 8.3 * 1 unit
defined
subjected ity
to
as SM activity
to isoelectric
of SM activity
to 4.4
pH 7.9
fraction
A minor
(Fig.
2) show that
the major-
one in the pH range
peak of activity
SM activity native
and had a specific
serum.
The basic
DISCUSSION:
The purification
the appearance component
of the purification
in the pH range
SM activity
protein
in 2 regions:
and 8.3.
the original
acidic
The results
of the progress
The isoelectric
than
in 1 ml of Supernate-l
6.0
was noted
to 6.8
in
the
pH range.
A tabulation
initial
focusing.
was located
and one between 4.2
present
229
and represented
focused
times
procedure
in
a recovery
described exhibit
the 6.0
1230
7.9
in this SM activity.
2.
2% of the
to 8.3)
more pure
initial
in Table
was 120,000
to 6.8 pH range
of 2% of the
presented
contained
which
(pH range
and was 350,000
which
to 6.8
activity
component
of two components that
6.0
is
times
possessed
than
native
report
greater 10% of
serum.
results
in
The slightly contained SM activity.
57 ug of This
com-
Vol. 67, No. 3, 1975
BIOCHEMICAL
ponent
more pure
was 120,000-fold
protein
analysis.
350,000
times
of
the minute
The basic
and accounted
amounts
of SM in blood, Thus,
the 98 ug of SM (350,000
pure
The activities
Both basic
exhibited component
theirs noted
is
slightly
in the
activities
of Sephadex
the course uptake
which
indicates
at least
two forms:
one which
unbound
(free)
and the
has a mol.
'free'
the material this
is
treatment
is
reassociation
acetic
acid
molecules
used
similar
to that
promoting
small
percent
to substances
peak.
in the
wt.
with
fractions
of the
SM activity
having
mol.
wts.
these
forms
in blood
SM exists which
wt.
This
during
of > 50,000 (3,4)
which most
prevents
of the SM
(3-5)
of
SM from
larger
chromatography.
chromatography
resided
This
as well
size
as other
is growth
by radioimmunoassay,
the effluent is
is bound
the concentration
the majority in
in
Presumably,
acid
to dissociate
similar
the
Furthermore,
conditions,
after
of
laboratories
of 5- to 12000.
having
1231
although
species.
may be that
The
multiple
In other
the
of SM activity
has a mol.
as measured
of i 1500.
(4),
of 1% formic
the range
was noted
steps.
group
the larger
noted
No insulin,
cartilage
and the other
SM from human serum
(1,2). Sephadex
in
segments.
reassociation
of SM activity
a mol.
observed
It
4% of
to chromatography.
was too low
or to minimize
substances
be detected
Under
in our procedures
having
prior from
procedure.
the majority
in components
acid
of
by costal
that
(11).
level
were
form
in the presence
in a single
effectively
Nonetheless,
out
this
is recorded
formic
7000
SM activity
carried
during
The bound
of ca.
with
dissociates
chromatography
activity
treated
wt.
of SM
contains
suggests
has accumulated
(3,10,11).
purification
The distribution
- 9.4).
Because
of the purification
by Van Wyk's
Evidence
molecule
activity.
the purification
hormone.
to a transport
initial
material)
chromatography
is
on the Lowry
of 7000 and a blood
throughout
(PI 8.6
based
pH 7.9 and 8.3 was
of fold
purified
SM-C isolated
more basic
eluates
fold
wt.
and 3H-thymidine
resembles
between
10% of the
a mol.
in
RESEARCH COMMUNICATIONS
material
the number
determined
parallel
initial
focused for
assuming
of 35 S-Na2S04
the
component,
10 rig/ml,
stimulation
than
purified
can be misleading.
SM.
AND BIOPHYSICAL
of SM activity. which
could A
corresponded
to an activity
which
Vol. 67, No. 3, 1975
we had observed
BIOCHEMICAL
previously
we have no direct or oxidized It
is
(10)
evidence
interesting
when purifying
of its
glutathione,
nature,
serine,
to note
from human serum which
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SM by different
experiments
glutamate,
that
Pickart
stimulates
means.
indicate
it
or the nucleotide
and Thaler
(12)
is not
reduced
triphosphates.
isolated
DNA, RNA, and protein
Although
a tripeptide
synthesis
in rat
liver
cells. Both MSA and SM activities peptides tinct from
(13).
It
naturally
is
determined
in chick
SM-C was assayed These
peptides
possessed pete
exhibit
similar
binding
and insulin
sites
augment
point,
it
SM could
be MSA or NSILA-S.
biologic
activity
majority
of the activity
assigned
SM-C to our
which
is
e,
glucose
that
similar focuses
culture
insulin
by rat
because
to that in
which
has been
(2).
(16);
Lastly, (15). SM and SM com-
membranes
(17).
cartilage
we isolated
NSILA-S
(4).
and refer
we have employed
region,
rats
insulin
and chick
exists
was
and purified
of Van Wyk's
the basic
arising
the hormonal
SM-A activity
and liver
the material
However,
are dis-
which
MSA activity
uptake
cells
of 35S-Na2SC4
possible
whereas
poly-
compound
of hypophysectomized
responses;
fat
with
Thus,
cartilage
of isolated
uptake is
systems
in monolayer
and increased
dependent
Some of the confusion
(14),
fibroblasts
the costal
MSA activity
for
At this
using
of a single
the purification. assay
hormone
MSA, and the SM’s
or products
bioassay
cartilage
embryo
NSILA-S,
procedures.
during
by a chick
assessed
molecules
to the different
is measured
to be growth
whether
purification
can be attributed response
not known
occurring
the different
appear
to as
an assay
and because
we tentatively
of
the have
preparation.
ACKNOWLEDGEMENTS: The technical is gratefully acknowledged.
expertise
of Yolanda
P. Lyles
and Lynne
Banta
REFERENCES: C. H., and Froesch, E. R., J. Clin. Invest., (1968) 1. Jakob, A., Houri, 47: 2678. 2. Pierson, R. W., Jr. and Temin, H. M., J. Cell Physiol., (1972) 2: 319. suppl., (1973) 175. 3. Uthne, K., Acta. Endocrinol. L. E., Hintz, R. L., Clemmons, D. R., Voina, 4. Van Wyk, J. J., Underwood, and Weaver, R. P., Rec. Progr. Horm. Res., (1974) 30: 259. 5. Hall, K., Acta Endocrinol. (1972) 163. suppl.,
1232
S.J.,
Vol. 67, No. 3, 1975
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Liberti, J. P., Can. 3. Biochem., (1973) 51: 1113. Liberti, J. P., Endocrine Sot. Meetings, r Y., (1975) Abs. 456. Finney, D. J., in Statistical Method in Biological Assay, (1952) Hafner Publ. Co., N. Y. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem., (1951) 193: 265. Liberti, J. P., Biochem. Biophys. Res. Comm., (1970) 2, 356. Society Meetings, N. Y., Uthne, K., Uthne, T., and Ellis, S., Endocrine (1975) Abs. No. 131. Pickart, L. and Thaler, M. M., Nature, (1973) 243: 85. Cohen, K. L., Short, P. A., and Nissley, S. P., Endocrinol. (1975) 96: 193. li;;ll, K., Acta Endocrinol., (1970) 63: 338. Van Wyk, J. J., Hall, K. and WeaverFR. P., Biochim. Biophys. Acta., (1969) 192: 560. Smith, G. L., and Temin, H. H., J. Cell. Physiol., (1974) 84: 181. Hints, R. L., Clemmons, D. R., Underwood, L. E., and Van W$, J. J., Proc. Natl. Acad. Sci., (U.S.), (1972) -69: 2351.
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