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Human pituitary thyrotropin: Isolation and chemical characterization of its subunits
Vol. 51, No. 2,1973
BIOCHEMICAL
HUMAN
PITUITARY
ISOLATION
THYROTROPIN:
AND
CHEMICAL
CHARACTERIZATION
M.
AND BIOPHYSICAL RESEARCH COhViWNICATIONS
OF ITS SUBUNITS
R. Sairam
and
Choh
Hao Li
Hormone Research Laboratory University of California, San Francisco San Francisco, California 94 122 Received
February
1, 1973
SUMMARY. The subunits of human pituitary and purified by countercurrent distribution The NH2-terminal sequence of the (3 subunit amino unit of bovine thyrotropin. However, of tryptic and chymotryptic digests as well chymotryptic peptides suggest that the amino is identical to that of the a subunit of human
thyrotropin have been separated and exclusion chromatography. is identical to that of the /3 subacid composition and peptide map as compositions of tryptic and acid sequence of the a subunit interstitial cell stimulating hormone.
INTRODUCTION Human
pituitary
by Condliffe
thyrotropin
(1) in 1963. of HTSH’
into
treatment
propionic
acid
This
communication
presents
the separation
of the two
characterization.
It will
predictions of human
(4) that
first
In a recent
the separation with
was
two
prepared
conference,
subunits
as previously
of HTSH
be seen
that
sequence
(3) for
and chemical are
form
et al (2) reported
distribution
the data
purified
chromatography
described
a countercurrent
acid
Pierce
by column
subunits
the amino
in highly
of HTSH-a
bovine
TSH.
procedure
for
data
consistent
after
for with
is identical
their earlier to that
ICSH-a.
lAbbreviations:
TSH, thyrotropin; HTSH, human thyrotropin; HTSH-a, a subunit of HTSH; HTSH-/3, p subunit of HTSH; ICSH, interstitial cell stimulating hormone; ICSH-a, a-subunit of ICSH.
Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
336
Vol. 51, No. 2, 1973
BlOCHEMiCAl
MATERIALS HTSH with
was
minor
Amino
analyses
according
paper
from
METHODS
performic
pituitary
performed
et al (6). acid
Other
oxidation,
separation
was
equilibrated
acid at 40°
was
of 40%
alcohol was
automatic such
digests carried
analy-
as dansyl-
and two
Edman
dimensional
out as previously
C.
8 M urea
with
lower
phase.
effectively
A small
to facilitate mixture amount
the upper Under
phases
on a rotary
bicarbonate.
then
amount
these
conditions
evaporator on Sephadex
It was
found
5-9
tube
The lower were
and dialyzed
necessary
equilibrated
for
2 hours
in centrifuge at the interphase along
with
of the hormone phases
of tubes (see
extensively. in 0.05
to chromatograph
337
-n-propanol:
incubated
retained
pooled
(8)
of 2% dichloroacetic
occurred
the subunits
(HTSH-P)
G-100
ml) further
was
system
acid:
to 9 transfers that
in the first
the two phases, of tubes
was
submitted
at 2Z” C, G-25
of the solvent
(0.2
which
in
50 mg of the treated
phase
of precipitation
phase
24 hours
2% dicbloroacetic
solution, was
was dissolved
of Sephadex
and lyophilization.
(60:60:27:33).
small
purified
acid
After
on a column
in 15 ml of the lower
between
and upper
(pH 2. 8).
by desalting
~SO~(W/V):O.
The
mixing
60 mg of the hormone
(NH4)
added
The
further
recovered
dissolved
tubes.
trated
containing
in 0. 1 M acetic
consisting ethyl
of the subunits,
0. 05 M HCl
the protein
HTSH
were
1000 fresh
obtained.
procedures
enzymic
(5)
(7).
For 10 ml
was
From
on the Beckman
chromatography-electrophoresis
described
(1) and Stockell-Hartree glands.
20 mg HTSH
were
to Spackman
of Condiffe
fresh
of approximately
Acid
degradation,
AND
by procedures
modifications
a yield
glands,
zer
isolated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure The
on
the
distributed O-4 (HTSH-a) l),
concen-
fractions
were
M ammonium HTSH-a
twice
on
Vol. 51, No. 2,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E 05E 8
. LOWER PHASE 0 UPPER PHASE
N
0.4,i
2* 5
0.3 -\
g
02-
2 ” E 0
0”
0
I’ \ •\*~o/Oo~~~p”
0.1 ( -- ,0---o-I 0123456789
I
0-a I
I
I
TUBE
Figure
1:
Sephadex were
,,-o
-o-o-e-
I
I
1
f
NUMBER
Countercurrent distribution of urea-treated HTSH lower phases of tubes O-4 represent the a-fraction phases of tubes 5-9 represent the p-fraction.
G-100
to obtain
the final
product.
The
yields
(50 mg). The and the upper
of the a and p subunits
13 mg and 8 mg respectively. For
two dimensional
2 mg each
of the performic
with
trypsin
with
an enzyme:substrate
the paper alcohol
were
each
acid
and chymotrypsin
was
95%
24 hours
chromatography-electrophoresis oxidized in 0.5 ratio
thoroughly
air
and allowed
to develop
was
air
dried
RESULTS It is evident in separating composition
Figure
of HTSH data
and its
at room tube
between
acid
whereas
the sequence
sequence
90 mine.
sprayed
-with
0.1% The
temperature.
C
acid
ninhydrin purple
1 M NH40H.
Table
These
values
et al (2).
in
spots
After
composition
determined.
procedure 1 presents are There
was
found
is effective the amino
in agreement are
the a and S subunits.
of HTSH-a
of the p subunit
at 37’
DISCUSSION
subunits.
in the compositions
separately
chromatography-electrophoresis,
containing
of HTSH.
by Pierce
amino
for
digested
1 that the countercurrent
reported
terminal
were
and the amino AND
the a and p subunits
the preliminary differences
from
After
and lightly
cut out and put in a test the eluate
material
M NH4HC03
of 1:30. dried
of HTSH-a,
NH2-
to be H-Val-Glx-Asx-Cys-
was H-Phe-Cys-Ile-Pro-Thr-Glu-Tyr-Met-.
338
with
significant The
acid
BtOCHEMICAL
Vol. 51, No. 2,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE Amino
Acid
Composition
(Molar
Amino Acid
in parentheses
of bovine
of human
same digestion
with
histidine
was
As pointed ably
TSH-8
similar
5.1 2.5 2.9 5.5 8. 2 7.6 9.4 6.2 4.6 4.6 9.2 6.6 2.3 1.4 4.0 3.7 3.8 0.0
human
(9) while
(4).
serine, amino
for
The
lysine,
acids
were
ICSH-a,
sequence
carboxypeptidase under
from
(5.8) (2.5) (3.0) (5.4) (8.5) (7.8) (7.5) (5.6) (4.3) (4.2) (10.2) f6.4) (2.5) (1.3) (4.0) (4.0) (3.5) (0.0)
7.8 3.0 5.9 11.8 12.7 6.5 9.6 10.7 6.0 7.8 14.0 5.4
2.1 9.7 8.0 11.6 5.0 0.0
from
human
A released
(4).
of HTSH-8
of HTSH-a A digestion
and histidine; released
HTSH-8
taken
the sequence
carboxypeptidase
TSH and Its Subunits
HTSH-cl*
that the NH2-terminal
ICSH-a
tyrosine,
of Human
12.2 5.6 8.0 16.1 18.3 12.4 19.4 17.0 12.4 13.5 20.6 14.3 4.4 10.0 12.0 13.8 9.0 0.0
It may be noted that
Ratio)
HTSH
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic Acid Pr oline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Values
1
is identical
is identical of HTSH-a
with
with
that
liberated
the same
condition,
these
ICSH-a.
In the case
of HTSH-8,
only
tyrosine.
serine
acid
content
of HTSH-a
and lysine;
not detected. out earlier te that
(2,4),
of the a subunit
the amino of human
339
ICSH
(see
Table
1).
is remark, The
two
HTSH - o(
Figure
ilECTROPHORESlS -
A
2:
0TO -
0TZ
h
PHORESIS e
Two dimensional chromatography-electrophoresis patterns of tryptic digests of (A) oxidized HTSH-a and (B) oxidized human ICSH-a. Chromatography in solvent system n-butanol/acetic acid/water (4/l/5 by volume) and electrophdresis in pH 2.0 formic acid-acetic acid buffer for 60 mins. at 2000 volts.
I IECTRO
5 LIC 8 3 & 46 r/
t
B
Vol.51,No.2,
BIOCHEMICAL
1973
AND BIOPHYSICAL
TABLE Amino
acid composition (molar Tryptic and chymotryptic
Peptide No.”
I’1 t T6 t T9
ratio) of peptidea isolated from maps of oxidized HTSH-a
Total Residues
LYSl. 9 His3.0 Argl. 0 Cya8. Thr5. 1 Ser4. 6 Glx7. 6 Pro3. Alas.0 Val4.4 Ilel.0 Luel.5 Phe3.0 Metl. 8
58-61
5 Asx4. o 5 G1y3. 1 Tyr2.0
T2
Alao.
T3
S-0
. 7 J-v1
T3a
SerO. 8 Lys2.
T4
Free
Lys
T5
ThrO.
7 Metl.
0 Leul.
0 ValO.
8 G1xl.
0 Lysl.
T5a
Lys2.
0 Thro.
8 Met0.
7 LeuO.
9 Va1l.
T7
SerO . 6 TYrO . 7 Asxl.O
ArgO.8
T8
Va12. 0 ThrO+8
Gly2.0
T10
Free
.0
2
40-41
0
3
40-42
1
42
0
6
43-48
0 G1xO. 9
7
42-48
4
61-64
8
65-72
1
89
8
31-38
Metl.O
Thrl.o
Argo
Le\il.
c2
Argo
c3
A=()9
Arg1.0
c4
ClY2.0
Phe1.2
C5
Tyr0. Figure 2. reference (4).
. 7 Ser1.0
7 His1.0
8 Alal
. o
Leu1.1
Arg1.0
Phel.O
Lysl.O
Peptides TyrO
.
8
Pro2
. o
2 Lys2.0 va11.9
Thr1.0
Metl.
Thro.9
Met1.0
0
39-44 63-68 69-71
Lys1.0
ICSH- a nos. )**
l-32, 49-60, 73-88
33-39
Thr1.1
Ser
7
Human (residue
7
Pro2.0
9 TYr0.9
SerO
the
Peptides
Chymotryptic
*See C Wee
2
Composition
Tryptic
Cl
RESEARCH COMMUNICATIONS
Ser1.0
341
86-89
BIOCHEMICAL
Vol. 51, No. 2,1973
dimensional
chromatography-electrophoresis
oxidized The
HTSH-a
same
that
and human
is true
the amino could
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid
for
from
from
From
finger
human
is nearly
identical
is not unreasonable
that
the a subunits
acid
sequence
indistinguishable
as shown
As summarized
and chymotryptic of oxidized
are
of
in Figure
in Table
peptides,
HTSH-a,
digest
2.
2,
which identical
to
ICSH-a.
data,
tion
prints
of the tryptic
pattern.
of tryptic
the above
of HTSH-a
are
the chymotryptic
composition
be isolated obtained
ICSH-a
patterns
it may
be assumed
with
that
the a subunit
as it has been
shown
of the TSH and ICSH
the amino
of human
ICSH.
in the bovine
molecules
have
acid
sequence This
pituitary
identical
assumpgland
amino
(10). ACKNOWLEDGMENT
We thank work
was
Jean
supported
Knorr
and Daniel
in part
A-6097
from
the National
authors
also
thank
of fresh
human
by the United Institute
the National
pituitary
Gordon
for
States
of Arthritis Pituitary
Agency
technical Public
assistance. Health
and Metabolic in Baltimore
This
Service Diseases. for
Grant The
the supply
glands. REFERENCES
72, 893, 1963. P. G., Endocrinology J. G., Liao, T. H., Howard, S. M., Shome, B., and Cornell, Hor. Res. 27, 165, 1971. J. S., Rec. Prog. T. H., and Pierce, J. Gx J. Biol. Chem. 245, 3275, 1970. 3. Liao, M. R. , Papkof f, H., and Li, C. H., Bioxm. Biophys. Res. 4. Sairam, Comm. 48, 530, 1972. A., Biochem. J. 100, 754, 1966. 5. Stockellzartree, D. H., Stein, W. H., and Moore, S., Anal. Chem. -30, 6. Spackman, 1190, 1958. J. S., Lo, T. B., Schmidt, K., and Pankov, y. t 7. Li, C. H., Dixon, Arch. Biochem. Biophys. 141, 705, 1970. H., and Samy, T.%A., Biochem. Biophys. Act. 147, 175, 1967. a. Papkoff, T. H. and Pierce, J. G., J. Biol. Chem. 246, 850, 19710 9. Liao, J. G., Liao, T. H., Carlsen, R.. B. L aaeimo, T. I J- B~oL 10. Pierce, Chem. 246, 866, 1971. -w 1.
Condliffe,
2. Pierce,
342
BIOCHEMICAL
HUMAN
PITUITARY
ISOLATION
THYROTROPIN:
AND
CHEMICAL
CHARACTERIZATION
M.
AND BIOPHYSICAL RESEARCH COhViWNICATIONS
OF ITS SUBUNITS
R. Sairam
and
Choh
Hao Li
Hormone Research Laboratory University of California, San Francisco San Francisco, California 94 122 Received
February
1, 1973
SUMMARY. The subunits of human pituitary and purified by countercurrent distribution The NH2-terminal sequence of the (3 subunit amino unit of bovine thyrotropin. However, of tryptic and chymotryptic digests as well chymotryptic peptides suggest that the amino is identical to that of the a subunit of human
thyrotropin have been separated and exclusion chromatography. is identical to that of the /3 subacid composition and peptide map as compositions of tryptic and acid sequence of the a subunit interstitial cell stimulating hormone.
INTRODUCTION Human
pituitary
by Condliffe
thyrotropin
(1) in 1963. of HTSH’
into
treatment
propionic
acid
This
communication
presents
the separation
of the two
characterization.
It will
predictions of human
(4) that
first
In a recent
the separation with
was
two
prepared
conference,
subunits
as previously
of HTSH
be seen
that
sequence
(3) for
and chemical are
form
et al (2) reported
distribution
the data
purified
chromatography
described
a countercurrent
acid
Pierce
by column
subunits
the amino
in highly
of HTSH-a
bovine
TSH.
procedure
for
data
consistent
after
for with
is identical
their earlier to that
ICSH-a.
lAbbreviations:
TSH, thyrotropin; HTSH, human thyrotropin; HTSH-a, a subunit of HTSH; HTSH-/3, p subunit of HTSH; ICSH, interstitial cell stimulating hormone; ICSH-a, a-subunit of ICSH.
Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
336
Vol. 51, No. 2, 1973
BlOCHEMiCAl
MATERIALS HTSH with
was
minor
Amino
analyses
according
paper
from
METHODS
performic
pituitary
performed
et al (6). acid
Other
oxidation,
separation
was
equilibrated
acid at 40°
was
of 40%
alcohol was
automatic such
digests carried
analy-
as dansyl-
and two
Edman
dimensional
out as previously
C.
8 M urea
with
lower
phase.
effectively
A small
to facilitate mixture amount
the upper Under
phases
on a rotary
bicarbonate.
then
amount
these
conditions
evaporator on Sephadex
It was
found
5-9
tube
The lower were
and dialyzed
necessary
equilibrated
for
2 hours
in centrifuge at the interphase along
with
of the hormone phases
of tubes (see
extensively. in 0.05
to chromatograph
337
-n-propanol:
incubated
retained
pooled
(8)
of 2% dichloroacetic
occurred
the subunits
(HTSH-P)
G-100
ml) further
was
system
acid:
to 9 transfers that
in the first
the two phases, of tubes
was
submitted
at 2Z” C, G-25
of the solvent
(0.2
which
in
50 mg of the treated
phase
of precipitation
phase
24 hours
2% dicbloroacetic
solution, was
was dissolved
of Sephadex
and lyophilization.
(60:60:27:33).
small
purified
acid
After
on a column
in 15 ml of the lower
between
and upper
(pH 2. 8).
by desalting
~SO~(W/V):O.
The
mixing
60 mg of the hormone
(NH4)
added
The
further
recovered
dissolved
tubes.
trated
containing
in 0. 1 M acetic
consisting ethyl
of the subunits,
0. 05 M HCl
the protein
HTSH
were
1000 fresh
obtained.
procedures
enzymic
(5)
(7).
For 10 ml
was
From
on the Beckman
chromatography-electrophoresis
described
(1) and Stockell-Hartree glands.
20 mg HTSH
were
to Spackman
of Condiffe
fresh
of approximately
Acid
degradation,
AND
by procedures
modifications
a yield
glands,
zer
isolated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure The
on
the
distributed O-4 (HTSH-a) l),
concen-
fractions
were
M ammonium HTSH-a
twice
on
Vol. 51, No. 2,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E 05E 8
. LOWER PHASE 0 UPPER PHASE
N
0.4,i
2* 5
0.3 -\
g
02-
2 ” E 0
0”
0
I’ \ •\*~o/Oo~~~p”
0.1 ( -- ,0---o-I 0123456789
I
0-a I
I
I
TUBE
Figure
1:
Sephadex were
,,-o
-o-o-e-
I
I
1
f
NUMBER
Countercurrent distribution of urea-treated HTSH lower phases of tubes O-4 represent the a-fraction phases of tubes 5-9 represent the p-fraction.
G-100
to obtain
the final
product.
The
yields
(50 mg). The and the upper
of the a and p subunits
13 mg and 8 mg respectively. For
two dimensional
2 mg each
of the performic
with
trypsin
with
an enzyme:substrate
the paper alcohol
were
each
acid
and chymotrypsin
was
95%
24 hours
chromatography-electrophoresis oxidized in 0.5 ratio
thoroughly
air
and allowed
to develop
was
air
dried
RESULTS It is evident in separating composition
Figure
of HTSH data
and its
at room tube
between
acid
whereas
the sequence
sequence
90 mine.
sprayed
-with
0.1% The
temperature.
C
acid
ninhydrin purple
1 M NH40H.
Table
These
values
et al (2).
in
spots
After
composition
determined.
procedure 1 presents are There
was
found
is effective the amino
in agreement are
the a and S subunits.
of HTSH-a
of the p subunit
at 37’
DISCUSSION
subunits.
in the compositions
separately
chromatography-electrophoresis,
containing
of HTSH.
by Pierce
amino
for
digested
1 that the countercurrent
reported
terminal
were
and the amino AND
the a and p subunits
the preliminary differences
from
After
and lightly
cut out and put in a test the eluate
material
M NH4HC03
of 1:30. dried
of HTSH-a,
NH2-
to be H-Val-Glx-Asx-Cys-
was H-Phe-Cys-Ile-Pro-Thr-Glu-Tyr-Met-.
338
with
significant The
acid
BtOCHEMICAL
Vol. 51, No. 2,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE Amino
Acid
Composition
(Molar
Amino Acid
in parentheses
of bovine
of human
same digestion
with
histidine
was
As pointed ably
TSH-8
similar
5.1 2.5 2.9 5.5 8. 2 7.6 9.4 6.2 4.6 4.6 9.2 6.6 2.3 1.4 4.0 3.7 3.8 0.0
human
(9) while
(4).
serine, amino
for
The
lysine,
acids
were
ICSH-a,
sequence
carboxypeptidase under
from
(5.8) (2.5) (3.0) (5.4) (8.5) (7.8) (7.5) (5.6) (4.3) (4.2) (10.2) f6.4) (2.5) (1.3) (4.0) (4.0) (3.5) (0.0)
7.8 3.0 5.9 11.8 12.7 6.5 9.6 10.7 6.0 7.8 14.0 5.4
2.1 9.7 8.0 11.6 5.0 0.0
from
human
A released
(4).
of HTSH-8
of HTSH-a A digestion
and histidine; released
HTSH-8
taken
the sequence
carboxypeptidase
TSH and Its Subunits
HTSH-cl*
that the NH2-terminal
ICSH-a
tyrosine,
of Human
12.2 5.6 8.0 16.1 18.3 12.4 19.4 17.0 12.4 13.5 20.6 14.3 4.4 10.0 12.0 13.8 9.0 0.0
It may be noted that
Ratio)
HTSH
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic Acid Pr oline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Values
1
is identical
is identical of HTSH-a
with
with
that
liberated
the same
condition,
these
ICSH-a.
In the case
of HTSH-8,
only
tyrosine.
serine
acid
content
of HTSH-a
and lysine;
not detected. out earlier te that
(2,4),
of the a subunit
the amino of human
339
ICSH
(see
Table
1).
is remark, The
two
HTSH - o(
Figure
ilECTROPHORESlS -
A
2:
0TO -
0TZ
h
PHORESIS e
Two dimensional chromatography-electrophoresis patterns of tryptic digests of (A) oxidized HTSH-a and (B) oxidized human ICSH-a. Chromatography in solvent system n-butanol/acetic acid/water (4/l/5 by volume) and electrophdresis in pH 2.0 formic acid-acetic acid buffer for 60 mins. at 2000 volts.
I IECTRO
5 LIC 8 3 & 46 r/
t
B
Vol.51,No.2,
BIOCHEMICAL
1973
AND BIOPHYSICAL
TABLE Amino
acid composition (molar Tryptic and chymotryptic
Peptide No.”
I’1 t T6 t T9
ratio) of peptidea isolated from maps of oxidized HTSH-a
Total Residues
LYSl. 9 His3.0 Argl. 0 Cya8. Thr5. 1 Ser4. 6 Glx7. 6 Pro3. Alas.0 Val4.4 Ilel.0 Luel.5 Phe3.0 Metl. 8
58-61
5 Asx4. o 5 G1y3. 1 Tyr2.0
T2
Alao.
T3
S-0
. 7 J-v1
T3a
SerO. 8 Lys2.
T4
Free
Lys
T5
ThrO.
7 Metl.
0 Leul.
0 ValO.
8 G1xl.
0 Lysl.
T5a
Lys2.
0 Thro.
8 Met0.
7 LeuO.
9 Va1l.
T7
SerO . 6 TYrO . 7 Asxl.O
ArgO.8
T8
Va12. 0 ThrO+8
Gly2.0
T10
Free
.0
2
40-41
0
3
40-42
1
42
0
6
43-48
0 G1xO. 9
7
42-48
4
61-64
8
65-72
1
89
8
31-38
Metl.O
Thrl.o
Argo
Le\il.
c2
Argo
c3
A=()9
Arg1.0
c4
ClY2.0
Phe1.2
C5
Tyr0. Figure 2. reference (4).
. 7 Ser1.0
7 His1.0
8 Alal
. o
Leu1.1
Arg1.0
Phel.O
Lysl.O
Peptides TyrO
.
8
Pro2
. o
2 Lys2.0 va11.9
Thr1.0
Metl.
Thro.9
Met1.0
0
39-44 63-68 69-71
Lys1.0
ICSH- a nos. )**
l-32, 49-60, 73-88
33-39
Thr1.1
Ser
7
Human (residue
7
Pro2.0
9 TYr0.9
SerO
the
Peptides
Chymotryptic
*See C Wee
2
Composition
Tryptic
Cl
RESEARCH COMMUNICATIONS
Ser1.0
341
86-89
BIOCHEMICAL
Vol. 51, No. 2,1973
dimensional
chromatography-electrophoresis
oxidized The
HTSH-a
same
that
and human
is true
the amino could
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid
for
from
from
From
finger
human
is nearly
identical
is not unreasonable
that
the a subunits
acid
sequence
indistinguishable
as shown
As summarized
and chymotryptic of oxidized
are
of
in Figure
in Table
peptides,
HTSH-a,
digest
2.
2,
which identical
to
ICSH-a.
data,
tion
prints
of the tryptic
pattern.
of tryptic
the above
of HTSH-a
are
the chymotryptic
composition
be isolated obtained
ICSH-a
patterns
it may
be assumed
with
that
the a subunit
as it has been
shown
of the TSH and ICSH
the amino
of human
ICSH.
in the bovine
molecules
have
acid
sequence This
pituitary
identical
assumpgland
amino
(10). ACKNOWLEDGMENT
We thank work
was
Jean
supported
Knorr
and Daniel
in part
A-6097
from
the National
authors
also
thank
of fresh
human
by the United Institute
the National
pituitary
Gordon
for
States
of Arthritis Pituitary
Agency
technical Public
assistance. Health
and Metabolic in Baltimore
This
Service Diseases. for
Grant The
the supply
glands. REFERENCES
72, 893, 1963. P. G., Endocrinology J. G., Liao, T. H., Howard, S. M., Shome, B., and Cornell, Hor. Res. 27, 165, 1971. J. S., Rec. Prog. T. H., and Pierce, J. Gx J. Biol. Chem. 245, 3275, 1970. 3. Liao, M. R. , Papkof f, H., and Li, C. H., Bioxm. Biophys. Res. 4. Sairam, Comm. 48, 530, 1972. A., Biochem. J. 100, 754, 1966. 5. Stockellzartree, D. H., Stein, W. H., and Moore, S., Anal. Chem. -30, 6. Spackman, 1190, 1958. J. S., Lo, T. B., Schmidt, K., and Pankov, y. t 7. Li, C. H., Dixon, Arch. Biochem. Biophys. 141, 705, 1970. H., and Samy, T.%A., Biochem. Biophys. Act. 147, 175, 1967. a. Papkoff, T. H. and Pierce, J. G., J. Biol. Chem. 246, 850, 19710 9. Liao, J. G., Liao, T. H., Carlsen, R.. B. L aaeimo, T. I J- B~oL 10. Pierce, Chem. 246, 866, 1971. -w 1.
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342