Vol.
89,
No.
2, 1979
July
27, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 721-728
*Dapartmant of Biochemistry Purdue University Nest Lafayette, Indians 47907 hepartnmt of Pharmacology ard Toxicology ard Hniversity of Rochester Cancer Center University of Rochester School of Medicine and Dentistry Rochester, New York 14642 Received
June 11, 1979
SUMMARY:Chemical and enzymatic treatment of a higtilecular raight fraction fran a frog brain extract resulted in formation of a 'V&like material" (TRH-i). Sequential treatment with trypsin ard carlxxypeptidases A and B, acetic acid and then chemical ennidation generated a quantity of WH-i equivalent to 25% of the erdogenous TFX. TFX-i was similar to TRH (rGluVis'PrcNH2) as assessed by molecular 'iaight estimations, radioimmunoassay m-d susceptibility to senxn inactivation. TBH and TRH-i also competed with [%I-'Hw for binding to TBH receptors, stimulated pro&tin synthesis and uridine uptake, ard "down-regulated" TRH receptors in pituitary cells. These results suggest the possibility that lRH may te processed fran a nncranolecular precursor. Thyrotropin
releasiq+xmone
(TBH), the &lest
of the hypothalamic hxnx3nes, has been iso-
lated frun porcine (1) and ovine (2) hypothalamic fragaxsnts anl its structure
Qtennined.
mane and nrmerou.3 analogous have been chemically synthesized and their biological gated (3-4).
Despite these advances, little
Biosynthesis
of s&l
peptides such as glutathione
been shown to proceed by a I-XX-ribosaaal tions indicated
is known about the biosynthesis
lz&hmay (7-8),
hxever,
Initial
these reports have not
eight
species that xuld
Although it is difficult
be enzymatically
to find supporting
precursors
(11).
it wxld
processed to its
precedents for the narribo-
scmal synthesis of IRH, there is extensive documantation that hDrn~3nes and proteins as larger
(6) has obeerva-
(9-10).
probably be formed as a higher rrolecular Edctive form.
investi-
paptide antibiotics
TRH is canposed of only three amino acids, anl if it wxe synthesized on ribosanes, biologically
'Ihe hx-
of lBH.
ma&m&m of anino acid polymerization.
that 'IPH was synthesized by a similar
been confirmed by other laboratories
(5) or bacterial
activities
are synthesized
In the present study we have determined that the frog brain contains a
macrarx&cule similar
that can te converted -via enzymatic arrl chanical nodifications to a canpound tich is to TRH as detefinined by physical characteristics, innnmoch0nical properties and biological
activity. * Correspondence and inquiries should be addressed to Jack E. Dixon, Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907. ml.9 research k~s supported in p3rt by Research Grants pM 18849 (J.E.D.) ard AM 19974 (P.M.H.) fran the National Institute of Arthritis, Matabolism arxI Digestive Diseases anl Cancer Center Core Research grant ~3 11198 (P.M.H.). J.E.D. is a recipient of a Research Career Development Pward (AM 00218). J.H.R. is an N.I.H. postdoctoral fellow (AM 0594601). This is journal paper rumber 7610 fran the Purdue University Agricultural Experiment Station. 0006-291X/79/140721-08$01.00/0 721
Copyright All rights
@ I979 by Academic Press, Inc. of reproduction in anyform reserved.
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
line 2,3-Q IRH ([Q-TRH) used f or radioimmmcassay bad a specific activity of 21.8 Ci/nmole. Similar material used in receptor hiding experiments had a specific activity of 115 Ci/mnole (New F@ Nuclear). Trypsin, carboxypeptidase A, carbcmypeptidase B, bovine thyroglobulin and bovine serun altin wzre purchased fran Sigma. 'I'RH and $lu'His'Pro (free acid) were products of Bachem or gifts fran Abbott Laboratories. Tissue culture m&a, Vnrse serum ard fetal calf senrn wxe from Grand Island Biological Gxnpany. Hy@h oid calf sennn (thyroxine l% I for protein icdination < 1 n ml) was frcm Rockland Fatms, Gilbertsville, PA. Carrier-free axl [f% ] uridine (27 Ci/mnole) were fran Amersham/Searle. 'Ihe G93 and GH4Cl cell lines wzre obtained fran Dr. Armen H. Tashjian, Jr., Harvard Medical School, Boston, MA. Protein concentrations were determined by the nethod of Lowry --et al. (12) using bovine sennn albumin as a stardard. Radioimmmoassay - Antibody to TRH ws prepared ard TRH concentrations measured by a nudificz tion of the procedure of Bryce (13). Radioimmmoassay incubation mixtures contained 10 ~1 of IRH antiserum (binds 5,0X c~xn [?I]-TRH), 5 ~1 [?I]-TRH (12,500 cpn, 0.25 ng:), 0 to 50 ~1 of sample of 'IRH standard and phosphate tuffered salirxa (PBS, 0.01 M sodium phosphate containing 0.15 M N&l, pH 7.4) to make a firm1 xx&mm of 5C to 100 ~1. After memight incubation at 4"C, 5rK) ng of 17nr irmnme rabbit setxr in 10 ~1 PBS =re added and the incubated mixtures wxe diluted with an equal volm of saturated anmonilun milfate. After 20 min, the precipitated antigerrantibcdy cmnplex mas collected by centrifugation at 8,0(x) g for 6 min. The supematant was discarded ard the pellet kas washed with 300 ~1 and 200 ~1 aliquots of 50% saturated mnmonium sulfate. The final pallet kas dissolved in lC0 ~1 of 1 N &OH ar$ transferred to a scintillation vial. Fach assay tube k~s rinsed with 900 ~1 of H20 which was canbined with the NaOHsolution. The antiserun Ws found to be quite specific for TRH. The structural analogues ProNH2, pGlu'His, $lu*HisPro, Luteinking Hormone-Releasing Hormone and GluWisPrcNH2 were tested at 10X-fold molar excess ard did not reduce the binding of [3H]-IBH to the antibody. However, $lu'His'Pro'GlyNH2 cross reacted with the antibody at a 2Ffold nolar excess ard pGlu'L-dopa'ProNH2 showed a canpetition for antibody that parallels that of authentic '!RH and suggests that the antibody discriminates poorly at the histidice residue. This result is notsurprising since TRH is conjugated to thyroglobulin via the imidazole of histidine. Preparation of Brain Extracts - Frogs (Rana pipiens), ~chased fran Mogul Ed, bshkosh, WI, wre sacrificed by decapitation and their entirebraixsed. The brains fran 59 frogs (3.69 g) were hwenized in 25 ml of chilled 2 N acetic acid using a Polytron at setting 5 for 30 sec. The pellet was dissolved in 10 ml of 2 N acetic acid, trmogenized arxi sonicated as above. The supematants fran these extractions wxe canbined arxi centrifuged at 40,ooO g for 5 hr. The supernatant *as concentrated by ultrafiltration using an Anicon UM-05 filter and passed over a 2 x 50 an Sephxkx G-15 column using 1 N acetic acid as the eluent. Fractions having molecular caights greater than TRH wxe pooled axi concentrated by ultrafiltration (Amicon H?W5). Enzymatic Modification - A 3.7 ml aliquot, representing 25% of the sample after removal of the er&xgenous 'IRH, +.mslyophilized and redissolved in 0.1 M zmnnni~ bicarbonate buffer at pH 8.2. k mg of trypsin wxe added and the reaction mixture ws incubated at 37°C. After 12 hr incubation an additional 2 mg of trypsin wxe added and incubation continued for an additional 12 hr; then the mixture ms &ted at 10°C for 10 min. 'Ihe pH ws adjusted to 7.4 with 1 N Xl, a 100 ~1 aliquot of carboxypeptidase A (18.6 mg/ml) and a 24@ ~1 aliquot of carbxypeptidase B (2 mg/ml) were added to the trypsin digest and incubation continued for 6 hr at 25'C. Chemical Modification - In order to cyclize NH2-terminal Glx residues the lyophilized proteolytic digest +as tefluxed in 2 ml glacial acetic acid for 2 min. Conversion of carboxyl terminal amino acids to their respective amides x.as achieved according to tk method of Frmnmnn--et al. (14). The cyclized proteolytic digest was dissolved in 2 ml of Ethanol containing 5 g HCl/loO ml an3 inbated at room temperature for 90 min. The sample ws freed of lKX by resuspending in nethanol followed by rotary evaporation to dryness. 'Ihis procedure mas repeated three times. The dried residue ws then dissolved in 2 ml of aethanol saturated with da at -5°C arxi incubated at r7xm temperature for 4 hr. The sample Was freed of armonia by rotary evaporation of the solvent. Ihis was also repeated three times. At each step of the various mxlification procedures samples were removed and the "JRItlike" activity (TRH-i) determined. Biological Assay - 013 and GQ,Cl cells wzre grown in monolayer ollture in Ham's FlO medium containing 15% horse serum and 2.5% fetal calf se-. where noted in the text, the cells were rinsed with se-free madium and the aedi~ replaced with Ham's FlO containing 10% hypothyroid calf sennn as previously described (15). 7Rl is not degraded by the culture media. For tests of receptor affinity and bioactivity, samples containing TRH-i wxe dissolved in 0.15 M I&Cl and diluted at least
732
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
250-fold before addition to cell cultures. The affinity of uulabeled TRH ami TRH-i for 'L7(H receptors was datenaked using a radioreceptor assay with CH3 membranes (16). Reactions contained in a final volme of 50 ~1, 44 pg mnbraue protein, 0.5 pm11 [+I]-IRH and 10 ~1 unlabeled TlW or extract. After incubation to quilibrim for 2 hr at O'C reactions wre diluted, pxsed through a Millipore filter and rinsed in order to determine the amount of [%I-YXH bouud to mmbram receptors. Now specific bimiiqg, measured in control tubes containing no mnbraue protein or excess mlabeled TRH, was 1%. The rate of prolactin synthesis was determined fran pro&tin in the a&m-e nedim; pro&tin is stable in the odium and the amount secreted is equivalent to the total synthesized over a 72 hr period (17). Pro&tin w3.s detenained by radioimunoassay using preparations and procedures fran the Hormone Distribution Program of the National Institute of Arthritis, Metabolism and Digestive Diseases. 8iuding of [%]-a(~ to G$ cultures was determined using 25 IW [~J-TRH in an exchange assay as previously described (18); nonspecific hiding GBS 15% and has been subtracted fran each point. The uptake of [3H] uridiae into GQCl cells ws measured by the procedures of Martin et In all tests of hioactivity 'R&i concentrations were determined by mdioimnmoassay. -al. (19).
Fonmtion like
of lRI-i
material.
remved,with
cnlyll
cular
fraction.
fran,
chenical
sponds
- Table
When the frog
I shows
brain
of various
'IN-i
will
be referred
or enzymatic
treatment
and c&xxypeptidases
treatments
was chramtograph.4
ng of radioimmmoassayableTRH-like
Table
alter
of
the
in,
or
of IN-i
nacramlecular of IREi.
fraction
of TRH-i
FX-i1 Fdvalents 2 N acetic acid extract Extract after sephada
of frog G-153
brain
33602 11
Tl-pSill
Trypsin + cyclization Trypsin+carbcocypeptidases Trypsin + cartoxypeptidases
2$ Aand B A and B
+ cyclization
Trypsin + cyclization TryFsin+carbolrjpeptidasea + cyclizatim
2 This vztIw oxrespads measurrd by radio-say. 3~
frog
brain
AandB
4 Each ha=
smiler
of these steps keen dtiplid
as the
to the TRH present
extractkes
of 828 equivalents in
8284
is defined
mbjected
rg of 1YH frm tk mcramlfxular with hypsin, carbxypeptidases result
If584 444
+ amid&ion
+ amidation
1 ore eqcdvalent of llu+i by radi olllmmssaay.
of 'IN-i. ammts
mIxnIt
of 7RH correspondi~
in the 2N acetic
to ChroMtography fraction.
A and R, follow4
Other of lRH-i.
treawnts
treatment
by qclization which
cdt
utilized 257, of the G-15 gel filtration by 4 to give tk total amnmt obtained
723
extract
acid
0nSephadex
Seqmntial
ore
to aw
rranogramasneasureii
of the frop
brain
G-15whIch rerwed of tk macrcmlecular
and midation or-more
of the
sample ard the at each step.
equivalents
as
allkut 11 fraction
results steps
formed corre
when the wcro-
I
Appea-
lRH-
in the nacramk
equivalent
the amouut
of
of the 'IRH WIS
present
tie
Digestion
not appreciably
appearance most
observed
mterial
fraction.
by mdioimnm~sy.
C-15,
(TRH-i)
to as radioimmmoassayable
A am3 B did
on the
on Sephadex material
of the nacramlecular
to 1 ng of TRH as measured
trypsin
the effect
extract
in formation
described
above
of ‘@H-i
by
Vol.
89,
No.
molecular (which
2, 1979
fractions will
formed.
digested
cyclize
Although
unique
in
dases
A or
the
all
treated
is
of tiw
wry
shown
iummoassay of
tmml
treatmnts
ws
treated
only
a srrall
additional
1.
[3~]-'IRH
that
increase
equivalents
carried
alter
in
the
wre
these
amount
carried
of 'IRH-i
- The
of
varying
[~H]-TRH
[3H]-TRHto
nreviously. [3~]-'IRH,
antibody
tin
ws
(Table
afford
analyzed
the
Unless, which
formxl
indicate
similar
expressed
by plotting (Fig.
to
I -
as that
a it
profiles
by incubating
wre
obtained
I).
?RIti
wmld
similar
ms
hen
extract
of
that
conlitions
line
radio+
a&Wed.
828 equivalents
also
the
I is
carlmxypepti-
mcranolecular
using
the data
a straight
ard
care
(carboxypzptidases
WIS mted the
acid
TM-i
addition,
&dated
of 'IRH aml TRH+
concentrations
that
In
acetic
in Table
trypsin,
proteins.
of properties
rsdioimunoassay
of
listed
fran
aml then
with
I has a m&r
tith
to demnstrate
of 'IRH-i
cut
in Table
birding
of steps
not arise
acid
Properties
'Ihe
series cut
did
B, cyclized
'Ihe
free
reacted
828
Aard
TRH.
described
further
ax-d acetic
cmboxypptidase
outlined
versus
might
COMMlJNlCATlONS
wre
each of the teen
RESEARCH
smidated,
'IRE-i
trypsin
with
procedure
that
then
artifacts.
experimnts
to authentic
antibody
and
have
fran
with
and Physical
in Figure of
various
treatments
similar
amnmt
expximents arise
withtrypsin,
IimmnocMcal result
several
residues)
did not
anitted),
otherwise,
had teen
dammstratd
bzen
BIOPHYSICAL
aml carboxypeptidase
has mt
fraction
A and B wxe
trypsin Glx
TRH-i,
R, or
with
AND
NH2-terminal
activity
macramlecular
stated
it
forming
mnoassayable
BIOCHEMICAL
as
a constant the 'IRH-radiw
the reciprocals Incuba-
Insert).
/ f ,
4)
4 .
pl 1
I 0.01
~adioi.mmoassay
preserxe
(0) to anti-m of 0.35 (upper)
1
0.025
TRHi I
0.05 ng TRH
I
I
0.1
0.25
I
I
I
0.5
I
25
of
'IRH (0) and TRH-i (0) (insert) Liwmwer-Burk the absence of l'W-i anl binding of [~H]-'IRH and 0.175 (lmr) equivalents of TM-i (0). in
724
to
J
plot. Binding anti-'IRH in
of the
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
tion of [3H]-.TRH in the presence of a constant amnmt of IRH-i afforded with
an increased
slope aad no apparent
change in the y-intercept
binding b&men [~I-'RH aodTPH-i. In order to estirrate the size of TIE-i, modified material
(containing
dex Cr15 colum.
A 25 ~1 aliquot
of cross reactivity
of mch 1 ml fraction
kere resolved.
The first
represented approximately 60% of the total volune of the colmm.
a 0.5 ml aliquot
130 of the 828 equivalents
(3:4)
of the enzymatically
peak kid the same elution
TM-i
The
material
secod
plot
canpetitive ard chemically '!J.mpeaks
xmlums as [-7H]-TRH and peak
elutd
tith
the
ws 94%. These results
has physical properties
tich
are identical
in degrading TRH (20).
Pl?S and incubated with 250 ~1 aliquots
with
irdicati~
WJS subjected to tadioimmoassay.
Nmnerous reports have shown that rat serum is effective ms diluted
a dcmble reciprocal
(V-)
of lP.H-i) was passed over a 1 x 50 cm Sepba-
cross reactivity.
Total recovery of imnuooreactive
that only 60% of the radioimnmxssayable
COMMUNICATIONS
mid
irdicated to TRH.
I&en rat sex-m
of smthentic 'RH (1.25 ng/lO ~1) or
TRK-i (1.25 equivalents/l0
~1) at 37'C for varying times, both authentic TRH aml TF#-i wxe degraded
by the senm.
20 min incubation,
Following
75% of the authentic
TRH and 40% of the 'M-i
care
degraded as determined by radioimnmcassay. Biological
Properties
by a radioreceptor
of 'IX&i - The affinity
assay in tich
[3~]-'IRH and different
pituitary
amcentrations
tion displacement curves wze obtained,
of 'IRH-i for specific
tmmr cell mbranes
of either
TRH receptors k~s detenained
(GH3) were incubated
unlabeled TRH or TRH-i (Fig. 2).
and TRH-i appeared to have slightly
dth
Parallel
higher affinity
tracer cmpetifor TPH
receptors than predicted by radioimnmmssay. cell
The bioactivity of 'IRH-icas tested using CX3 monolayer cultures. 'Ik material did not affect growth at concentrations equivalent to 10 nM by radioimmmcassay, altbmgh sane toxicity GBS
Figure 2: Affinity of TRH and lRH-i for TRH receptors. Canpetition displacement an-ves slow the means amd range of duplicate detem6nations for u&belled TRH (0) TM--i (0). The results are plotted using TM-i concentrations estimated by radioimmoassay.
725
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
IO
CTRHJ 0
or [TRH~ 0 (no)
Figure 3: Frolactin stimulation by TRH and TFWi. E&placate 35 mn dishes of GH3 cells here incubated with 1.5 ml hypothyroid wdiun containing either ‘JXH (0) or TRIti (0). The concentrations of TFZH-i wxe estimated by radioticassay. Pro&tin was measured in the culture medium after 72 hrs. Cell proteins were r‘d)t affected by ‘IRH or TRH-i and averaged 185 &dish. The wn and range of duplicate disks are shown.
noted
at higkr
levels.
Both
‘IRH and TRFfi
72 hr incubation,
and the dose response
(Fig.
knrmone
3).
kcwth
Another bation
long-term
with
caused
of lRH is the
min after
its
and ‘IX-i
action
‘IRH the nmker
TRH and TM-i effect
DISCJJSSION:
The physical, to TRH.
is
with
highly
traphs affinity
specific. are
also
tion
with
depletion
but affinity
(data
for
of ‘IXH receptors
by a subclone
‘RH and ‘IX&i
effects
TRH or TRH-i
increased
not
at 1 to 2 nM shown).
After
24 to 48 hr
incz-
not chaqqed
(18).
(data
r&
of the GH3 cell
the’ uptake
in a
TRH ws
of ‘IRH receptors.
decreased
uptake
hslf-wxkal
synthesis
of
[3~]
A rapid
shown). lines
In 90
(19).
uridiw
into
cells,
innwnochenkal
ard biological
presented
in Fig.
to TF# and to pituitary
Structural stringent,
requirements as tinor
of ‘IRH-i
in
binding
to TRH receptors
any
the
of
would
that
three
sumst
that
lXH and TRH-i
The antiserum
‘IF%! receptors. for
changes
properties
1 and 2 demonstrate
used
in
this
on thyrotrophs
amino
acids
can
it
bind
is caw
report
and l~amnr severely
reduce
(16). In pituitary
states
parallel
of prolactin
activity.
The results
antisensn
stimulation
by either
regulation” cell
of uridine
grater
wre
“down pzr
d-dependent
to cultures
very
similar
of lRH is
stimulation
addition
curves
a 3-fold
WB r‘d)t affected
of receptors
parallel
had slightly
petitively
synthesis
caused
tuner
of pre-pro&tin the
cells &NA
acute
uptake
IRH stimulates (21-22), of
causes uridiw
the a loss
synthesis
of prolactin
of receptor
by increasing
726
uridine
sites !&use
by increasing by an unknrxn activity
the
concentra-
mchanism (23).
‘IT&i
(18)
ard
exerts
Vol.
89,
these
No.
2, 1979
'IRH-like
curves
for
actions
leading
Results
than 'IRH.
(24).
In add.ition,
insulin
(24).
In the
absence
of
frog
increase
measure
TW-i,
cyclize
steps
Tissues
react
with
other
wi,ght
It
should
proteins
(30).
vex-ted
to TRH-i
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
than
precursor
the
brain
my
which
fact
(14)
also
possess
this
luteiniziog
that
the
amino
carbmypeptidaee and enzymtic
post-translatioml
in
Using
have
steps
this atlined
processing
Do the
this
sequence
in Table mst
can
of amino Ihus,
an appre
was
tich
used
are
anidation chemical
to
knmo (14),
a
aml eozymtic
conversion
of a wcro-
question.
been
(27-28).
I.
in
which
have
been reported
Glx'His.Pro
enzymes
for
mcramlecule
tissues
Treatamt
result
C-terminal
that
precedent
(25,26).
not
to answx
also
suggests
is also
prccedures
in
required
of
This
Ihere
to
of IRH-i.
to TRH, tiich
lmnmne
sequence
A possesses
I).
extensions
observed.
enzymes
of proinsulin
of 828 equivalent
chemical
result
necessary
a variety
acid
hxmmes
antibody
or a similar
of sxmtostatin
molecular
respective
(Table
acid
hormone-releasing
present
a greater
A amd B does the
ws
are
as
hwing
fomation.
amino
and also
the processing
dose-response
'IRH receptors
the conversion
formed
that
of IRH-i
with
to their
formation
TRH-i
to have
experiments
is
ms for
aml ProNH2.
for
species
by the chemical to specific
filu
concentration
tomrd
be ooted
the
to Flu
additional
immoreactive
It&ad,
for
residues
directed
also
reflect
I substitute
a mcramlecule
molecular
the
in
for
for
'5%-i
amides
a canpomd
is recessary
important
and
interacts
prohonmnes
amd cartmypeptidases
nay
specific Glx
Obviously,
Antibodies
trypsin
This
f-ran
several
of
is
COMMUNICATIONS
of 'IRH.
necessary
44 equivalents
with
highly
in Table
activities
enzyme
the C-teminus
extract
to 'IRH?
Iflike
the TRH-i
arises
B are also
RESEARCH
cm radioimnmwssy,
tiicate
lRH-i
as C terminal
increase
a&lined
molecule
T&I-i.
enzymes fran
Nl2-terminal
dramtic
that
A a&
in TRH-i. is
based
data
of biological
suggest
tetinate
brain
BIOPHYSICAL
has been shown to convert
these acids
&ich
of the
Ihese
a carbmypeptidase
of mine
peptides
rather
barein
Trypsin
AND
concentrations
spectrum
Carbxypeptidases
ciable
to
equivalent
are parallel.
to the full
presented
weight
for
at
TRH and TRH-i
an agonist
removal
BIOCHEMICAL
gives
rise
shown to
to
u-cm-
Similarly,
higher
(29). be
found
acids
hit
in is
the susceptibility
several not
corr
of
l-e of importance.
S&ally, A. V., Redding, T., f'owrs, C. arrl Barretty, J. (1969) J. Biol. Clam. x, 4077-4088. T. F., Dssiderio, D., Ward, N., Vale, W. ard Guillmin, R. (1970) Nature 2, Burgus, R., hum, 321. R. (1971) Hormones2, 193-203. Vale, W., Burgus, R., Imm, T. and tilltin, Chaq, J.., Sievertsson, H., Curries, B., Folders, K. and Rowers, C. Y. (1971) J. Med. Chem. 14, 484-487. Johnston, R aml Bloch, K. (1951) J. Biol. Chem. 188, 221-240. ~ipmm, F. (1973) Accounts of Chemical Res. 6, 361-367. Mitnick, M. aud Reichlin, S. (1971) Sciencez, 1231-1232. Mituick, M. and Reich&, S. (1972) Endocrinology~, 1145-1153. Dixon, J.. E. azd Acres, S. 6. (1975) Fed. Proc. 34, 658. Bauer, K,. and LiImann F. (1976) Emkxrinolgoy~, 230-242. Habeuer, J. F., Rosenblatt, M., Kmper, R., Kroenberg, H., Rich, A. and Potts, J., Jr. (1978) Proc. &xl. &ad. Sci. EA75, 2616-2620. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. ard R&all, R. J. (1951) J. Biol. Chem. 193, 26+275. Bryce, G.. F. (1974) Imnmochemistry&, 507-511. Enmann, G., Paler, J. and Folkers, K. (1971) J. Med. Chem.14, 469-474.
727
Vol.
15.
16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
26. 27. 28. 29.
30.
89, No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Perrone, M. H. and Hinkle, P. M. (1978) J. Biol. Cbm. 253, 5168-5173. Hinkle, P. M., bbroch, E. L. and Tashjian, A. H., Jr. (1974) J. Riol. Chm. 249, 30853090. Dmnies, P. S. srd Tashjim, A. H., Jr. (1973) J. Biol. C&m. 248, 6174-6180. Hinkle, P. N. ami Tashjian, A. H., Jr. (1975) BiochemistryE, 3845-3851. Martin, T. F. J., Cixt, A. M. ard Tashjian, A. H., Jr. (1978) J. Biol. C&n.=, 99-105. Taylor, W. L. End Dixon, J. E. (1978) J. Biol. C&m. 253, 6934-6940 ard references therein. Dannies, P. S. and Tashjian, A. H., Jr. (1976) Biochan. Biophys. Res. bmnm. 7&, 1180-1189. Evans, G. A. and Rosenfeld, M. G. (1976) J. Biol. Cbem. 251, 2842-2847. Martin, T. F. J. and Tashjian, A. H., Jr. (1978) J. Biol. Cl-m. 253, 106-115. Steiner, D. F., Kermler, W., Tager, S. ard Peterson, J. D. (1974) Fed. Proc. 2, X05-2115. Suchanek, 6. and Kreil, C. (1977) Proc. btl. Sad. Sci. BAa, 975-978. Lowry, P. T. amI Scott, A, P. (1975) Gen. Gmp. Fndocrinol. 2, 16-23. Barma, A. and Porter, J. C. (1975) Riochem. Biophys. Res. Cam. 67, 1346- 1352. Millar, R. P., Aehwlt, C. and Rossier, G. (1977) Rio&em. Biophys. Res. Ccmm 74, 720-731. Arimra, A., Sato, H., Sqmt, A., Mshi, N. ad S&ally, A. V. (1975) ScienceE, 1001-1009. Dayhoff, M. ed. (1972) Atlas of Protein Sequences ard Structure, Vol 5, p. D-126, titioml Biw med. Res. Fomd. Washington, D.C.
728
89,
No.
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July
27, 1979
BIOCHEMICAL
AND
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RESEARCH
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Pages 721-728
*Dapartmant of Biochemistry Purdue University Nest Lafayette, Indians 47907 hepartnmt of Pharmacology ard Toxicology ard Hniversity of Rochester Cancer Center University of Rochester School of Medicine and Dentistry Rochester, New York 14642 Received
June 11, 1979
SUMMARY:Chemical and enzymatic treatment of a higtilecular raight fraction fran a frog brain extract resulted in formation of a 'V&like material" (TRH-i). Sequential treatment with trypsin ard carlxxypeptidases A and B, acetic acid and then chemical ennidation generated a quantity of WH-i equivalent to 25% of the erdogenous TFX. TFX-i was similar to TRH (rGluVis'PrcNH2) as assessed by molecular 'iaight estimations, radioimmunoassay m-d susceptibility to senxn inactivation. TBH and TRH-i also competed with [%I-'Hw for binding to TBH receptors, stimulated pro&tin synthesis and uridine uptake, ard "down-regulated" TRH receptors in pituitary cells. These results suggest the possibility that lRH may te processed fran a nncranolecular precursor. Thyrotropin
releasiq+xmone
(TBH), the &lest
of the hypothalamic hxnx3nes, has been iso-
lated frun porcine (1) and ovine (2) hypothalamic fragaxsnts anl its structure
Qtennined.
mane and nrmerou.3 analogous have been chemically synthesized and their biological gated (3-4).
Despite these advances, little
Biosynthesis
of s&l
peptides such as glutathione
been shown to proceed by a I-XX-ribosaaal tions indicated
is known about the biosynthesis
lz&hmay (7-8),
hxever,
Initial
these reports have not
eight
species that xuld
Although it is difficult
be enzymatically
to find supporting
precursors
(11).
it wxld
processed to its
precedents for the narribo-
scmal synthesis of IRH, there is extensive documantation that hDrn~3nes and proteins as larger
(6) has obeerva-
(9-10).
probably be formed as a higher rrolecular Edctive form.
investi-
paptide antibiotics
TRH is canposed of only three amino acids, anl if it wxe synthesized on ribosanes, biologically
'Ihe hx-
of lBH.
ma&m&m of anino acid polymerization.
that 'IPH was synthesized by a similar
been confirmed by other laboratories
(5) or bacterial
activities
are synthesized
In the present study we have determined that the frog brain contains a
macrarx&cule similar
that can te converted -via enzymatic arrl chanical nodifications to a canpound tich is to TRH as detefinined by physical characteristics, innnmoch0nical properties and biological
activity. * Correspondence and inquiries should be addressed to Jack E. Dixon, Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907. ml.9 research k~s supported in p3rt by Research Grants pM 18849 (J.E.D.) ard AM 19974 (P.M.H.) fran the National Institute of Arthritis, Matabolism arxI Digestive Diseases anl Cancer Center Core Research grant ~3 11198 (P.M.H.). J.E.D. is a recipient of a Research Career Development Pward (AM 00218). J.H.R. is an N.I.H. postdoctoral fellow (AM 0594601). This is journal paper rumber 7610 fran the Purdue University Agricultural Experiment Station. 0006-291X/79/140721-08$01.00/0 721
Copyright All rights
@ I979 by Academic Press, Inc. of reproduction in anyform reserved.
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
line 2,3-Q IRH ([Q-TRH) used f or radioimmmcassay bad a specific activity of 21.8 Ci/nmole. Similar material used in receptor hiding experiments had a specific activity of 115 Ci/mnole (New F@ Nuclear). Trypsin, carboxypeptidase A, carbcmypeptidase B, bovine thyroglobulin and bovine serun altin wzre purchased fran Sigma. 'I'RH and $lu'His'Pro (free acid) were products of Bachem or gifts fran Abbott Laboratories. Tissue culture m&a, Vnrse serum ard fetal calf senrn wxe from Grand Island Biological Gxnpany. Hy@h oid calf sennn (thyroxine l% I for protein icdination < 1 n ml) was frcm Rockland Fatms, Gilbertsville, PA. Carrier-free axl [f% ] uridine (27 Ci/mnole) were fran Amersham/Searle. 'Ihe G93 and GH4Cl cell lines wzre obtained fran Dr. Armen H. Tashjian, Jr., Harvard Medical School, Boston, MA. Protein concentrations were determined by the nethod of Lowry --et al. (12) using bovine sennn albumin as a stardard. Radioimmmoassay - Antibody to TRH ws prepared ard TRH concentrations measured by a nudificz tion of the procedure of Bryce (13). Radioimmmoassay incubation mixtures contained 10 ~1 of IRH antiserum (binds 5,0X c~xn [?I]-TRH), 5 ~1 [?I]-TRH (12,500 cpn, 0.25 ng:), 0 to 50 ~1 of sample of 'IRH standard and phosphate tuffered salirxa (PBS, 0.01 M sodium phosphate containing 0.15 M N&l, pH 7.4) to make a firm1 xx&mm of 5C to 100 ~1. After memight incubation at 4"C, 5rK) ng of 17nr irmnme rabbit setxr in 10 ~1 PBS =re added and the incubated mixtures wxe diluted with an equal volm of saturated anmonilun milfate. After 20 min, the precipitated antigerrantibcdy cmnplex mas collected by centrifugation at 8,0(x) g for 6 min. The supematant was discarded ard the pellet kas washed with 300 ~1 and 200 ~1 aliquots of 50% saturated mnmonium sulfate. The final pallet kas dissolved in lC0 ~1 of 1 N &OH ar$ transferred to a scintillation vial. Fach assay tube k~s rinsed with 900 ~1 of H20 which was canbined with the NaOHsolution. The antiserun Ws found to be quite specific for TRH. The structural analogues ProNH2, pGlu'His, $lu*HisPro, Luteinking Hormone-Releasing Hormone and GluWisPrcNH2 were tested at 10X-fold molar excess ard did not reduce the binding of [3H]-IBH to the antibody. However, $lu'His'Pro'GlyNH2 cross reacted with the antibody at a 2Ffold nolar excess ard pGlu'L-dopa'ProNH2 showed a canpetition for antibody that parallels that of authentic '!RH and suggests that the antibody discriminates poorly at the histidice residue. This result is notsurprising since TRH is conjugated to thyroglobulin via the imidazole of histidine. Preparation of Brain Extracts - Frogs (Rana pipiens), ~chased fran Mogul Ed, bshkosh, WI, wre sacrificed by decapitation and their entirebraixsed. The brains fran 59 frogs (3.69 g) were hwenized in 25 ml of chilled 2 N acetic acid using a Polytron at setting 5 for 30 sec. The pellet was dissolved in 10 ml of 2 N acetic acid, trmogenized arxi sonicated as above. The supematants fran these extractions wxe canbined arxi centrifuged at 40,ooO g for 5 hr. The supernatant *as concentrated by ultrafiltration using an Anicon UM-05 filter and passed over a 2 x 50 an Sephxkx G-15 column using 1 N acetic acid as the eluent. Fractions having molecular caights greater than TRH wxe pooled axi concentrated by ultrafiltration (Amicon H?W5). Enzymatic Modification - A 3.7 ml aliquot, representing 25% of the sample after removal of the er&xgenous 'IRH, +.mslyophilized and redissolved in 0.1 M zmnnni~ bicarbonate buffer at pH 8.2. k mg of trypsin wxe added and the reaction mixture ws incubated at 37°C. After 12 hr incubation an additional 2 mg of trypsin wxe added and incubation continued for an additional 12 hr; then the mixture ms &ted at 10°C for 10 min. 'Ihe pH ws adjusted to 7.4 with 1 N Xl, a 100 ~1 aliquot of carboxypeptidase A (18.6 mg/ml) and a 24@ ~1 aliquot of carbxypeptidase B (2 mg/ml) were added to the trypsin digest and incubation continued for 6 hr at 25'C. Chemical Modification - In order to cyclize NH2-terminal Glx residues the lyophilized proteolytic digest +as tefluxed in 2 ml glacial acetic acid for 2 min. Conversion of carboxyl terminal amino acids to their respective amides x.as achieved according to tk method of Frmnmnn--et al. (14). The cyclized proteolytic digest was dissolved in 2 ml of Ethanol containing 5 g HCl/loO ml an3 inbated at room temperature for 90 min. The sample ws freed of lKX by resuspending in nethanol followed by rotary evaporation to dryness. 'Ihis procedure mas repeated three times. The dried residue ws then dissolved in 2 ml of aethanol saturated with da at -5°C arxi incubated at r7xm temperature for 4 hr. The sample Was freed of armonia by rotary evaporation of the solvent. Ihis was also repeated three times. At each step of the various mxlification procedures samples were removed and the "JRItlike" activity (TRH-i) determined. Biological Assay - 013 and GQ,Cl cells wzre grown in monolayer ollture in Ham's FlO medium containing 15% horse serum and 2.5% fetal calf se-. where noted in the text, the cells were rinsed with se-free madium and the aedi~ replaced with Ham's FlO containing 10% hypothyroid calf sennn as previously described (15). 7Rl is not degraded by the culture media. For tests of receptor affinity and bioactivity, samples containing TRH-i wxe dissolved in 0.15 M I&Cl and diluted at least
732
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No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
250-fold before addition to cell cultures. The affinity of uulabeled TRH ami TRH-i for 'L7(H receptors was datenaked using a radioreceptor assay with CH3 membranes (16). Reactions contained in a final volme of 50 ~1, 44 pg mnbraue protein, 0.5 pm11 [+I]-IRH and 10 ~1 unlabeled TlW or extract. After incubation to quilibrim for 2 hr at O'C reactions wre diluted, pxsed through a Millipore filter and rinsed in order to determine the amount of [%I-YXH bouud to mmbram receptors. Now specific bimiiqg, measured in control tubes containing no mnbraue protein or excess mlabeled TRH, was 1%. The rate of prolactin synthesis was determined fran pro&tin in the a&m-e nedim; pro&tin is stable in the odium and the amount secreted is equivalent to the total synthesized over a 72 hr period (17). Pro&tin w3.s detenained by radioimunoassay using preparations and procedures fran the Hormone Distribution Program of the National Institute of Arthritis, Metabolism and Digestive Diseases. 8iuding of [%]-a(~ to G$ cultures was determined using 25 IW [~J-TRH in an exchange assay as previously described (18); nonspecific hiding GBS 15% and has been subtracted fran each point. The uptake of [3H] uridiae into GQCl cells ws measured by the procedures of Martin et In all tests of hioactivity 'R&i concentrations were determined by mdioimnmoassay. -al. (19).
Fonmtion like
of lRI-i
material.
remved,with
cnlyll
cular
fraction.
fran,
chenical
sponds
- Table
When the frog
I shows
brain
of various
'IN-i
will
be referred
or enzymatic
treatment
and c&xxypeptidases
treatments
was chramtograph.4
ng of radioimmmoassayableTRH-like
Table
alter
of
the
in,
or
of IN-i
nacramlecular of IREi.
fraction
of TRH-i
FX-i1 Fdvalents 2 N acetic acid extract Extract after sephada
of frog G-153
brain
33602 11
Tl-pSill
Trypsin + cyclization Trypsin+carbcocypeptidases Trypsin + cartoxypeptidases
2$ Aand B A and B
+ cyclization
Trypsin + cyclization TryFsin+carbolrjpeptidasea + cyclizatim
2 This vztIw oxrespads measurrd by radio-say. 3~
frog
brain
AandB
4 Each ha=
smiler
of these steps keen dtiplid
as the
to the TRH present
extractkes
of 828 equivalents in
8284
is defined
mbjected
rg of 1YH frm tk mcramlfxular with hypsin, carbxypeptidases result
If584 444
+ amid&ion
+ amidation
1 ore eqcdvalent of llu+i by radi olllmmssaay.
of 'IN-i. ammts
mIxnIt
of 7RH correspondi~
in the 2N acetic
to ChroMtography fraction.
A and R, follow4
Other of lRH-i.
treawnts
treatment
by qclization which
cdt
utilized 257, of the G-15 gel filtration by 4 to give tk total amnmt obtained
723
extract
acid
0nSephadex
Seqmntial
ore
to aw
rranogramasneasureii
of the frop
brain
G-15whIch rerwed of tk macrcmlecular
and midation or-more
of the
sample ard the at each step.
equivalents
as
allkut 11 fraction
results steps
formed corre
when the wcro-
I
Appea-
lRH-
in the nacramk
equivalent
the amouut
of
of the 'IRH WIS
present
tie
Digestion
not appreciably
appearance most
observed
mterial
fraction.
by mdioimnm~sy.
C-15,
(TRH-i)
to as radioimmmoassayable
A am3 B did
on the
on Sephadex material
of the nacramlecular
to 1 ng of TRH as measured
trypsin
the effect
extract
in formation
described
above
of ‘@H-i
by
Vol.
89,
No.
molecular (which
2, 1979
fractions will
formed.
digested
cyclize
Although
unique
in
dases
A or
the
all
treated
is
of tiw
wry
shown
iummoassay of
tmml
treatmnts
ws
treated
only
a srrall
additional
1.
[3~]-'IRH
that
increase
equivalents
carried
alter
in
the
wre
these
amount
carried
of 'IRH-i
- The
of
varying
[~H]-TRH
[3H]-TRHto
nreviously. [3~]-'IRH,
antibody
tin
ws
(Table
afford
analyzed
the
Unless, which
formxl
indicate
similar
expressed
by plotting (Fig.
to
I -
as that
a it
profiles
by incubating
wre
obtained
I).
?RIti
wmld
similar
ms
hen
extract
of
that
conlitions
line
radio+
a&Wed.
828 equivalents
also
the
I is
carlmxypepti-
mcranolecular
using
the data
a straight
ard
care
(carboxypzptidases
WIS mted the
acid
TM-i
addition,
&dated
of 'IRH aml TRH+
concentrations
that
In
acetic
in Table
trypsin,
proteins.
of properties
rsdioimunoassay
of
listed
fran
aml then
with
I has a m&r
tith
to demnstrate
of 'IRH-i
cut
in Table
birding
of steps
not arise
acid
Properties
'Ihe
series cut
did
B, cyclized
'Ihe
free
reacted
828
Aard
TRH.
described
further
ax-d acetic
cmboxypptidase
outlined
versus
might
COMMlJNlCATlONS
wre
each of the teen
RESEARCH
smidated,
'IRE-i
trypsin
with
procedure
that
then
artifacts.
experimnts
to authentic
antibody
and
have
fran
with
and Physical
in Figure of
various
treatments
similar
amnmt
expximents arise
withtrypsin,
IimmnocMcal result
several
residues)
did not
anitted),
otherwise,
had teen
dammstratd
bzen
BIOPHYSICAL
aml carboxypeptidase
has mt
fraction
A and B wxe
trypsin Glx
TRH-i,
R, or
with
AND
NH2-terminal
activity
macramlecular
stated
it
forming
mnoassayable
BIOCHEMICAL
as
a constant the 'IRH-radiw
the reciprocals Incuba-
Insert).
/ f ,
4)
4 .
pl 1
I 0.01
~adioi.mmoassay
preserxe
(0) to anti-m of 0.35 (upper)
1
0.025
TRHi I
0.05 ng TRH
I
I
0.1
0.25
I
I
I
0.5
I
25
of
'IRH (0) and TRH-i (0) (insert) Liwmwer-Burk the absence of l'W-i anl binding of [~H]-'IRH and 0.175 (lmr) equivalents of TM-i (0). in
724
to
J
plot. Binding anti-'IRH in
of the
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
tion of [3H]-.TRH in the presence of a constant amnmt of IRH-i afforded with
an increased
slope aad no apparent
change in the y-intercept
binding b&men [~I-'RH aodTPH-i. In order to estirrate the size of TIE-i, modified material
(containing
dex Cr15 colum.
A 25 ~1 aliquot
of cross reactivity
of mch 1 ml fraction
kere resolved.
The first
represented approximately 60% of the total volune of the colmm.
a 0.5 ml aliquot
130 of the 828 equivalents
(3:4)
of the enzymatically
peak kid the same elution
TM-i
The
material
secod
plot
canpetitive ard chemically '!J.mpeaks
xmlums as [-7H]-TRH and peak
elutd
tith
the
ws 94%. These results
has physical properties
tich
are identical
in degrading TRH (20).
Pl?S and incubated with 250 ~1 aliquots
with
irdicati~
WJS subjected to tadioimmoassay.
Nmnerous reports have shown that rat serum is effective ms diluted
a dcmble reciprocal
(V-)
of lP.H-i) was passed over a 1 x 50 cm Sepba-
cross reactivity.
Total recovery of imnuooreactive
that only 60% of the radioimnmxssayable
COMMUNICATIONS
mid
irdicated to TRH.
I&en rat sex-m
of smthentic 'RH (1.25 ng/lO ~1) or
TRK-i (1.25 equivalents/l0
~1) at 37'C for varying times, both authentic TRH aml TF#-i wxe degraded
by the senm.
20 min incubation,
Following
75% of the authentic
TRH and 40% of the 'M-i
care
degraded as determined by radioimnmcassay. Biological
Properties
by a radioreceptor
of 'IX&i - The affinity
assay in tich
[3~]-'IRH and different
pituitary
amcentrations
tion displacement curves wze obtained,
of 'IRH-i for specific
tmmr cell mbranes
of either
TRH receptors k~s detenained
(GH3) were incubated
unlabeled TRH or TRH-i (Fig. 2).
and TRH-i appeared to have slightly
dth
Parallel
higher affinity
tracer cmpetifor TPH
receptors than predicted by radioimnmmssay. cell
The bioactivity of 'IRH-icas tested using CX3 monolayer cultures. 'Ik material did not affect growth at concentrations equivalent to 10 nM by radioimmmcassay, altbmgh sane toxicity GBS
Figure 2: Affinity of TRH and lRH-i for TRH receptors. Canpetition displacement an-ves slow the means amd range of duplicate detem6nations for u&belled TRH (0) TM--i (0). The results are plotted using TM-i concentrations estimated by radioimmoassay.
725
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
IO
CTRHJ 0
or [TRH~ 0 (no)
Figure 3: Frolactin stimulation by TRH and TFWi. E&placate 35 mn dishes of GH3 cells here incubated with 1.5 ml hypothyroid wdiun containing either ‘JXH (0) or TRIti (0). The concentrations of TFZH-i wxe estimated by radioticassay. Pro&tin was measured in the culture medium after 72 hrs. Cell proteins were r‘d)t affected by ‘IRH or TRH-i and averaged 185 &dish. The wn and range of duplicate disks are shown.
noted
at higkr
levels.
Both
‘IRH and TRFfi
72 hr incubation,
and the dose response
(Fig.
knrmone
3).
kcwth
Another bation
long-term
with
caused
of lRH is the
min after
its
and ‘IX-i
action
‘IRH the nmker
TRH and TM-i effect
DISCJJSSION:
The physical, to TRH.
is
with
highly
traphs affinity
specific. are
also
tion
with
depletion
but affinity
(data
for
of ‘IXH receptors
by a subclone
‘RH and ‘IX&i
effects
TRH or TRH-i
increased
not
at 1 to 2 nM shown).
After
24 to 48 hr
incz-
not chaqqed
(18).
(data
r&
of the GH3 cell
the’ uptake
in a
TRH ws
of ‘IRH receptors.
decreased
uptake
hslf-wxkal
synthesis
of
[3~]
A rapid
shown). lines
In 90
(19).
uridiw
into
cells,
innwnochenkal
ard biological
presented
in Fig.
to TF# and to pituitary
Structural stringent,
requirements as tinor
of ‘IRH-i
in
binding
to TRH receptors
any
the
of
would
that
three
sumst
that
lXH and TRH-i
The antiserum
‘IF%! receptors. for
changes
properties
1 and 2 demonstrate
used
in
this
on thyrotrophs
amino
acids
can
it
bind
is caw
report
and l~amnr severely
reduce
(16). In pituitary
states
parallel
of prolactin
activity.
The results
antisensn
stimulation
by either
regulation” cell
of uridine
grater
wre
“down pzr
d-dependent
to cultures
very
similar
of lRH is
stimulation
addition
curves
a 3-fold
WB r‘d)t affected
of receptors
parallel
had slightly
petitively
synthesis
caused
tuner
of pre-pro&tin the
cells &NA
acute
uptake
IRH stimulates (21-22), of
causes uridiw
the a loss
synthesis
of prolactin
of receptor
by increasing
726
uridine
sites !&use
by increasing by an unknrxn activity
the
concentra-
mchanism (23).
‘IT&i
(18)
ard
exerts
Vol.
89,
these
No.
2, 1979
'IRH-like
curves
for
actions
leading
Results
than 'IRH.
(24).
In add.ition,
insulin
(24).
In the
absence
of
frog
increase
measure
TW-i,
cyclize
steps
Tissues
react
with
other
wi,ght
It
should
proteins
(30).
vex-ted
to TRH-i
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
than
precursor
the
brain
my
which
fact
(14)
also
possess
this
luteiniziog
that
the
amino
carbmypeptidaee and enzymtic
post-translatioml
in
Using
have
steps
this atlined
processing
Do the
this
sequence
in Table mst
can
of amino Ihus,
an appre
was
tich
used
are
anidation chemical
to
knmo (14),
a
aml eozymtic
conversion
of a wcro-
question.
been
(27-28).
I.
in
which
have
been reported
Glx'His.Pro
enzymes
for
mcramlecule
tissues
Treatamt
result
C-terminal
that
precedent
(25,26).
not
to answx
also
suggests
is also
prccedures
in
required
of
This
Ihere
to
of IRH-i.
to TRH, tiich
lmnmne
sequence
A possesses
I).
extensions
observed.
enzymes
of proinsulin
of 828 equivalent
chemical
result
necessary
a variety
acid
hxmmes
antibody
or a similar
of sxmtostatin
molecular
respective
(Table
acid
hormone-releasing
present
a greater
A amd B does the
ws
are
as
hwing
fomation.
amino
and also
the processing
dose-response
'IRH receptors
the conversion
formed
that
of IRH-i
with
to their
formation
TRH-i
to have
experiments
is
ms for
aml ProNH2.
for
species
by the chemical to specific
filu
concentration
tomrd
be ooted
the
to Flu
additional
immoreactive
It&ad,
for
residues
directed
also
reflect
I substitute
a mcramlecule
molecular
the
in
for
for
'5%-i
amides
a canpomd
is recessary
important
and
interacts
prohonmnes
amd cartmypeptidases
nay
specific Glx
Obviously,
Antibodies
trypsin
This
f-ran
several
of
is
COMMUNICATIONS
of 'IRH.
necessary
44 equivalents
with
highly
in Table
activities
enzyme
the C-teminus
extract
to 'IRH?
Iflike
the TRH-i
arises
B are also
RESEARCH
cm radioimnmwssy,
tiicate
lRH-i
as C terminal
increase
a&lined
molecule
T&I-i.
enzymes fran
Nl2-terminal
dramtic
that
A a&
in TRH-i. is
based
data
of biological
suggest
tetinate
brain
BIOPHYSICAL
has been shown to convert
these acids
&ich
of the
Ihese
a carbmypeptidase
of mine
peptides
rather
barein
Trypsin
AND
concentrations
spectrum
Carbxypeptidases
ciable
to
equivalent
are parallel.
to the full
presented
weight
for
at
TRH and TRH-i
an agonist
removal
BIOCHEMICAL
gives
rise
shown to
to
u-cm-
Similarly,
higher
(29). be
found
acids
hit
in is
the susceptibility
several not
corr
of
l-e of importance.
S&ally, A. V., Redding, T., f'owrs, C. arrl Barretty, J. (1969) J. Biol. Clam. x, 4077-4088. T. F., Dssiderio, D., Ward, N., Vale, W. ard Guillmin, R. (1970) Nature 2, Burgus, R., hum, 321. R. (1971) Hormones2, 193-203. Vale, W., Burgus, R., Imm, T. and tilltin, Chaq, J.., Sievertsson, H., Curries, B., Folders, K. and Rowers, C. Y. (1971) J. Med. Chem. 14, 484-487. Johnston, R aml Bloch, K. (1951) J. Biol. Chem. 188, 221-240. ~ipmm, F. (1973) Accounts of Chemical Res. 6, 361-367. Mitnick, M. aud Reichlin, S. (1971) Sciencez, 1231-1232. Mituick, M. and Reich&, S. (1972) Endocrinology~, 1145-1153. Dixon, J.. E. azd Acres, S. 6. (1975) Fed. Proc. 34, 658. Bauer, K,. and LiImann F. (1976) Emkxrinolgoy~, 230-242. Habeuer, J. F., Rosenblatt, M., Kmper, R., Kroenberg, H., Rich, A. and Potts, J., Jr. (1978) Proc. &xl. &ad. Sci. EA75, 2616-2620. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. ard R&all, R. J. (1951) J. Biol. Chem. 193, 26+275. Bryce, G.. F. (1974) Imnmochemistry&, 507-511. Enmann, G., Paler, J. and Folkers, K. (1971) J. Med. Chem.14, 469-474.
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Vol.
15.
16. 17. 18. 19. 20. 21. 22.
23. 24. 25.
26. 27. 28. 29.
30.
89, No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Perrone, M. H. and Hinkle, P. M. (1978) J. Biol. Cbm. 253, 5168-5173. Hinkle, P. M., bbroch, E. L. and Tashjian, A. H., Jr. (1974) J. Riol. Chm. 249, 30853090. Dmnies, P. S. srd Tashjim, A. H., Jr. (1973) J. Biol. C&m. 248, 6174-6180. Hinkle, P. N. ami Tashjian, A. H., Jr. (1975) BiochemistryE, 3845-3851. Martin, T. F. J., Cixt, A. M. ard Tashjian, A. H., Jr. (1978) J. Biol. C&n.=, 99-105. Taylor, W. L. End Dixon, J. E. (1978) J. Biol. C&m. 253, 6934-6940 ard references therein. Dannies, P. S. and Tashjian, A. H., Jr. (1976) Biochan. Biophys. Res. bmnm. 7&, 1180-1189. Evans, G. A. and Rosenfeld, M. G. (1976) J. Biol. Cbem. 251, 2842-2847. Martin, T. F. J. and Tashjian, A. H., Jr. (1978) J. Biol. Cl-m. 253, 106-115. Steiner, D. F., Kermler, W., Tager, S. ard Peterson, J. D. (1974) Fed. Proc. 2, X05-2115. Suchanek, 6. and Kreil, C. (1977) Proc. btl. Sad. Sci. BAa, 975-978. Lowry, P. T. amI Scott, A, P. (1975) Gen. Gmp. Fndocrinol. 2, 16-23. Barma, A. and Porter, J. C. (1975) Riochem. Biophys. Res. Cam. 67, 1346- 1352. Millar, R. P., Aehwlt, C. and Rossier, G. (1977) Rio&em. Biophys. Res. Ccmm 74, 720-731. Arimra, A., Sato, H., Sqmt, A., Mshi, N. ad S&ally, A. V. (1975) ScienceE, 1001-1009. Dayhoff, M. ed. (1972) Atlas of Protein Sequences ard Structure, Vol 5, p. D-126, titioml Biw med. Res. Fomd. Washington, D.C.
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