Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 2a2~ ~018
AWALOGUES ~ ~ ONA~OTROPIN REL~ASING HOR~ONE.
~ _0~
Thi~5 in~arl ti~n ~el~t~s to novel p~ptides and to th~ir u~e~.
1, .
Gon~dotrop in r~lea~ing hormon~ (GnR~ a d~cap~p~ide whic:h
i~ s~cre~ ~d I by ~ hypo~l~ic r~qian o~ th~ brain.
M~ n go~dotropin r~le~ing ho~on~ ~as ~ ollowin~
~ino ~ic!l $~qu~nce p ~;lu- Hi3~ S~r- Tyr~ Gly- ~eu- Ar~-
Pro-Gly N H2 I There a~e how~ r va3~iant l~ m8 oi~ mammalian
GnR~ in ot he~ ~rQrt~ xate~, ~o~ Qxample ~i h and birds.
The ~llow in,g i~ a table in which the primary ~tru~u~es o~
GnE~Hs ~ol at~d from the brain~ of differ~nt vært~brat6!s i8
d~picted .
I
~l ~G~ r~ -8 ~T~
~alm~s~ p~ rp~ ~T~ ly~H
~1~3e~n ~ ~ s~7:s~ ~d ~ ~ Ty~
Gn~ and i~l~ an~logues ~timulate th~ ~el~aYe o~ pitul~ary
gonadotro~ ~ hor~c~n~s and 'ch~e in turn activ~te
~meto~en~ !55i 5 and ~teroid~gRn~ tha gon~. Incre~ed
r~ as~ oi g Dnadotropin hormoneE~ ha~ b~n ~hown ~o ~t~mula~
the rep~o ~u~ ,tiv~ pro~e~s ia~ animal~ and hen~ m~thods o~
inerea~in~ t I th~ ~elease o;E gon~dotro~n hor~aones ~a:s
~ppl1cn'cl-¦n in ~h- edical and agriauleU~al sphorqe.
:
.
21129ql8
GnRH i3 ~ ~il4 presen~ in, and ha~ e~f~3ct3 on, the pla<:enta,
~ntr~1 n~ ~rl )us o~yst~m, adren~1~, gonads, ~nd ma~ary tls~u~
in m~m~na1~ I a nd hence anR~ anA1ogues a1r30 h~v~ pot~ntlal in
rnedica1 ~ nd I vet~rin;lry ~pp1ications ln r~gu1ating the~e
tissu~s
It i~ th u~ ¦ de~ ble that fuxther and mox~a e~ective
analogue~ o~ GnRX b~3 found to increa~ or ~ll3cre~se ~ne
re1ease o~ g~nadotropin hormon~s in v~Lriou~ diffor~nt cl~6e~
of vert~b~ ~at~s.
~ttempt~ ~ la~ al~o been made to incrsas~ th~ gonadotropin
rel~!l3lng ~IC~ivitiQ'I3 0~ th~ various p~cimary v6!rte~rate GnI?Hs
in mamma~ .8 ! by incorpor~ting D-amin~ ~cid~ ~nto th~ir
~tru~u~
~n ~n ~rt lo~ in Endocrinolo~y, volume 12~, ,pag~o 18~0 tD
1840, 1~8~ I b~ J ~ X~ng et ~1 there ~ d~sclo~ed ~ ch~ erl~
~na~ o~ue ~ n~tural ly occurring ~erte~rate gonadotrop~n
~sleasing ho~one, pGlu-His-Trp-~er-H$s-~-Tyr ~u-Ax~ro-
aly.NH~.
Y ~1 ~
A~c:ording tol one ~3pect. of ~he invention ~her~ ~ pr~-vided a
peptide ~ ~hi~h i~ ~ chlmaeric an~1O~ue o~ ~ n~tux~lly
~ccu~ring ~ebr~ e gon~dotropin releaR~ng honmone o~ the
~ormula;
pG1u~ s-r ~rp--~r--A~B-C-D-P~o-E
wherein:
.
2029~118 ~
I i
~ t ~ H~ r ~yr
B ls D~Ar , ~-Ly~3 , D-Trp , D~Ala , D-Tyr , D N~l ~ 2 ), or D-tert-
but-Ser,
1~ ~eu ~ T~p ~
~ i~ Ar~, G~ rr, or IJeu: and
E is ~ly~l ~H2~ D-Al~.NH2, NEt or aza-Gly,
~ith the rgviEio that wh~n A ~ H~, C i~ L~u, D i~: Arg and
E i~ Cly.l 1~2ltl~ B i~ not I) ~r.
~y "chin~ r~c analogue~ i mean~ a vertel~rate gonadotropin
~elea~n~ hormone whereln one or more o~ th~ a~oino acid3 at
~o~itlon~ 5 ~ 7 or 8 are eubstitu~e~ with a di~erent am~.no
acid occ~ rr~ng in the corre~pondinq po~ition in anotht3r
natur~lly ~ r1ng vQrte~rat~ ~onad~t~opln rel~a~ing
hormone . , .
Pr~f~r~Rd pq¦pt~d~ accordin~ to ~h~ inv~ntion inc~lud~:
~Glu ~ Hi ~ Trp - S~r ~ H~ - D - Trp ~ ~u ~ A~g - Pro -
~ly N~2
' ~ '
pGlu ~^ Hl I ~rrp - Ser - Tyr ~ D - Trp ~ Trp - Arg - Pro -
Gly NH2
II
~Glu - ~i ~ Trp - ~er - Hi~ Trp - Trp - A~g ~ Pro -
G;ly NH2
,
2~29018 -1
1 ~ I
III
p~ H1~i - l~rP -~ ~er - l~yr - D - Trp - ~rp - Tyr - Pro
Gly NH2
~V
p~lu - Ri~ - 'rrp - Ser - q~rr - D - Ar~ - ~rp - q~y~ - Pro -
G~y NH2
`, V
pS~lu ~- H~ -I ~rp ~ Ser - Tyr - D - ~y~ - ~rrp - ~ryr - Pro -
Gly ~2
~X
palu ~ i - Trp - S~r ~ ~yr ~ y~ ~ Trp - Gln - Pro -
Gly NH2
I VII
pGlU ~i~ -I ~rp - Ser~ - ~yr - D ~ys 'rrp - L~U - Pro -
~l~r ~z l
I VI I
pGlU - H1E I ~ ~rP - ser Tyr - ~ - L~ Trp - Arg ~ Pro -
t;~ly NH2
I IX
pGlu ~ H~ Trp ~ Ser - Hi~i - D Arg - Trp - ~rq - P~o - :
aly ~H2
.
~n anoth~ r Lspe~t of ~he invention a pRp~id~ whi~h l~i ~n
~nalogue of ~ a vertebral:e gonadotrop~n rel~a iing hormon~3
having onl ~ qP the ~ollowirlg ~o~nulae i~ pro~idQd:
. . .
.
2029018 11
s I ~
P~31U ~ }~1~ ~ IT~ e~ - ~Y~ A1a ~ I~U - ~1n - Pro -
G1Y ~H2
I XIII
P~:1U ~ ~ S~r - Tyr - D ~ TrP - L~U - G~ n - PrO
N} :~
I XIV
,
p:;lu - Hi~ -` Trp - S~r - ~yr ~ D - P~rg - 'rrP - ~U - PrO -
Gl y NH2
~ XV
pGlU - H1L I q!rp - ser - H$5; ~ Trp ~ ~rrp - ryr - Pro -
~ly - M~Sz l
! XVI
pt:lu - Hi~ I ~ Trp - Ser - H1~ - D - Ly - ~ Tyr - P~O -
~ly N~lz l
XVII
~h~ p~p~i tleh o~ the învention have a~onlst propertie~
~lat~v~ ~o ~ a naturally occurrin~ vert~br~ gona~otr~pln
~le~ing h~ono .
In yet anc Ith-r a~p~t o~ the lnventl~ ther~ rovided th~
u~e o~ ~ . ~Qp~ld~ describe~ a~ov~ a~ a r~ul~tor ~or
~gul~tins I '¢h~ r0~ase o~ hormone~ in ve~ct~brAt~ ~nd ln
~arti~ul~ ^ ~h~ uE~e ~ a p~ptide of ~ny 0~3 th~ fe~x~ulae I~,
VI, ~7XII, I~ XVI or XVII ~ regulate ths~ r~lea~e Q~e
horr~or~e~ Ln !chlsk2ns.
1, '-., I 2029bl8 ~
Peptlde~ c ~ ~ho invention are dec: ap~ptidee havin~ A ~qUenae
c~X ten ~ no' ~c:ids. The ~ir~t amino acid in the sQquenc~
o~upie~ ~ o~l~lon one, the ~eoond zlmino acid in tho ~e~u~3nc~
oac1uple~ ~ ~ition two and ~imilarly for ~mino ac:lds 3 to 10 .
Ihe lnv~n :l~n provides ~ new ~et of ~n~H sn~logu~ wlt~
altered ac tlvity compar~d Wi~h native mammallan GnRH ~f ~oth
m~mal~n an~ non~amn~ n specie~. Some o~ the ~n~logue~
o~ 'thel in~ ron t~ on are chim~eric GnRH analogue6~ in which th~3
amino ~c:11 I ~ ,t one of the variable po~iti~ Alor1g tS~ ten
amino ~ d ~s~u~nc:e, nam~ly po~ition~ 5, 7 or 8, 1~
~ub6~itu~ Id w~th a di~feren'c amino acid whlch ~ac~urs in tne
~ame posi ~ic ~n in another ~ertebrate ~:n~ rhe ¢himaeric
~nalogue~s ~ the inventic~n ~ur~he~ ina;:rpor~e a D~ no acsid
at po~iti~ ~n 6 ~n ~h~ir amlno acid ~equenel ~he chima~ic
an~logue~ ~nay al~o h~ ~ui~stitut3d a~ the c:arboxy'l terminu~
~th N-~t lyl~ide (NE'c) o~r D~Ala.NH2 or zlz~-Gly ~o 6~nhancs
~l~eix ~c:t vi~y~ ~rhese are represe~ed in Table 'I .
~h~ inv~n :lon ~l~o provid~ a number o~ rlovel analo~ue~ o~
~atu~lly oc Icurr~ng gonadotropirl relea~lng ho~n~n~ wh~Ch
lnaluds ~ 3- u~$r~o a~:~ d in th2ir amino a~id s~ anc:~ ~ On~
~artic~ . ~n~logue o~ the ~ nvent~on h~ A ~ur~h~r
~ubsti~ut o~ I ~n tnat Gly. N}{z, WhiCh oc:~u~io~ po~3ition
along t~l I , ~ino ~cld ~ uence, i~ sub8tituted w1 th
~thyl~mld a ~NEt). ~rh~k ~re ~epr~nted iJI Tabl~
To delin~ st~ the func~ional lmpc~r~anco o~ ~he var~ ar~ am~n~
~cidQ in ~ ~o~ltions 5, 7 and ~, ~himaeric analogue~ ~c:cording
to the ln~ en~on ~re test6!d ~or luteinizirs~ hor~one tLdl) ~n~
2029018
.
~olllcl~ sti~ulating hor~one (F~) rRleasing a~t~v~tie~ a~)d
~or r~ l~or bindln~ acti~lty .t~ rat, she~p and chlolcen
pltult~r~ B8 ~
~ho pre~ç ntl Btudy ha~ vestigated ~he actiYl~ias of Gnl~
~nalo~ i4co~po~a~ing ~om~ination~; of the substi~ution~:
~1~5, Trpr I~U8, Gln~ and Tyra ~n n~amm~lian Gn~H ~o~o~her wi~h
th~ ~nc:o~ po~ation o~ 4 ~ Trp4, D-Ly~ ~nd D-~rg~ on r~cepto~
~indlns~ ~ ~d ¦gonadotropin hormone release ln ~he~p, rat ~nl3
~hlcken p. Lt~lltari~
tn th~3 lto 1~,rin~ ~xampl~ tho ~cruo~r~ peptld~
lndiaated 1~ ~ o~ ng an~ substitution ~hat it h~ in lts
amino acl~ I ~ ~quon~ relative ~o ~he amino ~ s~q~ence o~
n~cive ~ 1~ ~ n gonadotropin rele~ing ~ormon4. The
pO8itl0h, in ~he araino ac~d ~equenae, o~ ~he ~ul~titu~ion i#
indi~ted by a nu~nber ~n ~upersaript. I~ no ~lbstitution i~
ind~catçd il 1 arly parti~ular positi~n, ~u~h poE~ition i~
occupled I 1y l~he amino a~id, pre~nt ~n ~h~t po~ on, ~n
r~ativ~ a~ n~lian gonadotropin relea~ng hormon~. Therefo~e,
~c~r ex~mpl ~ Hi~5 ~ T~p~]GnR~ repxe~nts ~n ~nalogue ~f
aalian ~t~dot~opin relea:3ing hormone with ~h~ o a~d
tll~tidins ~n 1pos~tion 5 and the amino acid D T~yptophan i~
pD~ on
~ i
z~8
E~
T~ pcp~ld bl; ! wer~a prepareâ wlth an au~m~ed pxo~r~ on
Bcck=an sylt~ 990 pept~ e ~ynthes1zer. Prot-cted peptlde~
. .
2D291118
: .
. 1 1.
~rere as~ mblec~ ~y Mtand~rd ~ol id-pha~ method~l~y on p~^
~eth~rlb~n hydrylamine~l~ d~vinyl~enzene-styr~r~e copolymer
~esin ~3 , ~ 4) utilising ~0% triflu~roacot~c ~aid in
dlchlorom ~ l~ne for N-termina~ deprot~ctit)n and
d~ loh~ y] ~rbodiim~e for the coupling of tha ~ollow~rlg
~o~-px~'c0 t~ Id amino acids : Gly, Pro, Arg(Tos), ~ln~Xan),
~yr(C12-B 1 , Ly~(C1-~), 1,Qu.H20, I'rp, D-Trp~ Gl~l(OBZl),
Ni~To~) ~ ~er~zl~ a~ w~ll a~ z~pGlu.
~eptide~ Jele ~leaved ~rom the re~in ~nd d~prot~ct~d uslng 1~
~ni~ole 1 ~ ~distilled hydrogen ~luoride ~S)~ Th~ ~ptid~-
~thylamid wa~ cleaved with red~ st~ lled 4thylamins and
depr~tect d wlth hydrogen fluo~ide . ~h~ p~ptld~3~ w~r~
puri~ied o ~x~er 'chan ~6% homogeneity ~ba~ on the r~tio
o~ th~ i te~ Jrated area under the main peak v~. ~he tot~l
lntsgr~t~ ~rea recorded at Z10 nm) b~ p~QparatlvQ 18
r~v~r~ed- h~ ~o hlgh per~or~ance llquid c:hro~a'c~r~phy ~13i~g
~ o . 25M tr ~ethylammonium pho~phate pH ~, ~ 5/~oe~onl~rll~
bu~fe~ ~e~ wlth de~alting 1 n a 0.1% trl~luoroi3c~a~ic
~oid/i~cet nltr~l~. structur2Q o~ peptide~ w~3r~ con~lrmed l~y
~ou~in~ ~ ~i~o ~id analy~is.
~ ~
~nt~rior i~ult~le~ ~er~ ~emo~red from chi~k~n ho~d~ ~old~n
Grovç~ Pou t~y Cc~., c~pe ~own) w~ h h~d be~n k~pt vn ic:e and
wa~h~d w tn d~in~ectar~t ( ' Hibltan~ l; 0 . 596 chlorh~x~dlne
~luconate iin 80~ methanol), wl~hin ~ h o~ d~h. rrhe
pitultarl ~ ~wç~re collected into Dul~eoco~ Modi~i~d Eagl~
~ledlum (t; b~o, G~and Island, NY) buff2r2c~ wi~h 20 m~S HEP}~S
~pH 7 4) t ¦roo~ ~empera'cure, minoed w~h ~ or ~l~de, and
dige~e~ wi~h c:ollagenas~ ~or 1 h at 37~ whilQ undergoing
~ ,91~ 8
9 1
,
c3ontinuou agitation by a ~lowly r~a~ing s~ er. Th~
~oll~g~na a lolution con~l6ted e~f O.g9~ collagenns~ ~W~v) ~5$
~J/m~, ~ort hl~gt~n BiochRmic:al Corp., ~reehold, NJ) and l~mgfl
deoxyrlbo ual~a~e ~M~1~3s ~abox~tories, ~lka~rdt, XN) in HEPES-
BS~ bu~ containing (~ NaCl 137t KC1 5, Na2~P04 0.7,
~EPE~ 25 p~ 7.2), CaCl2 0.3~, glucose 10 ~n~ (w/v) BAS
tfat~y-a~: d j~Xee, Pen~ex ~raction V, Miles Labora~c:ries). At
10-min 1 ~Fval~ th~ cell~ wer~ triturat~d Wi~h ~ 5 ml
p$p~t~e. The c~ uspen~ion was ~nt~l~ug2d ~wia~ ~ 500g
~or S min and t~l~ pellet resuspende:9 each time irl ~u~er A
~NaCl 140; ~M~! K~l 4mM, ll1azHPO4 1.4 mM, glu::ose 8.3 mM, HEP~S
20 mM ~p~ 7J4) and ph~nol red 6 mg/l] to which 0.5 mM EDTA
antl o. 39~ w~lv) l~SA wa~ ad~ed. The c:ell su~penslon wa~ th~n
~ lt~r~d ~rough nylon gauze prlor ~o ~ilution lnto
Dulbeaco~ Y odlfi~d E~gle~ Medium with 10% foetal cal~ nerum
~:ib~o), P~ ni~il lln ~60 m~ tr~ptomycin tlOO mg/l),
~omycln (1 ~/1) and amphoteri~:in B (;~0 Rlg/l) ~nd
di~pen~ed ~ryto pla~tic tiE~SU~ culture well~ ~Falcon, oxnar~,
~A) at a e~ y of 1, 3 pituitary equivalant~ p~r well (
w~ll plat ~1. one pituitary equi~lent reprQ~nta 5.0 to 5.7
105 c~l~ . I T)~e c~ell~ we~e cultur~d ~t 3~t~ 5% C~2 for 24
h, after whiah the medil~m wa~ replacec~ w~th ~r6~
~mphoteri i1 B-free medium. C:ulture wa~ th~n cont~n~ed for
fu~th~r ~4 h.
Prlor to ti~nulation, the cells wer~ w~shed twia~ wlth bu~ar
A ~ontain n~ q CaCl~ ancl ~ SA an~ pre-incubat3d twla~
~or 5 min 1~ the ~;~m~ buf~er. Stiml~la~ion~ wer~ c~rried ou~
~or 30 ~1 ~t ~7C in bu~er A with 1 m~ C~lC12 and 0~1~ BSA.
rhe m~diu m Iw~8 c011~3ct.~d, ~en'crifuged and atored z~t -~0C
un~11 r~d olmn~unoa~:~ay o~ chick~r LH. Mam~Llar~ Gin~H wz~
included nl ~11 bioa:3says a~ a r~ference stand~rd ~or tl~
C~Oll-p~flti rile actlvity o~ the other C;nRH ~nalogu~
20%9~8
~1~ = =~D~ Y ~q;~ bl5;~
~he biol~ ~ l ac:~ivlty Of ~he synthetia GnRH~ WaE~ a~se~e~
usitlg cul turad ~h~ep anterior pltui~ry (~ 8 a~ prQviou~ly
d~cxi~ed ( 1, 2 ) . Ar~terior pituitari~ we~e ~ e~ted ~ra~
~l~eep wlt hln 30 m~n of ~laughte~, diced ~nd in~lbated ln
collagena ~ olu~ion ~ d~arib~d fo~ c:hick~n pituit~ries.
~he cell~ ~ re re~uspended in Minimum ER~ntial Medium -
~aglQ ¢MJ~ M) I ~Wlth Hank's s~lts) (Gi~oo, P~i~lo~, Saotland)
~ntainln~ ~ 110~ ~o~tal calf s~rum (Gibco) ~nd ~ntiblotic~ and
cultured lq5 - .105/dish) ~t 37C ih 5~6 C~2 for 4 or 5 day~,
~qith ~he ~ ~lum b~ing replaaed a~ter 3 days~
~rioX to ~ ul~tion th~ cell3 wer~ washed tw~c~ w~th MEM
a~nta~nir~ ~ ~0 ~6 ~oetal ~al~ BerUm and 4 times w~th serum-
~ree M;~;M I Pqpti:le stimulations (in t~iplica~) wer~
~onduct~ ov~r 2 h in 1 ml ~erum-~ree MEM at 37~ ln 5% ~0;~.
gO0 1 o~ n~sdlum ~rom each te~ was collect~d irlto tuh~
aontalnlnt ~ 100 1 ~ H~PES~SA bu~er, cen~xlfug~d an~ ~tor~d
at -20C u~ ~till radiol~munoa~say. Mammalian Gnl~H wa~ ~ncluded
in all b~c a~y~ a~ ~ referen¢~ ~tanda~d ~or the co~p~ativ~
~otivity e Ip ~hs o~h~r Gn~g ~n~loglle~.
Ov~ h~ L~ r~diaimmuno~says (~A) w~re pe~Po~m~d with
dupl~cate aliqu~t~ o~ conditioned mediu~ tor dllu~ion~
therao~) ~ ro~ stimulAtion ~xperiment:3 ac pr~riou~y ~e~cr1~sd
~), exce~ t ~h~t t~e ~econd ~ntibody wa2~ l~nXed to a~llulo~q:
(~ac~C~l I~70, Wellc:ome P.e~s~ents ~i~nited, seckellham~
Engl~nd). ~riPi~d ovin~ ER- 105~ , gi~t ~om L.~
E~eichert) ~- iodln~ted by the chlor~mine-T m~thod ~n~ ~ree
2029018
! l
I , 11
: I
lodid~ ~i l~r~mov~d on a oellulo~e CF 11 ~Whatman Inc.,
ton, NJ~ colu~n. A ~tandard curve was ~nerated wi~h
~nlabelle ~ ~heep ~H ~NIH~L~-S18) atld anti~um ~DN 15 (gift
fro~ G.D. Ni~hder~ Colorado State Univer~lty) whlch had
t be~n rai~ ~d ~gain~t ~heep LH.
! Chi~ke" L H ~a~ yed a~ previously de~crib~d ~7), RX~ept
that ~he ~e~ond antibody wa~ linXed to cell~lo8~ tSac-
~D70).
ov~ne FSH ~Idioi~munoassay~ employed anti-oFSH-l anti~arum
(~lnal d~ .ut!ion 1:1~0,000), radioiodlnated ~SH~I-l and oFSH-
~P-l as ~ tandard. ~11 rea~ents were ~uppli~d ~y S. ~t~
tnat~on~l ~r~on~ and Pituitary Progr~m, NID~K) a~ utili~ed
in~tru~ :~d. Separation o~ bound ~nd rree 125I-oF~ w~
~chieved i ~tp ~ ~e~on~ antibody conjugated to ~allulo~e tSa4-
70) ~11 ao~pArative studi~s b2tw~n ~n~l~gues w~e
ormed i~ the ~me radiolmmunoassay ~nd th~ lntra~a-~say
coe~iclQl Itlof varlation f~r ~ad~oi~munoas~ys wer~ : 13.7
(chi~en ~ ~1, 4~% ~she~p LH~, 8.3% ~h~sp F~H).
~D~Q~ ~
~h~ ~$ndil ~gjof ~ynthetic vertebr~e gonado~ropln r~le~ing
h~rmone~ ~d An~logues thereof to ~onadotr~in ~eleasing
hormon~ rl ~cqptors in rat ~ntsrior pituita~y m~m~rane~ WA~
i~va~t~g~t ~d¦as previously de~xib~d (l, 2) . In ~he pr~s~nt
~tudy a ~h~p p~ ltary gonado~opin r~ 3~ ng hormos~
~eaept~r ~ ~n~ln~ ay wa~ also dev~loped,
~nterior ~ lltlultarie6 fro~ ad~lt mal~ rat~ ~Long-Ev~n~) o~
a~trated ad~l~ m~e ~heep were homoge~i~e~ in.i~e-cold lO m~
Tri~-HCl b ~fr~r, pH 7.4, ~ontAlning 1 mM dith~t~itol, 1~0
.
, ,. I . l
1~
snM E~A nd O . ~ 9s BSA. The rat pituitz~xy homogenatQ ~a
cent:~iPuc edl at 1~, Ooo x g ~or 30 min ~ 4C to yield a
~nam~ran~ ellet whlch wa~ re~u~pendQd in the ~ri~l{cl bu~fer.
~he ~hoeE pltuitary homog~natQ, however, wa~ c~ntrifug~d ~t
100 x g 5 ¦ rain, 4C) and the ~;up~rnat~nt rec~ntri~uge~ at
3soO x c ~or 10 mln ~t 4C. Th~ r~3nultlng p~llet wa~
re~u~penc ed ¦ in the ~rl~-H~l bu~er. 'rh~ b~ ndlng aE~8~y
compri~q ¦ incu~tiDn of the membr~n~ prepar~tion tO.16 rat ox
o,O~ ~he ~ p~ultary equlvalants/tu~ o,ooo cpm 125I-[~-
~la6-N M~-L~u7, Pro9-NHEt~;nRH ~radioiod~nated by a
m~dl~lcat on ~ the ¢hloraluine-T method ~8) - l3peclPic
~ctivlty yithe se~-di~placement method, lOOO l~oO ~i/ g]
~nd incr~ ~ilng ~on~ent~a~ion~ o~ the te~t p~3ptide~ for ~o min
~t 4~C~ hf a~G~y was pexfo~ed in gla~ te~t-tUb~B ~ 12 X
~5 m~) wl h a tot~ olum~ o~ 0.5 ml lo mM Tx~ lCl ~u~er pH
7 . 4 cont~ ning 1 m~l di~h~ othreitol, 1 mM EDTA and 0.1% BSA.
~h~ lncub ~on w~ t~rminat:ed ~y dilu~ion with 3 ml ic~ co~d
phospha~ bu~f~red salln~, pH 7~ 4, containing 1~ BSA (PB~-
BSA), ~o~, o~ed ~y ~m~ecliate vacuum ~ltrat10n ~chrou~h ~las~-
~ibre ~11 te~ tGF~; Wh~man) pre~ksd ~n PE~S-~SA. The
~ilt~r~ w rq wash~d twice with 3 ml ps~s~E~sA, and the r~ta~ned
~dloac'ci ~1~y ~ount~d. Non-speç~ic bin~lng w~ d~rmln~ad
ln ~che pr se~e c:~ lo 6 M unlab~lle~ Ala6, N ~e~ u7, Pro9-
~IEt~n~H nd 3ub~tracte~ ~rom all samples (~ 1n r~ and 269c
~n sheep3
l~ç~lu ~L~
~1)50 valu fi~ ( concentr~ ns requir~d Por h~lf maxim~
g~na~Otro] )ir~ r~l~a~e in bioa~ays or ~or half m~xim~l
inhibi~o v~ 1~5I~ M~L~u7, Pro~-N~t~GnRH b~ne~lng) o~
¢nE~H ana ogues werc~ determih~3~ uslng a ~our-pa~me~3r,
nonlinear ~q~ve ~itting pro~ram (All~lt), ~oralng ~urv~ to
,
.
20290~8
1~
1~ 13
l l I
~hare ~s ~al and m~ximal value~. ~ value~ wexe ~lculat~d
~rom tho ~50s oy ~hR method o~ Ch~3ng and Pru~ f ~ 9 ) .
stand~rd e~or~ of ~he mean were dRrived from A~ t
~i~ata~ . I Rel~ e potenci~3s wer~ calcul~t~d ~ro~ EO50 and
~d value~ ~ tive to those ~or mammal.tan anE~H. All v~lula8
prQsen~e~ ~r~ th~ mean~ o~ severdl independent experimentf~.
~1~
All o~ :h~ an~o~ues tested had enh~nc~d L~r~3lea~ing
ivity inl c:hlcken pituitar~ ll cultures when comp~r~d
~rith ~na~nm ~llan GnR~. sin~e mammalian Gn~ and chicken Gn~H
in (thQ rel~slng n~rmone ln the chi~k~n h~p~halamu~ have
ilar a :tivi~ie~, ~he analogue~ are ~ mor~ ~tive than
chlcken G nR~ I. Analogues IV, VI, VIII, IX, XV, XVI, ~nd
XVII had r~ative a~tivi~1e8 500~6 or lligh~r compared with
~aal~an ~,~RX an~ most o~ ~he~se ~r~ char~a~r1sQd by havin~
~-amino ~L~ ~id~ p7-Tyr8 ~ubstitution~. In ~h~ep pitu~t~ry c~ll
C:UltUrss, ~L~Llogue~ I, II, XIII and X~v di~pl~L~ enh~nc~d ~-
~e lea~ ing adtivlt~ .
X~eceptor ~ ~inidlng potency of ~:he ~;n~H ohi~eri~ ~Lnalc)gu~ in
~heep arl~ r~t pltuL~ta~y wa~ in general aorre!laLt~a wi1:.h, I~-
~elea~ g p~ter~ in the ~heep pituitary, howeve~,two
classe~ o~ ' ahalo~ueLs emerg~d in wh$s:h l:kL~ r~l~tive ~ynituda
0~ the~ 1 LC5i~f~t~ d~vi~ted signlf~c~antly:
~ Hi ~5~ rp6~ç~nx~ exibi~d highe~r r~ ptor 3: indlng
~tlvlty 1 .hal n I~l-rel~a~in~ act~vl~y. (~ble ~)
2 ~ h~ I analogue~; wi~h a ~rp7-Tyr~ su;:~stitut1on
character ~tically displayed I~-r~ in~ ~Lct~it~ which i~
1 - I 2~9~8 ~1
14
hlgh~r thar ~ ~Lei~ r~l~tive SeCeptor bind~n~ potencie~ ~TablQ
I) .
cortain o~ t~ 1~ an~lo~u~, as ln~ia~ed in T~bl~s 1 and ~,
~howsd pot~ ~nc i0~ f~r FSH rell3ase whiah w~re ~ ar to their
pot~n~le~ ~o ~ L~i rel-al:~, n t c ~heep pltulti~ry.
. . .
,
l~
..
a~~
1. Mill r,¦ R.P., and King, J.~. ~1983) ~g~l~ 113,
1~64 1369
. Mill ~, R.P., ~llkon, ~.C.d~L., Foll~e, B.K., and }~lng,
J.A. ~ i~ 119, 224-~31
I .
3, stew r~ , J, ~., and Young, J . D . ~ 4 ) in ~l~
~, 2nd ~d1~ion~ pp. 55- 5~, Pisrce
~he~ ~ 1 Co ., ~ockf ord , I l l ino ~ ~
4 . ~qerr ~i~eld, R. B. ~ 63~ h~m. SQ~ 5, 2149-
21S4
. ~ivl~ IX/I J., Ka1~er, R., and Galy~n~ 78)
aiQ~ ~ 17, lg27-193B
ll r/ R.P., ~nd Aehnelt, . ~1977) ~ng~ ~ 101,
76~- 6~1
. Poll ~ , ~.X., Scane~, ~.G., and cur~nin~ham, ~ 72)
151~ ~, 52, 359-378
. Nett ~.M., ahd AdamM, T.E. (1~77) ~D~D~CQ~
101, 13 5~ 4
g. Chen ~, ¦Y.c. ar~d Prusof~, W.H. (1973) ~9~4L~
22, 0~9-3108
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