ISOLATION, CHARACTERIZATION ANDS~RUCTURALSTUDmS OF THE

.I.

"-

:y "

Macrozoarces amer

icanus '.~

:' ' " . 'BY' ," '

~~

ISOLATION, CHARACTERIZATION ANDS~RUCTURALSTUDmS OF THE

ANT~!tEEZE

POLy?EPTIDES

FROM e>CEAN' POUT

....:....©

XlAOMAO

LI, 'B.SC .

/

^{.. ..}

A TBESIS SUBMITTED IN PARTIAL FULFILMENT OF THE- REQUIREMENTS FOR THE DECIt£E OF'

<.JdASTER OF SCIENCE1

/

. DEPARTMENTOF-BIOCHEMISTj!Y MEMORIAL UNIVERSITY Of:.NI',WFOtMDLAND

ST,.JOHN'S, NEWFOUNDLAND .MARCH· 108&

I

~

^..

\

. \

perm Ls s tdn has been,granted to .the National"Library of Canada"to'mi c r o fi 1m th"is thesis:and' to le nd or sell copiesof the film •.

~The'">-a,uthor\ (copyright o",:ner)· has; res e rve d 0 t-h e r pubLf ce e Lo n rights, and neither 'the t·besis·nor· extensive extracts' from it may be printed or ot he r wi s e repr cduced without hls/her wr i t ten permission.

L'autorisatio n a~t~ accord~e"

:a La.Biblioth~que na.t lonale

~~tt;a~~g:e ~~ 'd:i~~~~;;m~~

~~1~~ndr1\les;exemp~aires du• ...L· au~eur (ti t u l a ire du droit'

d' auteur) sa raee eve le s autres.drotts de publication,' n L ta enaee n I de ,l o ng s extrait s d e cel le-ci rre dolvent.·~ t r e imprimtls au autrement reproduits sans son' _. autorisationflcd.te'.

ISBN 9-315-31044_8

-..

) - ^-

.j.

ABSTRACT

.Antifreezep~lypePtideS'tAFPj^we're'se,P3ral~~u('('l'$.~1ully^{lrom tht'SNI;'11lIIr} OCt'aDp.outM(l..,."o~oCln.c.1Im..CriCG"U~^usingsePh3deX~~7':;gel·rillr sl ion.QAE-

. .- • . -,.J.I .

Sepbadex and SP ·S ephadex icc -excbecge chromate aph}' and re\'('r~n.'il'

", " )

HPLC;' -.;r . /

.

..:.~ .

Itwas found

"> .:" .

lbat

'-.

the.ocean

-'. - -

pou~

.

^...-,,\FPcOd;;istsof a

.. .'-

multipie

': .

ra~ily

, , ' ,'

^{:pr at}

".

I;ast.twelve active components.ornearlyident icalsizl(ab?~,t..6.000 daltons] , .whichcan

be _.

c1assil'ied_. -

into

two

separa~e

^groups

.

^..

n~med QAE: ~Dd' SP. .

^{on-.~ -~}

~h>

^.

r,

/

i

b~isattheir aminoa~id compositions~^andtheitbebevieer-un.·loD.eX'cbang~·-t ...

chromato gra phy.. .

. . ... J .

A partialamino-acifsequenc,e.o rSt>~l'A,orle or themajorocean pout-Afro

. . /

. . .

. . .

"wasest&blishe~up to,41~esidues~~()ndts"chYi1mt:ryt>ti-c-~-epli~eS\iSin-gaprolt'i-n-.- -:-.:-

~equenator. ^SP,.hA~'''ll.~omparedwith.tw~othe r majorcompo nents, namt'ly .

.'

. . .. . ^{' ,, '} - , '

^,

SP· l· B and SP- I-C ,bY'trypticaI dC'hymotry pll~~eptldemeppmg andarm nuari d analysesTh.y

,~o.~j(,~r.'turnl

similaritieswith minordillerenees .•

AcDNAcodin g fot.tbebiosynth eticprecursor of.c neof the ocft n pout.Afp.-l-

. ~:s is~lated ~ d' pur'iried.-trom-

the plasmid

pB~,

^r22

in ,~: a~,

^{,HB IOI .}

1 <

^was'

then'c1~nedinto'phage MI3 mpS,Tbt'~lle.nee

o r

the<'D>1Aw~d~termined^by:

~ . ... . ' , I

't'bedideoxychain.termi~a~ion ~elhod.Ithaduntra~.~lat\d,~egions^at,~tth^3'an.d ~.

5',an~'ac~ingregion for astgn!LP,tp1ide:'(',ontainlng 22amino'a~d'r(>~iclue~'and.

a maturepolypept idecontaining65-residu(' s. The s·equenrt'/

Or

the mature~

poly~ePtide'mat<:hed

^tbeprotein data Irom

bot~ SP.l~Band~P~~.(,.

^Th;'

'1.~ul·l!i

- ."

·ii-

hom bott.'proteincbe~istr~and'mcleeular-biology'~bo\\'t'd^{thatSP- I·'-\,SP-j-D} and

sf-I-e

have very .similaramino acid eompositjons

and-::;u~n('('s ~'ilh onl~"

.rinoj."·"~irrerences. For~xample,SP-I-Acontains lie and AJa at"pos:itions53and 62,wbile".botb >51'-1-8and SP-I-<?conta in respectivelyLeu andValat thesame positioli~.^{'S.P. l.:C-blUian},additi?~al,?)Y^{at its}C:~>ter,~~rialcompared to both~P-l-^..I

.',:,,-~AandSP.i-li '". ^>

The ami noacid>analysesshowedthatSP-l>~A.^SP-I-BandS!'-l·C>contain lall i-teentyp es ora~iDo^{acids'with_mod est}"conte~ts^{or Ala}aDd "l1~-'halr.('ystin('>

Thereisno obviousrepea tin g

str~cttire

^{,in ocean}

p~ut

^AFP.These. resultsconfirm that.oculipoutAFP representanewtype01fishantirre~2eprot eins-

OceanpOut,AFP Iromindivid ual fishcaught inbothwinter"and summer

~-~----':"--~-hom:-N!iwrouDdlaDd;

and-in-wintermonths

rrom -~eW::Bru-nswick-\\'ere-an-arrl)"le,;d~ol<r'

^{- - -}

reverse pbase HPLC>.T hereslllt~didnotshow"a~li.rSig-nm'faiil""~ll1at~·-'--

>~easOn.al.p'~I;'morphisms"

-iii- } ,

ACKNOWLEDGEMENT~

/C,\ributedtothe~uteeS5f,u lcompletionof_t,hi~in\ C;stigation,

. These,peopleare"Dr.C.· L.Hew'for his

ex~elle~~ ~upervi~~ dir~lilln

^and

vaht~ble

'assistatlce:,.Mr.,

~.

^B.Joshi',for peptide

~SeqUencin~

^and'

r(.a~ing

^and

·.correctiog this manu.,.cript;Dr..M..H.Kao .foranti~reezeactivuymeasurement s;

· \. - \ I "

Mr.K.Y.Trinh,Dr,N.,C,Wang andall other membersin the laboratoryfor

'discu~ions:' SU~geSt\ons '

andassisla noe; Dr G. L. FIl'lche r. Dr.

·\\5.

Anabthan'araya'~an

^{an'dDr.'W.s,Davidsontorhelpful}

di~cussion; a~~

^tbc

" I .. . '

membersofthedh'i~gfacilify,MarineSciencesResearch.Labcratoty,Memoria l Universityo( j'l ewf(;lUndland,for the colleetion.orexperiment al material.

- -Financialassistancewp.'i providedby!\'SERC'.Canada..

-- ^.

o

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^·iv-

CONTENTS

'

....

AllSTllACT ACXNOILEDGDa:KTS

COIl1EllTS

"

^{L!ST0'.TABLES}

j ^r

LInOF FIGURES

"

"' ,.

.dii

"

^.

~.

LIST OF ABBREVlAnOIfS zi

.. .ImoDOcnolf ...-..

1. pr opnt i.. atIlacro llloll cdarAFOP.aDdAFP 2. Dbtrlbut1~DudSt ruct ureof AFOP

I

3. Tb or l. .of th'~. cb&DlIlU ~fAFGPAct~~ltT 4. DlItrlbvtioD.and St rue't un ofAFP 5. S. . .ollalVa.rlatlolll atAFP a. Co ~tro1'of AFPBl0.JU,th. .l.

7. Po. lil hleYeehU1...of AFPAclt't'ltJ 8.'Itatl~Dalefor th. Ptl ••I1.~^Inl'_•.~t1iatio.D

"

. .

,

CHAPTER 1 ISDLATIDJIAlID ClWlACTERlZATIONOF OCEAH POUTItFP 11

1.1 WATDIALSAXD wEnlODS /

1.1.1 S~Pb"adu:'0-76~.:.f11tr.tloDCllrouto graphi '" 14 .1.1:2-'

QAE.S.pb&d.~s:

(A26) 1011.·. .'11.&.111.

C1lro_togra~bJ .

¹⁴

1.1.3 SP-S. pba.4u (C60) Io.-n:chug. chroaatogr apbi

'.. 't . . .

^.16

<,

1.1.4 Rinne Ph .... HPLC 16

·~ ^. \ '

" ' \

1.~. ~

~. 7

1.1.8,

1.2.2

WOhc~lar .e1lh~ ~ltllUtl~D

^'bJGtl-f lltrt:t.l 0DHPLC 10

.and Sp SPolyaeryluldlSlabG~lEhctro phoril l .

AmiDO Acid and N-TerminalADaIr'" 17

'" . I . . ' .

i7_._._' ..'

J.I. . .~t:.III.llt .of.~. nul"fltn uhof OClan PoutAFP

r;

Pl\rityof HnSerltl 18

1\

-RESULTS. .' .8'

.pr.. .

m !uc~ l " \

^{of'., ..}

,ulti~h

CompODlII,torUily 0

.

.'

"O~.anpout. -'-

¹⁸

\

cor~d.tl~1I.

^{of 0-76 AFP .ith}IDd1vldua;lCOIllPOllutlfroe 23'

oIOII.-:ISChuglChromatographyUdDIRnlnl Ph...HPLC

I

~TER

²

'S~UC'IURAL STUD~ES

^ON

$p~i':;;~SP-;:.l:'B-·

^AND

SP ~l-~

1.2.3 Ch~~actlrl' ~lC'of,OCIaD PoutAn 1.2.3 . 1 lIo1~cu1l.tlt1g lltE.tl~tloli

1.2.3 .2_Am1~O.ACld

^AIlalyle'

I

.. • _{_} "j.' _{. '}

_.

1.2.3.3 1.2.3.4 1.2.4

1.2.6

1.3

2.' 2.1.1

)

,H-T~.rall1.&lA:-illOAcidAnalf.1I

Antjfrlul!cthit,Mu su rement ,

COlD arhoD of OClanP~~t^Anfr oID. Newf oundlandaDd

N"'IBr~U.lct" _

COIDP.U 1l 01l of R,wfoua.dlud,Oc...n Pout-An111. SWIlIIIr eel

\ 1D

IlDter

I'

6UlIlIARY

I

WATEIUALS AJlD·MS:11IODS

i .

:.::.tt l01lof Ch,motrypti cP,ptld...of SP-l-Afor

\ D.g."'"'' St."" . J' .

23 23 31

....

"

"

..

..

SI

,r .,

....i·

3.1,0 O!1Intat101llof the II DNAby FigureH Format.ionTellt 3:.1.4 ,1 Lis ation of thl cDNAand IU3 mpH

.3,!}.4.2

T~ai~forutioll.

^{of B.Coli.JJ,(ui1.}by Lisatld '113 mpH 3.,1.6 Purificati OIlof Sinsll-.trande'dDNA e..DNA)fr olll thl Colollrll. .Tr&u f orml d Piaqu i . - i

CHAPTER 3 ,SEQUEHCI NO OF THEeDNACODING FORONEOF THE 0CEAJi pour AFP PJtF.CUiSORS

i

3.1' YATERIALSAH~,UETHODS

'3.1.1 Preparation of t~1~~a'lIIidDNA,with thlcDBA burt.

3:i1·.2 PU~iflcatioll^{of the'Pla.mi dDNA} 3!1.3

I'~J.atl0D.

^{ofthe In . .}

rt~d

eDNA Fragmlnt 3:1.'" Clolling_, oftt .cDNA Inurt

. . .

ill.t6 W13

.

~pH

RESULTS

61

62

62

62

~

62 62 68

~8

70

70

73 t

"

76

. "

77 77

77

"

7g "

/ !

.

.

Pep tidlWapptll.g &ll.d Amill.O·Ac id.lI1a l y"l01SP·l- A•. SP-1-B~d SP~1-CbyTrypli'n Digut-ioll. . P'lpUd.·Yappill.g of SP-i-A,SP-1- B AHDSP-l - C by Cb.r-otr~dllDigutioll.

Th.I~lydll~lg.•'t i oD.~D.sp-i-A

, .

Purity of Rlasn,tl

, i ! ,

IilqUille. .of.ChJlllotryp. trc Plpt idI . . f. .rolllSP-i-:A IWIlI1t.o~thl.nll1llolY.d.Il-.Disu.tio D oD;Sf-1-A' TrypticPiptidl w&pp11lg:and billoAcidAnalyl" of SP-i-A.SP- i-B &ll.dsr- r-c

Ch~trJPti1::P!lptidl Yappi il:S ofSP-1-A. SP-1-B'ud

SP-1-C .

2.1.2

2.2

2.2.2 2. 1.6

2.3; J'-2.~

,

2.2 .~

}l~...1

t -...

3.1.7 Pnl1alllarySCHU h!of t.h. II DHAbyShg h LUI 7~

Trackic&

3.1.8 DNAS.qlll~ C 1Di^bJD14.0 %}Ch"~11T.,..lut.1ollWIt-hod. 80 3.1 .8 .1·AIIIl,eal1.D.& Reac t.l n of tilePr ill eru4tb.l ••DNA 80

T••plat..

3.1.8 .2 ebb. Ezt.llld oD.bact-l oll' 3.1.8.3 -Cd

Ei'~~~80r"1'

3.1.'11 Pu1tyofR".'D~'

/ .

3.2 REStn.T&

/ .

a.

2.1 p;ur 1fl cat lon'of the Pla.1I1d DNA 3.2.2

I n.

^Sfze'

~t

thl ID.eru4cOMA

•.3.2.3! ".R.l1llt.ofcOifA S.qUe1lcing

3.3/

""",-",y

r

/

\

BO 02. 03 03'

8•

o .

.... ..

10.

1. AminoAcid Andy... ot the Oc...n.Pollt'AFP

--

,, ' 2 AntHr. .ZI Act1Yity01thlOCtaD. Pout AFP

:J

^{3 Amino}

^AC~d

ADaly. . , 01

t~e

ChJlllot r yptic Plptid..

• 01SP~1-A -. _ _ 0 •

-, 4 Cblll.pari.oA01 the Amho Acid SequeAce of CloAINO 77 aad the Partial Sequnc.. of Sf-i -A .

, . ---=---' , -

.. 6;- :~~~/Cld'An.ly...o~thl~eI1llolyti cPeptide.01

IS. Amino Acid ADaly,. . 01 thiTryptic-~ept.id .. \,

7,.·AU pml ntof the TrypticPeptid.. of 5P- 1-A, SP-1 ·B '

."aDdsF- 1·C .1th theAmino AcidSequlnce of CloDeNo.77

..

....

.

B·. The cDNA SequlDCI of .clonl·No.77 aDd It:1IMinoAcid

SeqUlnCl, I ' ; ~ ^{. . . , .}

9. ~e quellc,COlDparie611 of Cloll.lNo.77aDdNo.~9

iO. AmiDo AcidSeqlllllc" ·ofS~1Il1IAntiftu z.P~IJP.ptid"·

.'

\

»,

\

33

ss .57

-"

. ..

"

.• 7

•

^•100

' .

^{._ -' ,..-., . -j'"}

··ix·

LIST or ^FIGURES

^Jr,

. .

"';

, .

,.

'2"' 1, Chrou"'ography'of ...h. OClanPOll'"'1Dt.~;5erlim011.a 10

SIPht.~';.a-76,C;;~l-til...r.uoll. C.rtumn . .... _"

2~. ChrOU.t.Oll~aphY 'ot'

^0-7,6

m

^{Oil. •}

~-s.phadn-

^{'(A26) ;,.. :'.}

' ~i:~

COl,umn

I , ~ ~.

^{" " :}

~

^{.., •}

"1 '

.2bc,Chrou"'osraphy of QAE-ull.boU dcompoll.l~t.O,ll. aSP;Sip~adn ''2i/,

'(eso)Colm ,. ' .' .~. ,• " .

" " ,, '.' .0 ' .

3" ~~::...ographiot0-76An,011.'aRn',:~ !.:Pha.. C18'~LC

-

' ' ,

---:r-:

^{Chroma...ographyot 0-76'An:'.QAEA.}sp~i "SP-2.5P-3~ud

.SP~•.""'" Rei" r. . Pha.. C1B HP:Cc01~ , "

6, Eet1 "''''1on.&t ...hl Wolecular IlIlghteat - OF' u Pou'"AFP 011.' Gel-tll...;auoll.KPLC .

~'. -

37

e. polyacrJ.1uid.,G,"1Ele~~~OPh~r~H'ot

?cui " :

AFP.

~ .; j

Ch;outcigraphy of the l1ater

~.ra

from Wntoundland ed' H."Brunldclr:'Oceall. Pou...011.,'aS-'ph~u G-,7~.:C01WU

8, Chrou...ographyO~haterG-76,AFP1-r.om·~n(oundlaDd ,- 30"

• 'ind,HI" BrunelleII': Oc.o Pov.!-.o_lL-a.C-18C~l;=:'-

O. Chrolll;,&tograpAy'otthl'.iD...r:ud S,nmmlrSira trom 42"

HnfoundbJldOCUli.P~lItOil.~S.ph&de:r 0-76Col~ j'

'.

^'~

10, Chro~toi:rapby ~f'the 0-76 Ano,fthe·N. .foIlJld1aDd Dc•.an Pout fro. linter aDd'Swmnl rY~nth.on a CIS Colwu .

'

..

11, Chrou...ographJ. of thl0-76'Anof the .Nntoundlud Oc.'aA ,48 Pout froa linter aDd Swmnlr Youthe011.a ReYer•• Ph••• C3

HPLC ColUlllD. .'

12.,?~ou ~gr.Ph,1of 5P~i ~~aReY~rllPh... CIS,~LC'C~l~" -'""' 0 13. Chtou

ogr.~~f

the ChJmOtrtPt.icP.pt1du of

SP"'l~.A

^on

~ . 6~ ,

' .

/

... .

^{~. '}

j ' J;"

-:

,'.,t

"

"

..

e1~

.

·"""1.

o

",'."

/ ( , ~ ^{. .}

V C ; \ .·

""lor..

P.... C1. ';'LC

~.h..

^{. . .. .}

1 .' Chr . .tosr~plly01,t~1 T1l1I1RQlytl~Jl pt 1d..of SP-l -A on a 59

~ , R IN,'Pll.••• C18 HPLC.ColUlllIl • '

lS! Chroutoiraplly of thl,Ttypt, 1cP.ptidee of SPL-l-A.-61'-1-8 ,ADdSl'-l-C~n ~':R...~r..PhanC18HPLCColUIIID

lB. ChrolUtosraplly01t,b a- cIiT-Ot r n t,1c Pept1du of Sl'-l-A. i l SP-.l-BaDd sP-I-Co~.aA.n~ri, Ph~. . C18 HPLC ColWllD.

17.'baird,of tll.i Unpuri1'11d&lidPurified~·l..illll.idDIfAOD.a ,0.8IAs~ro. .:O, ~'I n TBE'Bnf.fh -

, . if ' .

18. balyd',of thl PltII!lS..tl d phlmld UNA011a

e.e . -

~ .... Asaro..011^,ill

mE.

^{BU,f.hr I}

L·

..,.l~_,.

, I":

^"ADa I Y' i l01,t ll.l,eDNA'Fraplll.t,'in Parallil -tth

),DNA-Hl ad Inyat~INC).JI....0.8 .. Agaro•• Gil·1D.nE~

Buffer '

20~ ^{b.j:rd;'0'1'III'DRA}~f,t.h'^{eDNA011.a 0.8• Agaro.'Gu,in 88}

TB£Bul1'1r, '

21'.Rudtatthe Figure 8 Forutioll.~T;. t, 89

22..Autorad~olraph^{of Partoffob'}Seqnlneillg

CI1

tramClan. 91 Ho. ~l

. .

, . 0 \

LIST OF ABBR EVlAT l o"NS

MGP antiru'ue

s:lyr~pto:ltl"i~

I.. MP antiht>e n~rotPin{s)/pf'Plidt>l$1.

Bis r-;,N;~m;\'h)'IPD~bisa('r}'1:rmid i' ^to

- ,

_BSA "bovinesl"~umalbumin

CD eireulardichroism

.rONA ecmplemeararyD:'\A

CM ri.r~J:yml"thy l

_d dNTP 2'.a'. didMxynurifOS idt>'riphCbphalp DNAase dec r yribouuelease

DN5-CI dansyl ebloride

E.CoH -E.dar'~ hiCleDli

HPLC highperforma nceliquid ehrornato graphy

1.0. inte rnaldiamet er

!PTG isoprop)'l-'~l)..tbiog21~ttopyranosidl'

M.W. •molecularweight

PEG polyJthylenp"glycol'

QAE dielhy1-(2.bydro~y.prop}·1Jaminoelhyl 'Rr replical!:'eformI

R~Aas('

ri~oDucle"

RP~I revolu tions perminute SDS sodiu.!!'dodl"c)')sulf>hal t"

' -

I.

) .

Sp 55

TBE buner

~bliffer

.TEAP

TEMED TFA' TLC TLCK

TPeK X-gal

-xil-

sulphcpropyl shorthornsculpin single-stra nded

O.089~1Tris.O.089~1boric add,O,OO:?:-'I EDT..\.bnrrl'T

IOmMTris-HCI,pHS.ImMEDTAburrer, trietbylamleephosphat e

.N. N,N':N'.tetram~.tbylethyleneEian\ine

trifluoroacet icadd

"thinlayer chromatogra phy ; _ 'K.o- p-tosyl~L-h·sin('-chloromethylklolon~

-

^'

r;1-tosylamide--2· pbt'noll.'thyl·cblorom~lh)'1ketone 5.bromo-4-.('bloro- 3-iridoly l'd-D.g ala('tos ~de

\,

.j,

Introduct ion

Inthepolarand subpolarr~gioll~.

;'::1

~'3tNI('mlwrat uf''''(':UII",flS1,,\\;I~

·l.SOC(II.To surviveinsucbaharshenvironment.somv marinellfll:311islIlS h:ll.',-

.-

developed vari,<:>us typesoradaptanon.ThemO~1intr iguing oneisfhr-IIn 'IITh'lI n '

of~ uniqueclass of serum proteins•.which spl'<'lfi r ally lowers till'{rl·t-zinK

.temperatureoftheplasmatoapoint thai isbelow

ib nl

~rthesurroundingwat er.

These (reedng p~int-depressi~gproteins are'commonly.known3Santirrt!~zl' proteins.

" . ' '.

\

^.

In 1953, Scbclender and.colleaguesfirst.reported^:1trl~hloro^3('('11(',~rit soluble macromole cularsubstancein the serumora~?r~hern^polarfishW~ith could lowerthe serumfreezing temperature(2.3).This~ub~tancl.'.W:Lo;roundIll'l.'r tohave a proteinaceousnature.Since then,mll-n)'spe~offishhavebeenfound to"conta in these interestin gprotein s.Based on their amio add andcarbohydrate co~positions,^(reezingpoint-depressingproteins -can beelass jfied intotwo'd i!>tinct groups. Theyareantifree ze glycoproteins(AfGPIandantif reezeprote ins(AfPI

1.PROPER T IE SOFMACR OMO LECULA R AFGPAND AF P_.• I.,;)

~1aI;Urreezeproteins sharetheIcllcwing(:hatacllOristic s, L-Their elleet on freezingpoint-depressionisncneolligatlve.tba ti~.thl-')'

lower the rreezingpointmuch morethanwould be expected{IDthe basis of the.osmolalityor their~olut ions. ' 2.They showthermal hysteresis,thatis,thereis adiUl'rl.'DCpbetween

the appar€'Dt freezin g and meltingte mperat ures.withtheIattvr b('ing eq u ivalen t to what wouldhi.'expec tedIromtheosmolality flfthl'

anufreeeesolutions. . '

3,Plotsofthe rmalhyst eresisversusantilreeae eoneentr atice ereconvex.

r:

\

rathertha nlinnr.whiehmeansthatthereis:asaturation errl"("131 (b ig~~rprote,in(,oDc:entr~hoDs.••

•.The onra llrr~lingpeiut-d epressionbyantirr~ze~lb. sumo[its

~::t:~:r~;:::r:;~t::~, ~~:~:~::U~:::~i~~~~~l~~lig~lh'e p roperty o f

2.DISTRIBUTIONANDSTRUCTUREOF M GP

AFGP hav e heeD found in antarctic fisbes. such..~.TrI "'OComU' ...

bore"',,,vi,,ti ..Dillodic"'" ·", ow. o",',IS). inarctic:rebes.liketbepola rcod

Bor.ogo~;,

^u.ida{61"

sa((ro~~d

^{EhQinu •}."oe;li.{ii,"and mo;erecent ly,

t~e

tomcodMierGll'odtuf~eo~ (8),'atl~ntieco~Godu."'0'''''0(gl,and Labrador rod God...GerGCI(10)

i~ Newro~~dland

^{andLa br adorwaters.} The primary

str~('tu~h

oftheAFG?~romT.hreA,..~vinJ:i.D.m.o"'.!.'lJUr-B••aida,M.'_e~dandG.

_or"". _{~~ve b~D}

wellts"blisbed (11.12,13,

5~'

^{8,'II,'T heyha vesimilar,irnot}

ide ntical,struer ures. AFGPare a mixt u reofclose lyrelat ed compo nentswhith dirterstructurally in sizea~dthe presenceorProInd / orArg in the sm aller ecmpcn e ats. Fo r examp le.tbeArCP from botbT.bord~"rni...I:iand D.

",....,"-,"consist orei&htpolypeptides.madeupo(• repea t ing-tripe ptideunito(-"

Ala·A la-Thr witb thed~accharideg.lactos)'I-~acet)"lr;alactosllmineglycO$idically linkedto Tbr residues.Theinter nal Jinkage~etw~eDthe suga r mon om ersis"1·3, The tri pept ide unit isrepeated seventeento'filty times,giving the eight

"\ "" ,"

.

componentsofmolecularweights 2,600to 33.000 , The,smalle r components6-8

- r , \

dilfuItemtbe1)iggttODesin tbe .rep!aremt>ntorProfor Alaat ('{'rJa inpositi ons.

S;~;I'dr. ; f. ^I_~~'.

^AI,

"p;'",

Th,

;~ ^{s cm e.cr} t~ ^es ~.u" ^lo mpo"n,::

\'iseosit\andcire,ulardtehrclsmst u~ i~son the AfGPfro mT.f,orc Ag r",.,i "ti

,,:,- \ \

/ ~" )

\

/

.\

i. _-3-

,

suggestedeither3Dexte ndedroeform at ion er-a-fomp l('h>(ynt'~iblt,fand lll jl('oil second arystr uct ure with out any indica tionof·~.helix^or,i-str'~('~url'^·PI.-.Murl' reeee n y. a ,",,1.1CDstudy,",,~,t'da 3-lo1d lett-handedhelix

t

thv

'''11;"."

type asthe.mcs tlikely'conform at ion of this AFOP(14). )

Aninte restingcbsevetia e-has been the cooperative junct ion between AFGP (1&). The.P~o-contai~ingsmallercomporienta(AFGP 7and AF:GP8) exhibited

-

weak antifreezeactivities,~ut.ifDoeoft

.AFGP .·the largercomponents

(AFG

1.5), a very

l arge

(2:8 fold) potentia tioncif

antifreeze 'a~tivityhad.been.o~serve. u,was also reported. thatstruc tu ral similarity was eecesseryfOrthecooperativerunc"tion~b('l:aus~the 'antihe..ere activi tydid notpot ent iatewhenthe sma llerpeptide wasmixedwith flounder AFP (16). However, tb\eseobservati ons have recentlybeendisputed by other. laboratories (personalecmmunjeationwithDr.Hew).

..3.THEORIES OF THE MECHANISMSOE-AFGP ACTIVITY

The so-called -int erfacial m'echa nism- isthl;

m os l

popularone (3 ), Tht·

mainideaoftbismodelisthatactiveAFGPfunctionat anin.tf'rraria l regio n betw eentbelee-:r

ice.nude~s

^{and the}

soluti~n

^to'inbi it the growthor thein•.

Someexperi menta l datasupporting themodel include:

I .

. l.T b.

~""~

^{growthorth' leewas'' 'ally}

^;m,a;'td i,

^thepreeenee "I active AFGP(~P1-6);inecnues t, the a-alilS growt h wes.ncemel in the presenceorinact ive'Mep(AFGP!07-l~ot.a-nonantifreeze glycop rote in. "The suggestedtba t the activ AFGP shieldthe irE' nucleus. 'I:berewere alsoobserva t ionsthe-activeAFGP',became -entra pped-asicegrc wtbccnfiuu ed (31. •

2.Almost allebemles l modifications

o~

^{the:i echande}

~roups.

^{such ne,}

. gradedremov~1of the.sugarsidechain byjelimination, acetylationlIf

...

the hydroxylgroup sand positionin g a negative chargeonCoG. inact ivated AFGP aseeurreeaereagents(I i,12).Thissuggestedthat therole-of_AFGP disacchar ideis todisru pt ice surfaceand that thisis ledtspee elbletorthe antifreezeact: vi/1Y, .

Ot her mecha nismslike-vliquidphaseinte raction-or-lee phaseinteract i~m."

'have .also beenpost ulat ed,·thetormer suggesting,thatcerb inhydr~philicgi~up~'

of the ArGP mayinteractwit~wat ermoleculestodisrupt the localtetrehedrst- c~rd iDati.on01 theliquidwat er•..and tbelatt er suggesting,th'at:~GP_ c~p somehow enterinto some form ofsolidsolution withwat ermolecules and thus

c~mph~tely

^alter'the

stru~tu;~,

^and

properties~h~

^system

'l~om't~OIie

^'or,

p~r/

w8tet I.3).

4. D1STRmUTION AND STRUCTURE OF AFP I,

Res,earch carried ou t over the past decade has led to a lur th er. underst andingof AFP,l'nt~ow,AFP have be'enstudied inIoura pecles of fish

, .. . '3 '

118,10,2.0.21,22),They are winte r flound erP#cuttopcurOfUtlc.,amcri~anu~,. short bo!nsculpinMl/OcDecphldu, .eorpiu••sea ravenH,mirri pf, r u, am,rica;;.. ,' and oceanpoutMacro.oare.. americo"",.

Affi~"DgtbeSt', IlcunderAFPhavebeen studied in most det ail,OeYries and

. .

eowcrkereirepcrtedthree compon ent s ofmolecul ar weights'6,000, 8,000and

. "

12,000in the AFP homflounde r(lS), At abou t the sametime, Hew an'd colleagues report eda major,compon ent,separ~tedfro'ma~ep hadexG·j5colu m n.

of molecular weight 10,000(10),Lat er,It'he~the sing.le 10,000dalton compon en t was anal}" zedon reverse phase HPLC ,i'tresolvedinto at least sevenccmponenrs or molecular,we,ight s3,300to·1,500123 ). Thetwnmost·abundant("ompOnE'nl~~

.;.

No.6(or.~Iali~^No.8{or BI. wer e 3,300daltoll~.eal'h:'^Diltt'rl'1I1frolll Ill\';\1"(;1' discussedabove,thesetw o1'1Impo~ (,1IIScomprised nined~rrt'rl'ntt~·Pt.,."ramino acidswithAJaacco unti ng for60 molr-;.(23. 21. 25).Doth orlhl'st,AFI'"I' h'37 -residuep';;'i;.peptides contAiningthree

f."1(,~·(,1l _

^{amin? arid}

r('si~l: r"'l"~ats

^{of Th,-}

. ." . I .

...(~a.AJa-polaraminoacid.Ala'A1:L•.-\la'Ala-Al~'A~~Ab. ItisdearthatI.ht· All!

content

\9

^{AfGPandt.he AFP}rro~winter.^f1ourJi.('t.'aresim ilar lnspiteof.~ht·^rar"

thatother amino ' acid ecmpostnous:are different and that AFGP

han"

., 1 , ' , ." " . , ' ,. ' .

dis.accbarides.,..The_dirtere~ce bet ween_th~A_~nd.Bcomponentsinwinu-r .FlounderAfP";;8.s.duetothe'r;'pla('ement~{from AtoB)

in

fositiOhI'~ (Lp;~:\laI, position22 (Glu+ Lys)and posit ion26(.A1a+Asp )., CDstudiesshflwedIbul -".AOunde.r'AFPpossessedalarge prc portic n(abou tSSC:Iofe-heliculconrcm uulon in.aqueous~olutiona~-i-c.Viscositystudies at _loCindicatedanasymmeu-ie shape (26).

Flou nder AFP are'synthesizedlndlvidua llyvialarg er pmllrsors,I;!I.2;.1

"'\..Compledte DtaryDNAmadeto themR~'A^{ofthe A.and}B componentprer-u rso rs wereclon ed ill' theplasm idpB~322andtheirsequences wert'dt'll' rmim'dII):ill"

methodof.Maxam and Gilbert12~,251. Thl''p_rt'p roprnlt'inSl'qU('fI('l'~

or

bull.l compone ntsare 82 residu eslong. The'prepro' par isof thl'1w0prl't'lIrSf)r~an- ident!cal. Themat ure sequences.ofbothA and Bcom.poDelllscon tai n

3,

^amino

acidresid u es.The differencebetween thetwo is'at threepositilJn~ns nll'nliu oNI 'a bove.Alsother eare sev eralsjJe'!tehengesinthe nud "ot idt, s('quenC'I's"l'n(,l)din~

the maturep.r01 ~ins(24,251.

""

· ..

G.j5gel- filtrati o n and QAE-Sepha dexjA2.i lion-exchange columnswiththe major one(BIaccount ing for76%of thematerial and~.!!lJ!1?rone"(A.I for2-1'C of-the mat erial (21). The'c:omponen~B hod manycharacteristicsincommonwith flound er AFP.

suc~ ~,

^siae'(as

tima~sephadex

^G-;,')

~olumn. abo~t

IO,OOO-ll,~dalMns. [aminoacidecmposition. theabunda nce-or.Ala(ab out 60 .~ol.%);ili'ermolysin:l>eptidemappingand high(l.heli<ial-ci.m~e~ t{ebout

sslt.- 3t

qOCI.butithadt~elve^{types ofaminoacids}i~stead^or~ine^f21_,27).'Recentlr. thetwomost.abundantcompo~.elffs'f~S.8;^'55-3}~ave ~e~n.isolated^by7.ephild~x '.G'15· columll.~Dd,'_E'versepbaseHPLC.S~.8'andSs;.3arepolypeptiJ"es('(Qta!ning 45 and 33aminoa~id.residues~espertively,.Both'or thesehaY~blockedK~,

!erminals·(28), Thepresence of rbree repeati ng.st ructures·ofeleven aminoacid residues illsculp i.n,\FPwasconsis~entwi,~.

""

of Oound er.AFPandsupported ,tbeideathatt,beybelongtot~esame group .

The AFP fromsea raven,00theot herhand.have-differe ntcharaete nsues 'Cram thoseoCthe AFPdiscussed above.Onemajorcom~nenlwasisolat ed'using SepbadexG-; ",andQAE-Sep~adex1:\25) colu mns. T~e.mOI:Jllrweight oftill' componen t was larger(ap proximatel y1·I,OOQ-16,OOql,Itcontairi?delghte en~~:pes ofamino acidswith ahighconte ntofhalf-cystin e[aboutBmolCCIandonlyan averageamount of Ala(about?l-IA molC:C:J. Fur thermore,itwas sensitive to sulpbhyd rylreagen ts, "Circular dichroism studiesindicatedtheabsenceofa signiflca n.l amo unt of c-belix and a possible presence of ~st ruc l u r(', Immunologicalstudiesal500showed'thatson ravenAFP doesnot<'t~~.rea(:t^{wit h} antibodiesraisedtoehheeflounder M sculpinA!P

(2?'.

' -)

,.

AFP Ircm winte rno~odl"rand shorthor nsculpinnppvar1"11I'1"1;j!; 1'"th., same group,

wh~b@

,AFP rrorl!

s~a

^{ra venrop re-ent anol lll'r'nll'}

Ii fi~h ·

5.SEASON.~LYAI\IATIONS^{OF AFP}

.with mosttor'the Iishes stud ied,theoverallfree zin g point-de pressio n.

th"~~1

h"t. m;,\and:ml 01AFP.;0 the

blo~d

^PlIn:,shew ,",,,I "<II,, with themaxima'mwmt er andthermmmaIn summer,":InNewfoundland wmtcr

I

nouDde.~.'^thema~iraappe a red IrcmJanuar y~o^{A,ptiland themin!ma.lrcmlui)'}

to Octoberwhen~be^{wate r}te~peraturt's^were·1.1to~~IAo('^andtotoUOC, respectivel~; .The AFP beganto appea r iii·the serum in Novembor and disappearedduring,~laywhe nthe water temp erat ures wereoj106"(' and·1.01(, 3,OOCrespec tively . There.was verylittleor nqAFPrrom,\la}'to ;'\ovt'mlwf, The above cycleoCthe....AEPcorrelatedcloselywith that.ofthe :-;"("wrollnd lnrll\

,watertempe~ature.(29)..wnbanot herpopulationor thesa ~espeeles.thl'N;)V:1 S'CotiaIl--fl')unthr,.tb~AF~'levelincreased approx imately onemont hI:lJ~ra~d declined two mont hsearlier thanitdidinits co unt erp art ;\e ...IoundtnndHounde r.

'This cyclealsocor re latedclosely withtha tof;\0\'3Scotia nI'ia"'r tl'm pl'r a.tur::

(30). In"short lio'rn sculpi n,theappeua:neeof AFPin'til l"plas maw,:~.^fit

e "

^{the -sam etime'or theYe~ras thatof};\{'wro~ ndl3 n d rloun~er,^butthe AFP disapp ear edlat erthanin NewfoundlandHounder', Sig nificantamountsor

I ." . .

\FPwe,re.s.tillpresenti.nNewfoundland5~orl horn^sculpindllrin~ JIlI~'^when-no AF Pequid be'det ectedin,'the'plasm a of the Xvwr" imrlla ndJlcundi-rl:!tI A~otb.er pcpulatic cofsculp iu-.thearctic"sculpin.however,showed thai till':

couce ntra nc nsof AFPinthe plasm a.even duringsummer..wore ClllI11lIHa bl..III thoseob~erved'

iit

tbe plasm aor:"e... ro undian d sculpi nduringwinterltil!

.//

<;.'"

.Il-

, /

.:»

"

.j.

Fro':"the aboveobsen:at ioDs,n.seemsthatthe annualeyele orthe Af P

. . ' ... ' .

·level.isilecmmcefeature amongthe subpolar fishes,buttherei~:a'tt'ndt'nt~'ror flShesip,habit,iY,morepolar()(UDStomain~ainhight'rconterratioDs ofAFP eYenibsummera'DdtheirAFPlevel5 declinelaterthaDtb05~,krless polarIishes.• Tb"... .D"";'PI!,Dr

hD~ h~~"I ~,g~D;';';' '~"lhl,;ro.m

^en

~

^_

"O.CON T R O L OFTHEAFPBIOSYNTHESIS

The,annualcyeln oftii~^AFPindi~ate.lbat^{enviroDmenialfadorslnrluenee'}

•.the appearance 'fodt~e-'di.sap.pnraneeo~theAFPfromtheplasma. ,I,thasb~~n' roundthat photoperiodplays~melc r rolein thetimingofthe appearance~rthe t AFP 131),Longday.le~gths(>12hjdelayed theons,eto!~t~~,,~~~e~raDeeofAFP, Wat ertemperatureaffected ooly the rate~rthedisappearan~epCthe AFP(4).

Winterfloundermaint ained under longday-length had bothidefayed appearance anda.decreaseda;c:umulationor theAFPmR:o.:.-\(321. Itwasfoundthatth··

pit uita ry glaod regulates the level of AFP vte'a negative mechaa isn. Hypophyseeto mlaed~i~.~~~uDdercce tia uedtosynthesizetheAFPiD.,ummel or undertheconditio~sof,warm wate!{6to12~C1aDdlong day· length(18hi.

"

_wbtletb~sbam-operatedcontrolgroupdid 'not(331.Anothtrexperimentrevealed thathypopbysec:tomy of

wiDte~

^flounderresulted in thein('reas'in

liv~r

^weigh;

tota l

Ji~er

^polyA+

~NA

and the AFPmRNA13-'),Itwas'alsofoundthatth;

level orA£f.JnRNAin winterfloundershoweda,nannualcyele,similar10~ut prec:eedingslightly th~t of AFP (351, Alloftheseresultssuggestedth:lt photoperiod~ndwatertemperatur~may 8('tastheinitialcuesfor-t-he ronirolor theAFP biosynt besis,while a pituita ry hormonemay be~bemediatora~dthat

·thebor~nerna)'regulatetheAFPbios~'nthesisb~'thanging the leveloftheAFP

·mRNAUS~Dga negativ etra Dstripttonal ('ont!olmecha nism.

J,

·g.

Evidencehas beenround lila.tr~nsb\ion;)l('~;n t rolm..('han~,rnstll...r~h·I"

optim ize the production0,'A.f'Pinthe "'lol(> T Ilounder1361. TOlaltR :".-\!r"lll theliJ.n-o~[nt er~ishsboweda~ aprtO~im:lt(ll,·-lO'ejnm':I"'·in Ala:1"r l'I'I"r capacity over tbetRNA from thesummerfish:inecntrasst•Ih~'t('('('ptnr(";w:lfit i\~..

fOT

otbe~

^{ami noacids}

sh~'wed

^01seasl)nardin e-Troct'S. Biosyntheuc studie-, suggestedthat Alanyl. tRNA,syntbe.IlL5I'.

r~nrtion(>d

^{bestbetween0",oq',1)(>('whirh}

is~be

»r:..

te~per8tllre~ben.AF Psynthesis occurs.whilE"Pr.?I~·1andral}'I-"

tRNA syetheteseswerem~t.active between 20and.30°(' . So.lb~AI3 JH;A acceptorand theAlan~I.I~NA,sY!lthet ase both run,eli? " I'spec::ia.ll)' in winft'r

'0

increasethetr an slatio,;,a! efficiency ortheAFPmRNA,

Recently ,in winterIlounde r.threenonove rlapp inr;, thro mosomal"region1<

coding for the AFP have beenreported{3il.EQ.("h region containstwoA.FP gcnt'1<

spaced from 3 to': kil?basepairsap~~t,I1ut3.11of thethe e.. regionsrepresentunl}' 1().20%

o J

the complementof thismJ!"ltigenl'fami ly. One ofthesixP;l"DI'Shas b~~D sequenced.and identified as ava riantofthe gene eodinglor themost abund antAFP,componentA,It,is 1.0 kilobnsl'pa ir s long,a nd. contains an ioterven iogsequenceof0.6 kilobasepairsbetweenthe codingre g ionsforthe h'ulk or the signalsequence(p resequence)andfor the proprotein.

7. POSSIDLE MECHANISMSOF AFP ACTIVITY

(

.

^.'

^.

Co ncernin g the mechan is ms or the AFP rueeucn.no'directevideneeis

&v!liia ble,De~';iessug gestedam~elinwhich the a-helical notifr"ezepolypl'ptidt, oriente din sucha way thatall.the polar residu es,likc

T he

^nnd"A~p~wereIr~at l'd 00oneside, whilethe!lo opola r'residues . Iik{1 Ala..were-Iocatedon theother slde.

- ,0-

\ ".

Tbt"·pol.arresidu es eouldlormhydrogenbondsuiththe watermull'1'ult"Sinthetee laUit't".InotherIIo'olds.thrwaltr'moleeujes in thetr..('rY~lals ~d~rbt'dIhr.\F'P

l~l. Tbt adso~ptioD o r

^theaDlir,,.lt'toMop

tr~'!il.1bi ':'OUI~ ron(,l'i\':bl~'

^)C'3dtoan

. .

^~

mere ase in tbeJurfart".,nwithonly asmallinereeseinl.hl'\-ol~ ~t..~hirhcould. 'u use"aoiDt'tU5(,intbt";urfa ct' Ireeenera_1~·6rdutorfreE-zing"toO(('UI,lht'.

' .

. .

\ .

.

. . '

.

^.

.

nugy~Ilstbe removedf~~th,systemandI~wouldbedcne by'lowe ringthl"

".ft.p'ui n g

temper~tlir,. ~~,is m~J '

^{explained':the'}

Don('~lIigatin

^{lowe' ing}at..

. ' ~

- . - .

....ft l'f'zing trmpera t urebyAFP. _ •

The 'mod el d:Ser ibedabovecould not explain the high"('0011'01

~r

^{Ala in}

fIouDd l'tand'seulr P. Tht con served elevee aminor":l.cid residu erepeal. pres:n t in bot h Iloundet andsc ulpinAFPwhic.b..h~...highcont entor:\13. implies tbatitmay playa cru c ial roleintbe/lnli.rte:le.rllnrtion.·A~yn~hetirpolypr",tidt>

conta ining.~5 ~OI% Alaand35 mol

rc

Aspflh~omly distribute dshowed lDtirretoleutivitYI3Q). TIlisresul\ stronglyemph3siz!,:d_~e.roleof Ala. On the otberhand.th e

,. .

In raven

. . AFP

eeetai nsao averageamo unt or Ala^'

.

^. (14molt'f.1. bata higbbaU·cJ stioecooteot(8~I.%).This sugges'ts tbalhaU·cysti n e·maybt>

invoivM.in theranetion..There fore...searavcn'AFPma yrep~esehtanothertype 01 me chanism. ~indderitly,aauther batl·cystine·rODtaini~gAr~waS\Wlated

I . . •

rtien tlyfromtheseco ndinst ar larvae,of .thespruce budwcr mChori.tOtl;U.FQ . -'",mi/.reflt.. LikeseeravenAf~,^{it only}c~ntl1int'd^amod~st.^{amountor f a.(8} mol

%!

^(40).Itw~i~tere~ting .1o--,!bsef.7e^{th,!tthe re was3'D}immunol?gi ll31cross- tnct iv it}·bet ween this AFPand!be antibodiesra ised10~earavenAFP1401.

;

...

·11·

·8. RATIONALEFOR THE PRESENT INVESTIGATION

, .

Oceanpout(Mocrc!'(lGrc u Gnuri ClInu.),\ FI'have bor-n Iound','arliNh~

.

^{~. .'}

..

"ou r labor at orytodinerlrom allofthethree.\rrty~('''mentio ned;Ih, h-" Or,i'a n' pou tAFP,

~avt'

^at

l e'~t

^{eightact in-}

COrRPo~.!'nl~,

^whi('h

~3t1

into 1"':0 dist illt"

.

. ^.

gro ups based onth ~ i rbehav iouronion-exch angechromato gr aphy:tnat is,till' CM-bini:lin ggtOU~and the QAE-bin,d'ing groupt221.Amlnqaci danalys esshmH;,I' thatoeean.pout AFPt~n tai n!Jlost , .,i! nol.all"l}:pesof aminoad~s wi(h,mn~l'S~I,

Alaeonte n tsand,!lo balf·('ystin e at all. How ever.the ao!in.o.addcom positio ns,,!

· .

thepeptide s in onegroupwer equit e simila r. The ctrc ulae. die hrots mSpt't;,t t a"f

'. ' ~ "

oce anpout AFp'aio-cresem bled thespectra(rom',-\F GPin thefarCY tt'gion.·

• • .F" .. ' )

.Unlike the latte r,however,theyexhibi(ed llro~aliC'CD,bands" inthe,2~o-3~nm region.Un derdiffere ntden'a t u'rill$('. -dltio ns.the CDspectraofo(,E.'a~^{'pout AFfI}

" ~..

..

were diffe re nt,e.g.a sigmoidaleonfo rmano naltransit io nwh ~nheate d106~':(',a

·

typital,speclru~

^of

d~natu·.id

^{prole in}

~ith,6M

^{guanidi ne}

hy~~c~d_t'

treat m ent

"a n da chang e-over to e-belic alspect rumrht'n treated~ithS:o~TakE.'.n.togt'lht'f, these tl;:su lts'indicait'tb'at ocean pout AFP has a."w elldl',fint'd,~e('ondary'and"

ter ti ary str uc ture, whichdiffersIromthoseor"theot herkn-owp'AFP 1,111.' •

, I

~ Tbe levelofoeeae poutAFP alsos~o...e,danann.ua t cycle,.,I,owe n r,iJ~nlih

·~be.tbreypeeies^{discu ssed}~bove, oc~,an.p.outcont ai ne d asignifi("a~t^{emount.e l}

AFp'evt'oinsummeri2Zl.

J

! ro!'1 tbe.i.n f~rmati~nabove. it canbe seentha tocean pout:\f Phev«many~

uniquefea t ures~nd^{it basbeen}.suggl'Sl~d^thaitheyr~~t'~~nt^{a'bewtypl'(If}Tis~

AFP.

"

'r

, - \\

-I:?-

,

~

a

^{Iurt her}~leptoundersland-1 h~slrurlur~andmerhunismofaction.~~

r:

.these AF.P.aprojectwasplan ned...irhthefoRowing objl.:ljlin's: I L furtherrhararlej.izati~northe'·multiple ocean poutAf' P components "

wlthion-exchangechromat ographyandreverse phaseIIPlC':

2.elucida t ionor the primar ystruc tureorODE'

o r

^themajorO('~II^pout AFPromp?n('~t,s us.i.n~rhniqu ('sinproteinchemist ry:• 3..

eluridati~n

^{of the}

slrue~ural l~lati~nship

^ofthem('mbt rs ofthe AFP .,.. ('mily. ,byp.epti~e~ mapping and" aminoacid analyses:

4.in'Ye~tjgatilElofthestructureor the biosynt het lcpreeur scr ts jot the

-:- AFP; - -

S,iDYe5~lgatipn^ortb~seasonal andpop~latiof'1a1\';riationsof ;;;'npout

AFP: ' " - , '

- _"I

;' )

.-

~

..

,

.

^,

r

·13-

\

Chapter- 1

isola t io n And Characterization or

Ocean Pout,AFP f

" .

Previousworkcarried outin ourtabora~oQ'^hasshe wn thatO(,I':l.lI·,,,,UI..\1-'1'

. ,

.can be'isolat ed aod separatedsu~(,~s~uVr~.by Sephe dex'·G· j',.,gd.n1t r~l i~n.

('hro~a;ographYI^QAE,eM, SP·S@'p'ha~ex'lon-I'X;tiangl'chro matography:l~d analytical Ieversephase HPLC(22).Oeeen pout AFPcan bedividedinto atIt·a...'

~Iigbt.active

compo nents'orapproxima tel yide'ntit':lJsize[about6.000dninm-]

I;~

\

the above procedures(~21. 111thepresentstudy"sepb adex(;-;1;gl'].£ih r:lIiun

cbromatogr~pb):, ^QAE, SP.Sephadex ion-exchange rhrc.mato gra p h}: und"

, ,

pr:p~rative reverse 'phaseHPLe WNt used wilh some m(jd,i ri~'a l ions nnd 'im provements. The purit;as welllIS'tift:seasonal and (!;(·ugr:l.phir varinti.u... uf

. " \ '

ocean poutAFP

~were

^{examined. .- . '}

1:1.MArERIALSAND METHODS

"Ocean pout.were''t ollected

~

divers from ...atersaround the

:\\'aIOl~

Peninsula,N~WrOUlIdl~naandwerekept' at ambientse3wal~rtl'ml)l'ral~rt'snnd

• ' 1 "

pbotoperj ods."Serum sampleswere preparedbylo\\"speed('{> ntrifugaliu ll IHIOOIt

x

is

'mi~)of clotted blooda.no.stored.at

.20

oC,

\

/

Thepr~<:d.Jlrl.'usedtorthepur iri("all<>n"rIh.·"c~anpOUI AFPi~ d.·~.-r ih..<1 b...IOw.Anhough thisisessent i:l.">·th..sam",3Sus..d 11I'iorl.'1:!2).modif..ario us in theburttrsystems and grad ien tsweremad e. Ined d ruon.theC\I-Spphaol" xion- exchangecJ:lromalogra p hystep~·asemitte d and thMIP LC'wasusedonboth analyuea landprep arauvescales..

1.1 .1 .S~P~ADEXG-76 GEL-FILTRATION CHRC?MATOGRAPHY

O~ean^poutseru~ t·~mil was loadedon.u Sephad ex·G.;."colum n 12.·')~m I.D.,x 511c m.l inO.lM l\'H) 'C0₃buffer. fractionseramllor SOdr,ops)each Wer eeo~l ected-a n dpooledaccord in g 10th eirubsoebeneeat230.n01 . ~\~opt"ab.

Wer eresol....ed.Th e(irst one corresponded tomost of the serumprot eins'andthe seco n dpee k ecnr al ned:l.nt i fre~epolypeptides asdemonstrate dea r lier inour '!aba r at ory(22),

Fra euoescorres~ondingto the AFP peak,were'pooled ,lyophilized and .rechrcrmrt ogrs pbedob the samecolum non ce . The'mat eri alwasdesignated9-,."

AFP.

1.1.2.QAE-SE~HADEX(A25) ION·EXCHANG E CHROMATOGRAPHY

.sO·m gofC.7, AFPin:r ~1^{ofstartingbuffer15}m~tTris,.ff(~,^{pljg,4) was}

dial )'sed,against alitre of thestarting buff"r at 4"('overnig ht usillgdialysis 't ub ing(~(;W,cut.o H'3,0!?01. Afterapplicat ionofthesample.eluuo n v"as car-r-ied ou r'witb thesta rt inghurr~;. Fift >·rra rlinnswer eecllee ted140 drop s or2."ml per rube].thena lineargradlcntof 0100.2~t:\'a("1intheslarii ngburreewasnpp li-:d .Twopeak swereob t ained; therir~t-on ewas.fh ..unbou ndpea kandthesecondOIL~'

"'as named.QAEA,

-15·

1.1.3_SP.:SEPHADEX (CGO)IO N· EXCHA N G E . CHROMATOGRAPHY

Theunboundpeak homQAE·Sephadc"I.\ :!,', )("hromatng ra ph~'1',0mil;in 2 milwas dialysedagains t,Ilitreof sta rtingsolution10 ,OI'M~/lC'1.pllajal 4"C overn ight. Aft('rsample loading,thecolu mn waswashe dwit h JlOmloftltt' st art ing.soluttou.A lin urgradie~tof 200 ml 0.0 1~lNaC'l.pll3nud200 ml0.2 M NaCI,pH3 was employedafterwards .Freetjo nswerecoliectedin3,0 ru],;iZt,,;.

. .

Based'on'absorbanceat230om, '-I pea kswereoblt\lnl"l1andlh-!,ignall'dSl' - 1:

SP-2. SP ·3an dSP~ 4 respective ly. Theywere lyophilized and(!<.:.S3Itl,d 8.'l·in

1.1.4.i REVERSEPHASEHPLC

Sepa ratlonofpeptidesbyrl'\'ef'!fl"phaseIIPI.Cisha ~,'dunthl' diff,·rl·n...·in .theirhydrophob icities . Wit ha solvent gradien t {Ifin("tl'a~inghydrop hnbil 'il)' "

peptides elute fro mthecolum nin orderof inctl'llsinghydrophobie hy. TIll' gr.!~iw'tcanbe fo rmedbyincr easingthe JU'r5t:ntag'·oftheorgnniephaSl'B(sueh

By Sepba d ex G-j5 gel-Iilt muo n and QAE. SI' ion-l'xl· hanll;'·

~hromatography.oceanpoutAPP had beens<,parat.·din t"c.grulIl's:QAEA, SP·1..SP:~.SP-3andSP·" They I'oN,<'nnalyn-d,hyfl' \"' rSI' IIhas••111'1.("

\

·I~·

r: » :

1.1.6.MOLECULARWEIGHT ESTIMATIO NBY GEL-FIL TR ATI O N HPLC ANDSDSPOLYACRY LAMIDErLABGEL ELECTROPBORESI~

Ithas beenreportedtbat,on gel-filtrationbyHP LCwith:ITSK·G300Q.PW

. .

column and an eluantcontaining0.1

cr,

TFAand 45c;(, acetonitrile,.theelut ion volumesof proteins_or~iideswerelinearly related.tothelogar'ith molecular weights. This orrersarapid andsensitive method(ort h weightestimati~nof'polypeptides o'rproteins (421. I

. . '

\ :;t . . .

. In_~bepreselJ.tstudy,Altex berogel_TSK-3~Wcolumns'were Two

col~'mns

^{(7.5mm LIT: cmj wert'}

connt'ctt'~

ⁱⁿseeles. Tht' eluantused was 0.100TFA in 45%CH3CN with a (low tate or 0.5mlpermin.

A 50Spolyaerylamideslabgelofgradient 9·25'Cacrylarnide was employed (-t3). A 3COstacking gel was layered on the topofthe running gel. Samples,were mixed with 2~50S,5.7%~merc:lptollthanol.IO~l'rolandbromophenol

~!:boiledfor 3 minandthE' DleadedonLthe gel.

Initiallythe electrophoresiswasearn ed outct60 V in 0.6

cr

Tre.2.88

cr ·

glycine and0.2%80S buffer.ACtt' rthesamples hnd entered l·herunning gel;the

•volta gewas increasedto120 V.Following elect rop horesis. the gelwasstained'

with brilliant blu:iCoomassiE'R·250jand destainedwuh methano!I·')"cr~' I \'j 3nd arl'tif acid {7.S

re.\"/\").

I ,. '

I

1.1.6. AMINOACID:AND N-TE RMINA L ANALYSES

50109ofsample werehydrolyz edin 200"Iof(l:-'1HCI:11110"(' (or:H h.

ThehydrolysisW3.Star riedoutinaelcsed ampul!"whichwns initia lly!"\';If'\I:ltt,,1 Aminoaddanal ysiswaspt'~formedon aDururn Amino Acid..\nal~'lNh~'lhl' AminoAddFacility,TheHospit alforSickChildren.Toront o.

ForNaerminelaD~lysis,10Ilgofsampleweredissolvedin20"I of 0,2 t\1 ,NaHC0 a. and2~,,~ ~~,DNS-CI^(2mg/ml

in

^acetone].MtNi~(,llhntion^at37°C.fnr

1-2b,th esamplewasdried and hy dr oly zed wit h6MHel. at110"('forru-ru h.:

,

Poly~mid~ ^{'T Lt } ?}

^emx5ern]

~~,

^{used toid en tifythe}

.~~I":S('~nl

:-:.tl:rnina\ r.

aminoad dbycom par in g thepositi on ofthesamplespotononesideoft .':pl:lh' wit h thos e

c r.

tbe aminoacid

~"tandards

^{onthe ot he rsidI'aftl' r1\\'0dim:}

.il~ilJnal

^.

runDing (44)-.

The solven ts usedinTLC wert1..) '(. formicarid forthelstt1iml'n~ion,. benzen e: a~etic acid IU (\'/vl for the-2nd dlrm-nsinn (bl ~Cll\"l'nlj, und etbylacera te.met henol.aeetic ad d20:1:1(\'/\'/.\"1

ror

tln-2nd dimi'li sin/l(2nd solvent].

,

,

1,1-7,MEASUREMENT SOF THERMAL HYSTERESIS'CFOCEA N POUTAFP

The rmal hysteresiswas measured by '. ,

~L

^II.nan llsin,;1\ nnndlilfl' osmo!"lE'ler (Clift onTechnicalPhysics.Hartford. CT,I'~,.\). Cry-rnl,;rnwlhor dis~olut ionwasobserved underalightmicrl)S('IIIll'. Theo~llIl1l1l1'lNw:.,rir,l calibra tedwith :'\a(,1solutionsorknown nsrnolnlitie s. The l"m(Jla lit i,·~·firth,·

AFP solutionswere then obtainedand convertedintothermalhystereses(T H.Iin

_!-1.8.PURITY OF REAGENTS'

SephadexG-i5, Sephadex G-25,QAE-Sephadex A25 'andSP·Sephadt'x ('.')0 were,pureheeed from Pbarmaeia [Dorval.,.~foDtreall. ^{Reagents for SO,SI} polyacrylamide gel elect rophoresis, such ,as, SDS, aerylamlde, ammonium persulphate, TEMED,hromop~e~ol blueand,Coomassiebrilli,anl blue Wer e

"ElectrophoresisPurity Reagents"andpurchased from Bio-Rad [Richmond,CA- USA): Acetonitrile was"HighPuri~~Solvent~end purchasedfrom Burdick5.:

JacksonLaboratories,Inc.(Muskegon,MI,USA). All other~~emil'alsw~reof reagent grade.

L2.RESULTS

1.2.1.PRESENCE OF A MULTIPLE COM PO N E NTFAMILYOF OCEAN POUT AFP

Earlie~researchfromOUtlaboratoryhadshown thattheslower·running peakfromSepbedex G75 ehromatog'raphyhad antifreezeactivit ynnd thaiWh~'ll

~

^{thiS AFP}(designated

Is.

^{G-75 AFP')wasfurther}fractionatedby'ion-exchange chro matogra phyI andreversephaseHP~C,it was .resolvedinto'at least eight

act iveecmpcneau(22).

Inthepr~entstudY:theG.75AFP (Figure I] wasresolved intoIivegroup~, i.e.QAEA,SP. I;~P~:l,SP~3aDdSP:"on QAE andSP.Sephadt'xion-exchange chromatog ra phy(Figure 2).The G·75,.\F Pcould be resolvedannl~'lienll~'into at

.J'

F';~:'

^1. co"ma'Og, ; p01

. 01 , J..

^pO"

^.'ot"

^mum" • '.pO""

0,- 15 ,2';1- tU t nt,10DcolulIlD ill0..1W_HH4'HC03buff er at.,DC. .- Tbi umple ,"olum• . .. ^I)mI. Th.'U n topUll: bad ao. t of tb. . . rum ptot dn.lJl.dthl . .eoadau CODtall1l dtb. allt UruupolJp.ptidu

~d"lgDat.d..C·76AFP).(Un tfllll.:_ ;r.cbro lUto l rap llJ _ ).

-co-

2.0

Ec

?l

N

.... -c w

_1.0

() Z

-c

(IJ

a:

0 rtJ(IJ

<

0 0

0.0

3J 40 50 60 70 60 90 100

FRACTIONN.JMBER

·::1·

Figure 2.(a):CbrolU.t.ographyofG-76 AYP OD a QAE-Sephadn: (A26)coll1llll lD 6mYTri.-HCI bUf te r, pH 9.4 at.rqolltemperature. The folWllll.

wa.

elutldwlt.h a 0.0 to 0.2U HaCIgradieDt1D the .ame bufter. The Unt puk wa. QAE-unbound puk.ud the ucond one·

wa.

dUignatedQAEA.

(b):Chromatographyof QAE-ullboundcompountonSP-Sepbadn: (CSO) col Wllll.

in 0.01'yHaCl,pH3 at room temperatu re. Thecol l1llll .... elutedwitba 0.01to 0.2NH~lgradient,pH3. Thef~ur'pe.lu obtainedure de.igutedSP~l ,SP-2 ,~SP-3andSP~4ru p ecUve!y i li order of elUUOli

tilDe. •

... .. .

.:!:!.

2.0

(a)

1.0 0.2M

0.1M

0.0 O.OM

E

_{60 70 80 90}

z

0 0 10 20 30 40 50 Q

C')

'"

^.~

f- 2.0

a:

-c

^f-_Z

ei o ^w

z 15

<

_CD

(b)

o

f5 '" ^u

~ z '"

0.2M

0.1M

O.OM

60 70 80 90 100 110 120 130 140 150

FRACTION NUMBER

leasttwelve componentsce reverse phase HPLCIfigurt>3).Theser" ~ult s"llnf,tlil anear liersuggestion thatoeea upoutAfPis amultipler~mil)'urrlns"lyn,la';·,l

\ .

components(22)

1.2.2.CORRELATION OF G.7SAFP WITH INDMDUAL COMPONENTSFROM ION-£XClIANGE·

CHROMATOG~PHYUSI N G REVERSEPHASE HPLC' A$shown inFigure4.there was a goodecrrelencn.betweenG·';SAfp and peaksQAEA.SP-I.SP.2.SP-3andSP-4.TheseTivefractionstromion-exehange

.

' , ~

-'-e-cbromatograpby couldaccount Ice allthe componenta-Iromthe G·, :' AFPon .analyticalHPLC. indicating thatthe.lracno nationproceduresweresuccessful ReversephaseHPLCalone canIractiouateallthecomponentsseparated by QAE andSP;oo'i.,.hang•chromatography. Itappearstoh.theh."way to prepare

puremate; Is. .

.1.2.3. CHARACT ERI STICSOF OCEANl'OUT.AFP 1.2.3.1.MOLECULAR WEIGHT ESTIMATION" .

Ongel-Iiltratio u by HPLCwithtwoTSK·3QOO-SW colum ns.themolecular

. . .

. .

weightsofSP· 2,SP-3andG·75AFP wen' allnearlyth,·same.'r.a~ p;ingIrom around 5,700 to6,500(F igure51.

It was alsofound thatthemolecularweigh'testlmauon using1?K-;J()()().SW columnswas quitereliable. A mixtureof insulin, SP-Iandnprotininewas- resolved;",0

t h'.~

^peakswith aprotimne .I,, ;,g\,\:,SP· lsecond end

;""Ii~

^th.·

IM t.

~ ~~

~,

-.

-.

7

· >/

-24-

--- --:'

, \

F1Sun 3." Chrout.osraphy of C-76ATP,Qn r ....n. pha .. HPLC~.06"

TFA'at. roolll t'lIlJl.ratun.Suph.wen duted w1th&l1.e.ton rU e sr&dlent

a.

indlcated q,dnWllbered 1t.o12rupectlYdyInor er~t

elution till.. Tb. colUIID u..d

wa.

"BoDdapalen: (7.8lllIIlI.D. ··

ti-

:Iao em) (I.ten).

n.

flow rat• . ..11Il1hdn. -

-2["

12 0.7

50 11 0.6

E<:

0.5 ⁰

4 ^C')

'"

~40

004^f-<:

c: t::

w

z o

0 0.3

z

...

<:

UJ III

()

a:

~30

0.2 0

VlIII

-c

0.1

20 0.0

25 30 35 40 45 50

ELUTION TIME (min)

·,1>

\ -,-v•

. .

Fiiurl4.. Cilroutolupb,rof 0"'76~. Q.AF.A..SP-1.SP-2;SP-3and~':'4 OD.

r" ...

ph....HPLClao 0.05" TFA at toOlOelmplnt.ura. Th, coluu..

~...t..W"~~lldapa.tTJIC18(7.8,. I.D.x3~,.clI) (latlt.>. Thl otoll':cODd.1t10D.'. , ntobt.. .... a.in"Filufl~.

.r : ....

.. I. '

.;

. .

"

0.2 50

40 30 0.1

20 10 E 0.0 0

.§

0.2 50

'"

1

^40~

'"

I-

«

QAEA ₃₀

Ii: _~

w0. 1

20

~

o

Z

-c

w

10

~

CD

a:

'"

0

~

^{0.0 0}

-c0.2 50

B 40

A SP-1

0.1 30

20 10

0.0 0

35 40 45 50 55

ELUTION TIME (min)

0.2

0.1

§

0.0 00.2

'"

N f-<:

w0.1

o ::;:

<:

m

gs

gJ 0.0 -c

0.2

0.1

0.0

SP-2

~

SP-3

~

SP-4

- J~

35 40 45 50 55

ELUTION TIME(min)

50 40 30

20 10 .

o

50 40~ _a:

30~ o

20t;j 10~

o

o

50 40 30

20 10

o

,-;-

·2ll-

G

.

. /

F1sur.5, E.t1aatioll of t.i.mol.cular'...lghfo. of oc._pou~AFP on gel-u'hrat.1011HPLC. Flgur." (a).(b) and (e) anfoh''pr oU I ..of G-75, . . AFP.SP~2&Il.d SP-3 ill 45 I actton1t.rU. (collt a l nl 11g 0.1"TFA)011tf0.'

SpherogdTSII;-3000-S1 (2':17..6IllII.1.D.:I:30 cm)(B.cbI&lI) c:olulII%II. 'Th.

flo..

rat• • • •0,5 .1/.,111.. F1guu Cd) 18tb plot of~lut1ont1m. vu.u.

Le:mol.cularwligbt. Th• •tudard. w.re B (DSA.6BtD.).C(cJ't0chrolll.

C.13tD). A(apr'ot11111ll. e.stD.).I (1DlUl1D.5.7·tD).S,(.horthorn IculpiD AFP.4..3 ltD.). F (floullder-AFP3.3tD.).1A(1l:I1~l1D/-cha1n. 2.5tD.). •~ .t~d'for,De"'D'poutAFP.

'.

(

..

0.2

Ec:

o<'l N

~

UJ0.1 UZ -cCD

a: o

(f) CD

..:

0.0

4.5

;:

~

Cl4.0

..J

3.5

-.1(}..

(a) (b) (e)

I~

^<,

^{.-.J ' - -} [ - - J ' - - -

30 35 40 30 35 40 30 35 40

ELUTION TIME(min)

oS

'

0 (d)

I\.S

I F

·IA 20 25 30 35 40 45

ELUTION TIME(min)

, ,

-3 1-

By SOS polyacrylamideslab gf'1 eleermphoresis.,hi'estimatedIlInl"fuhlr weights of the' G·7SAFP,QAEA, SP- I,

sp-a.

SP-3nndSP-.jwl'n'allunder u.sco (Figure6).

Based,on these determinations.the molecula r\'it'ight:> of(;·;5Af'P.QAEA, SP-IlSP-2,.SP~andS.P.4 areall similar.,.Th(>~'are between~700end e.soo.

Thesevalues wereingood agreement withthemolecularweights estimat ed using an analyticalultracentrifuge(22).

~.

.

1.2.3 .2.IOdINO ACID ANALYS ES

. . *'-

There'areflleast threecomponentsinSP-I, namelySP-I.A ,SP-I-B and Sp·I-C (see Figure4.1. T~were ecllected.tromSP~Iand reehromatogrephed individuallyonHPLCwith a less-steepgradient. The resultsof the aminoadd

a~alYses

^{ofQAEA,SP-I-A,}

~P-I-B'

^and,SP-I·e areshown in Table

t:~A,

SP·I·A, SP-I ·Band

sp -t- c

allconta inedfourteen to fifteentypesof d!ffert'Dt amino acids. Arg was present in QAEA,but~otin any ofth~Spotcomponents (SP-I-A,SP-I-Band

sp:i'-Cj,

Theamio~aci.d compositions~fSP-t-1-, SP.I-D andsP-I-ewere veryaimilarto eachother. The ral.'ttbattheAlaconten tisvery

' '-

modest and that thereisno half·cystineatall would suggestthat unlike(lound~r AFP which

ba.~e

^{a veryhigh}

c~nteDt

^{ofAla (60 my%). orsearavenAFP}

Wh~/

have an extraord inarilyhigh contentof half.15ilne(8 mol%),oceanPPurAFP

.

^{, " '. ,/ /}

represent a Dew type offish AFP.These resultswere[n gooj,J.greemt'ntwith the previousobs~rvatioDsmade in pur labora tory (22J.

Itshould bepoiDted'~~~hattbeealeuleue e cr.t he numberoraminoa{"~d­

'residues'Cor a givensampl:'wasmade-fn-;rat her arbitraryway. The avetage

),

ABCCDEFGABDE FGA

+

Figure

e.

Polyacrylamid . gil d.ctrophoruhof OC. 1Jl poutAFP. Lu.u A. B.C. D. E. F and G corrupoDchdto .t.a-nda.rd •. (i-75 AFP.QAEA.SP-L SP-2. SP-3 and 5P-4rup.ctively. Th. Itu.d,ard. cOIl..ht.d of ovalbumin (43to.), C:hymotr 7Jld ll.og ' DA(25.7to.),1110zym. (14.3to.),a.protinin.

(6.5to,) a.ndiuulinAchain (2.5to.) (not .ta.iud).

"3-

Ta bl.l. Amino AcidAnalp l'SofthE'O('(':lRPout AFJ>

t~'il'ldinnmcl]

SP·14A SP-t-B

sr.i.c

'QAI:::,t

k,

'.17(4 1' 1.81(41 ".80("). 15.76

Th, 4.62(6 ) 3.28(7 1 0.s.1(8 ) 1.1.1':'

S" 1.°"121 0.8O('} :U1{2}

.

i.O-1

GI" ^8.06(7) 2.42(6} O.B"(8 )., 14.82

Pm ^{5.24(6 )} '.50161 7.10(6) 21.4-t

Gly '." IS) ^{2.0 5(5)} '··\81') ^IUD

Al, '.21(6 ) 3.0-1(7)

7 'P

^IB.DO

V,I

'. 17(7)

^{3.13(1) 6.i6( I 22:.01}

- I

M" '.12('-) 2.2:8(5) _6~51 13.71

II. 3.62(") .2.001 ::'1 5.46(5) 10.58

Leu 1.60(2 1 1.47(3) ~.6O(3t 16.12

'"

Tyr 0.7b1l) 0.6211) 0.04(1) 2.23

Pb. 0.86(1) 0.30( 11 OJI..jll 0.18

Ly. 3.20(4) 1.7 5(4 ) -l..4':'(4)

' ot'.

Alg ^1;.60

...--

Total (60) (6') (64 '

M.W.

630'

^{6511 '6i 61.}

omol/r es.* 0.85 0.45 1.1l0

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