- Email: [email protected]
A conformational study of normal and dicoumarol-induced prothrombin
Volume 32, nurnbex 2
I~EBS L E ~ E t t $
3tme 1973
A CONFORMATIONAL F=TUDY O F N O R M A L A N D DICOUMAROL-INDUCED PROTHROMBIN Ngema~ B ] 0 R K and J0han S T E N F L 0 ]nsElu2e of Medical .ChemUrgy, University of Upp~zIa, Upps,L~ :rod~l~e ~et~rt~nent of C~n~eat Chemistry, Uni~,ersity of Lund, Mz~?,,IOGeneral Hospital, Malm.~, Sweden - ~ e e e ~ e d 1_9 NIa~ch ~ 9 7 9
0.002 M EDTA. They weIe flxen dialysed against the same buffer w2thont EDTA for abo~t 16 h ~ before use. abnormal protb~,ornbkn ~ n d u c , e d by d ~ c o n r n a ~ o ] ~dmird~trat~on ha~ been pmified flora bovine plasma I[1, 2]. Tiffs abnormat Wotl~ombin h~s the s~me main anl~igerd.g de'tenminants as normal prothromMn, but no p r o ~ r o m b i n acdvity l t , 2]. While normal p l o m r o m bin has th~e~ to four high-affmiIy binding sites for calcium ions per molecule, .flae abnormal protein binds at most one ca]cimn ion [3, 4]. F u r ~ e r m o r e Ca~+ ~ duce~ a confo~mafional change ha nonna~, b u t n o t in ab.norm~l, prothzombin, as shown with immunochemlea] ane,thods and suggested by equitibadum dialysis I3, 5]. Since norm~i Ca=+ b i n d i n g 5s neeessar~ for the phyMological activationof prolbJombin, a more detailedinvesfigatlon o f the conformai~on of th.e two prothxombins m the absence and ples0nce of Ca ~÷ seemed wz,~xanted. Tiffs paper reports such a study v~th the use o f opfieai rotaiory dispersion fOP,l)) and cilculaI .dichroism (CD).
L~+rds w a s n e c ~ s s m - y ~to r e d u c e
3. R e s u l t s
2. Materials and methods
NOllnal as well as dic,oumarol-induced p l o t ~ o m b m was purified asdescribed ,exrlier ll]+ Protein .concentrations were measured from the absolban.ces at 280 n m with an extinction ,e~f~cient ( E ] ~ m at 280 iwn) o f 14.5 for b o t h 9rotlar.onab~,s [5], Optical x.otatory dispexsion and circular di~lar.oism ~ ~pectra were measured with a Jasco Model J 2 0 zpeel roi~g!arimeler (JapanSpeetr0scopic Compan], Toleyo; J a p a n ) . T h e protein~ studied weredissolved in 0.02 M T ris-HC1 buffe/~ p i t 7;2feontairhng0.1 M NaC[ ,and + . -
"-2
=-
- %
•
.
-
2
-
-=
"
Z
-.-
~-
+
_
t h e !ig]~t abso+L-pficr~ a t
lower waveaengths caused by the EDq?A. In some experiments CaCt~ was added ~o a Final concentration of 0.005 M. The spectra wele recorded at room temp. (25°--27"), with protein concentrations of 0.4--0,8 mg per ml and ce~h ha~dng 10--0.5rnm paflflength. ~ c Iest~?ts ale given as plots of ~eduo~d mean Iesklu~ rotation, [m']. o~ reduced mean ~esidue el~fpticiay. [0'], against w a v e ] e n g , . The parameters [m'] and I9'] were c o m p u t e d in the usual manner [6, 7]. The mean reAdue weight o f ~he two prothrombins was c a l c u l a t e d from t h e ~ n ~ n o a c i d compoMtion ~5] a n d was f o u n d to be ~ I4 for bo~h of flaern.
The ~,onfo~na~n of non-n~1 and ~counaBrol-in+ duced proth~oJ ~bin ~n the absence and p:c~cnce o f ealc,Xum ions was investigated by optical lolaI0r¢ dis_pers~on and ck, r a h r dichroism, It is c.]ea~ from fig. a that, in .the abs:nce of Ca =*, ~.e ORD spectra showed no confonnati~,na! d~feren,ccs between the two pro~i.ns lhat cannot be accounted f o i by experimental error. Bofla cm';es have ~hnirna at 232 n m with lm'] O values .about --,..DO0, anti bohh have a shouader m o u n d 220 n m . The t~}o spectra agree well vAth the spe~trn m ofnoTmal p m t h z o m b i n measured by ln.gwail and Scheraga f8], b u t only flown to a wavelength o f a b o u t 230 rim; for untmown reasons ~ m e differences a r e evident a~ lo~erWivelen.gihi. No tabiy, .fl~epoint 0£~eio rotation is at-about 220 rim-in~he+spec.~mmo£~,gwall
<
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+.
.
.
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.
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~,43 i
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Aro]'urneB2, number 2
June 3[973
F'~BB L[ETTYERS
mO~" A
19
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Wov~]er~gth. t~m
,.f 0
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k
~1
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.~ - 2 0 ~
Fig. 1. Optical r~alozy d~sp~ision zpeclra o$ norrna.~ ,( ) and ~eounaaro]-indu~ed (- - -3 p z o t ~ m b i n in ~he ab~en~ of calcium ion~.
,~" -Z,OOD
and Scheraga [8], b u t at 206 n m ~ oua" cu~'es. The flax,ough at 232 nrn is indicagve o f some ~-hel~x in b o t h normal and abnormal pro~h~,omb~n |9], b u t the ]ow magnitude means that the ~-he]i~ cont,en,~ mus~ be very smal],~nd calculations of percentage me there. fore meaningless. A low ~-helL~ c o n t e m of n,o~mat prolhrombin was ~]so suggested b y ]ngwal] ~nd Scheraga [8] on the b~sis o f analyses o f ~.heir OR,D-data b y .flae Moffitt-~ang ~ quadon. Like the O R D results, s t u d ~ s by CD in the absence of Ca 2÷ did not show any conforrnatkm~d differences outsi.d,e ,experimented error be~we.en normal and a b n e r ma] pro'thronabku: either ~ lhe aromatic region (fig. :LA'~ or in the peptide b o n d region .(fig. 2B). In the latter ~egion the t w o cur~ez have minima b e t w e e n 202 and 2 0 4 n m with reduce0 m e a n residue ,elhpticities o f a~ound --5000~ and also have broad shoulders between 2 2 0 mad 2 3 0 .rim. Taese feztur~s are n o t compatible With any o f flae spectra of the three mode] .conforma. tions ~or plOtein ' z ~ C ~ l . e ,or combinafion~ xhereof ~9, ]'0]; specifically no minhnz ,characlefisfie of the ~x-hdix wele found, which also suggests that fhe conten~ .of this structure in proth,~ombin is very low.
~o
-~5000 I
I
200
2;0
-
1
230
I I
2z,O
I
250
Woa~eJe~flt~h, nrn Fig. 2. ~ c u l a r dickroism sp~eaxa of normal px~thr~rnb~ ~he absence { ) and presence {. . . . .)~f cale~lam ictus, and of dico~nmol-Lu,da~eed pro~hsombm als.~ ha Xhe absence & - -) and presence @.-..) o; ealeimn i~n~. A) A~,o~naficre#on; B) pept~fle-bondre.on.
_qaae conformaiion of the t w o plotekus :,anthe pl,e~ n e e ,of C a =* w~s s.-~adSedonly b y C D (fig.2). No~real p i ~ h T o m b h a ex2fibits a m ~ k e d change ha 1abe aromatic reg~n of the ~pec ~ m on binding ,ofcalciu m ions (fig.2A), w]a~reas no su,oh confoTrnadona] change was found ha the case of the ,dicoumaro'l-Ludueed protein, lu contrast, no ~iff.erenee ;m the pep-
~dde b o n d xe.gion o f the 'spectra were f o u n d t o aCCOln-
p.any ealgmm-bhading by neither protein ,(fig. 2B).
3,b~ J
~
220
-
q
Vo]gme 32, number 2
FEBS LETTERS
4. Disedssio~
Earlier Lnvestigations have shown that the properties o f normal and dicoumaroMnduced prothrombin k~ she absence o f calcium ions are very similar, al~laouo~ not identical. The abnon'nal protein has a somewhat lower electropholetic mobility than normal p r o t h r o m b ~ I t , 1 ] ], and the two proteins can also be disth~maished by quantitative immunoprecipiration 15, 2]. It has been suggested that these differences are due to a ~=~Jnorstructural d~fference, e.g. in d]suhqde pai~ng, Ln a prosthetic group or in conformation. Attempts to reveal a conformational difference b y titration o f phenolic ~ o u p s and by detemfination of fluorescence emission spectra fai]ed kn that identical rosa]is were obtained for both proteins 15]. Neither did the conformafional probes used in the present investigation, i.e. O ~ and CD analyses, produce evidence o f auy difference m the c o n f e r m a t i e n of oNclam-free, non,hal and abnorma] prothrombin. The inference o f these resuits is that the strueturM difference between the two proteins, of whazever kind it m a y be, must be so small that neither o f the latter methods is capable of detecting ~t. A result pertinent to flais conc]usion ~ that recent experiments, where pepfide maps were prepared f r o m NH2-termina] fragments, which constitute appro~dmately one t,hird o f the intact prothrombins, revealed clear differences between the prothrornbins. On the other hand the Cterminal fragments had identical e]ectrophoretic mad irnm~nochengeal properties [11]. Furthermore, N d s e s m e n and Suttie have estab'li~hed that activation o f normal and abnormal prothrombin by trypsin gives rise to the same amounts o f thromb,'m !2], ~ d i c a t i n g that the major varts o f the two molecu]es are identical. When c~deium is added, normal and diconmaroMnduced Nothlombin behave~ differently. The normal prolh~ombia molecule brads three to four cMcium ion~ with l'Agh affinity, wh2~le ~he abnormal pxote~ri binds a~ most on~ calcium ion with comparabN bindins strength I3, 4]. Simultaneously, a change occurs in lhe antigenicstructureof normal pr,othzombin but n o t ~ ,that o f the abnormal protein 13, 5]. This ~ s been interpreted as reflecting a conformafional c~hange induced b y t_he binding ,of ealemm, a conclusion .raft_her ~ p p o r t e d b y the anomalous shape ,of the Scatchard pl0rl:s .obtained Ln equilibrium ~'alysis experiments 13], 'Our cixcular diclaroism data eo~roborrate these results.
J~ne t973
The CD ~pectra ob~dned with and wiLhout Ca ~ in@kate float ,flais ion induces a conformafional transition ~ nor. real p r o t ~ o m b i n but n o t 5n the abnormal protein. The change is expressed only ",anthe aromatic region o f the spectrum and is therefore presumably o n b, z local chin, ge in ~he nei~shbourhood o f some aromatic amhuo ae~_d. This is ~ accoxdance wiLh earlier resuhs, which indicate that the catcium-b,ar.d~g groups o f high afFmiW reside only m the iNq-12-termma! p~rt o f the mo]ezule [11]. However, the experiments do not permit any conclusion about the exact na#ure o f the conformations] change. In conclusion, all tradable da~a indicate that the main functiona] difference between norma~ and dO. cournaroMnduced prothrombin is in their calciumbinding and ~ the properties i,'duced b y this ion. Normal protrarombin binds an opfimN a m o u n t o f calcium ions, which causes ~ e protein to assume a conformation that allows its physiolo~ca! aciPvation ~o thrombin to ~ake p!-~ce. Abnormal prothrombin, however, lacks the ability to bind a comparable a m o u n t o f calcium and @~refore carmot be activated in the normal way. He ¢¢ever, under physioaogical conditions the calcium level is such that the active conformation o f n o r t o n p r o t h r o m b i a is presumably a~]ways maintained in the circulating blood
~Aeknowledgements This irtvestigalion was supported by ~ a m s from the Swedish Naiural Science Research Council (Project No. 3055-]) and ~JaeSwedish Medical Research Counc~ (Project No. B72-iJX-581-08A),
References ~ ] J. Sten.qo an~ P.O. Garao% J. BioL Chem. 247 (~972) 816I). 21 G.L. Ne;~stuen mad J.%V.Sultie, J. BioL Clhern. 247 (1972) 8176. ~3] J. SlenEo anti P,O, Gm~,iot,Biuch~rn. Biophy~ Res, Cornmun. 50 (1973) 98. 4961. 15] J. S*enlqo, 3. Bi~t. Chem, 247 (1972) $a67. ~6] KJ , Dor_~ing~n, M.H. Zaflengo and C. Ta;ffol~ Pxoc. Nail. Ac,ad. SoL U.S. 58 {1967) 996. 345
Vdlume 32, nam~oe~ 2
FEBS LE~ITERS
17] ]a..E. Calhou, A. Kxalezyerad,Jx. and E. HzTaex, ]3ioehemistry 7 ,O96g) 3958. [8] J.S. l~agwall and 1 ~ . Sehe~aga, ~B~o~hemis~ry~ (:t969) 1860,
34.6
JuneT973
1'9] N. Gxeen~eld, B. Davidson ~md G.D. ~Fasm~xl, Bioche,m~sLry 8 .(2[967) 163~. [ 10] N..Gxeen~e]d and G.D. ~Fasman, B~oc~h,emistry 8 (.:196'9) 4t,08. ].11] J. Ste,nflo, to be l~ubl~shefl.
I~EBS L E ~ E t t $
3tme 1973
A CONFORMATIONAL F=TUDY O F N O R M A L A N D DICOUMAROL-INDUCED PROTHROMBIN Ngema~ B ] 0 R K and J0han S T E N F L 0 ]nsElu2e of Medical .ChemUrgy, University of Upp~zIa, Upps,L~ :rod~l~e ~et~rt~nent of C~n~eat Chemistry, Uni~,ersity of Lund, Mz~?,,IOGeneral Hospital, Malm.~, Sweden - ~ e e e ~ e d 1_9 NIa~ch ~ 9 7 9
0.002 M EDTA. They weIe flxen dialysed against the same buffer w2thont EDTA for abo~t 16 h ~ before use. abnormal protb~,ornbkn ~ n d u c , e d by d ~ c o n r n a ~ o ] ~dmird~trat~on ha~ been pmified flora bovine plasma I[1, 2]. Tiffs abnormat Wotl~ombin h~s the s~me main anl~igerd.g de'tenminants as normal prothromMn, but no p r o ~ r o m b i n acdvity l t , 2]. While normal p l o m r o m bin has th~e~ to four high-affmiIy binding sites for calcium ions per molecule, .flae abnormal protein binds at most one ca]cimn ion [3, 4]. F u r ~ e r m o r e Ca~+ ~ duce~ a confo~mafional change ha nonna~, b u t n o t in ab.norm~l, prothzombin, as shown with immunochemlea] ane,thods and suggested by equitibadum dialysis I3, 5]. Since norm~i Ca=+ b i n d i n g 5s neeessar~ for the phyMological activationof prolbJombin, a more detailedinvesfigatlon o f the conformai~on of th.e two prothxombins m the absence and ples0nce of Ca ~÷ seemed wz,~xanted. Tiffs paper reports such a study v~th the use o f opfieai rotaiory dispersion fOP,l)) and cilculaI .dichroism (CD).
L~+rds w a s n e c ~ s s m - y ~to r e d u c e
3. R e s u l t s
2. Materials and methods
NOllnal as well as dic,oumarol-induced p l o t ~ o m b m was purified asdescribed ,exrlier ll]+ Protein .concentrations were measured from the absolban.ces at 280 n m with an extinction ,e~f~cient ( E ] ~ m at 280 iwn) o f 14.5 for b o t h 9rotlar.onab~,s [5], Optical x.otatory dispexsion and circular di~lar.oism ~ ~pectra were measured with a Jasco Model J 2 0 zpeel roi~g!arimeler (JapanSpeetr0scopic Compan], Toleyo; J a p a n ) . T h e protein~ studied weredissolved in 0.02 M T ris-HC1 buffe/~ p i t 7;2feontairhng0.1 M NaC[ ,and + . -
"-2
=-
- %
•
.
-
2
-
-=
"
Z
-.-
~-
+
_
t h e !ig]~t abso+L-pficr~ a t
lower waveaengths caused by the EDq?A. In some experiments CaCt~ was added ~o a Final concentration of 0.005 M. The spectra wele recorded at room temp. (25°--27"), with protein concentrations of 0.4--0,8 mg per ml and ce~h ha~dng 10--0.5rnm paflflength. ~ c Iest~?ts ale given as plots of ~eduo~d mean Iesklu~ rotation, [m']. o~ reduced mean ~esidue el~fpticiay. [0'], against w a v e ] e n g , . The parameters [m'] and I9'] were c o m p u t e d in the usual manner [6, 7]. The mean reAdue weight o f ~he two prothrombins was c a l c u l a t e d from t h e ~ n ~ n o a c i d compoMtion ~5] a n d was f o u n d to be ~ I4 for bo~h of flaern.
The ~,onfo~na~n of non-n~1 and ~counaBrol-in+ duced proth~oJ ~bin ~n the absence and p:c~cnce o f ealc,Xum ions was investigated by optical lolaI0r¢ dis_pers~on and ck, r a h r dichroism, It is c.]ea~ from fig. a that, in .the abs:nce of Ca =*, ~.e ORD spectra showed no confonnati~,na! d~feren,ccs between the two pro~i.ns lhat cannot be accounted f o i by experimental error. Bofla cm';es have ~hnirna at 232 n m with lm'] O values .about --,..DO0, anti bohh have a shouader m o u n d 220 n m . The t~}o spectra agree well vAth the spe~trn m ofnoTmal p m t h z o m b i n measured by ln.gwail and Scheraga f8], b u t only flown to a wavelength o f a b o u t 230 rim; for untmown reasons ~ m e differences a r e evident a~ lo~erWivelen.gihi. No tabiy, .fl~epoint 0£~eio rotation is at-about 220 rim-in~he+spec.~mmo£~,gwall
<
--
+.
.
.
" -_
%
;=-.
-~+
-:
.
2
/.7+-i "
-
_ __
:
_
:
~,43 i
::
Aro]'urneB2, number 2
June 3[973
F'~BB L[ETTYERS
mO~" A
19
..~
|
..-
./~
i
~
-506
'il
•i
I
e..~\
,
-,
-
"x
/li
%
/--
\ \\
,i'7 .'J
,eft
,i
.%
/
/ . -2DDO
,
•
fl
i
q
250
250
270
2~0
II
300
Wov~]er~gth. t~m
,.f 0
h, I/
k
~1
2-~
~
2~5~ Y'/o~elen.gt,% n m
.~ - 2 0 ~
Fig. 1. Optical r~alozy d~sp~ision zpeclra o$ norrna.~ ,( ) and ~eounaaro]-indu~ed (- - -3 p z o t ~ m b i n in ~he ab~en~ of calcium ion~.
,~" -Z,OOD
and Scheraga [8], b u t at 206 n m ~ oua" cu~'es. The flax,ough at 232 nrn is indicagve o f some ~-hel~x in b o t h normal and abnormal pro~h~,omb~n |9], b u t the ]ow magnitude means that the ~-he]i~ cont,en,~ mus~ be very smal],~nd calculations of percentage me there. fore meaningless. A low ~-helL~ c o n t e m of n,o~mat prolhrombin was ~]so suggested b y ]ngwal] ~nd Scheraga [8] on the b~sis o f analyses o f ~.heir OR,D-data b y .flae Moffitt-~ang ~ quadon. Like the O R D results, s t u d ~ s by CD in the absence of Ca 2÷ did not show any conforrnatkm~d differences outsi.d,e ,experimented error be~we.en normal and a b n e r ma] pro'thronabku: either ~ lhe aromatic region (fig. :LA'~ or in the peptide b o n d region .(fig. 2B). In the latter ~egion the t w o cur~ez have minima b e t w e e n 202 and 2 0 4 n m with reduce0 m e a n residue ,elhpticities o f a~ound --5000~ and also have broad shoulders between 2 2 0 mad 2 3 0 .rim. Taese feztur~s are n o t compatible With any o f flae spectra of the three mode] .conforma. tions ~or plOtein ' z ~ C ~ l . e ,or combinafion~ xhereof ~9, ]'0]; specifically no minhnz ,characlefisfie of the ~x-hdix wele found, which also suggests that fhe conten~ .of this structure in proth,~ombin is very low.
~o
-~5000 I
I
200
2;0
-
1
230
I I
2z,O
I
250
Woa~eJe~flt~h, nrn Fig. 2. ~ c u l a r dickroism sp~eaxa of normal px~thr~rnb~ ~he absence { ) and presence {. . . . .)~f cale~lam ictus, and of dico~nmol-Lu,da~eed pro~hsombm als.~ ha Xhe absence & - -) and presence @.-..) o; ealeimn i~n~. A) A~,o~naficre#on; B) pept~fle-bondre.on.
_qaae conformaiion of the t w o plotekus :,anthe pl,e~ n e e ,of C a =* w~s s.-~adSedonly b y C D (fig.2). No~real p i ~ h T o m b h a ex2fibits a m ~ k e d change ha 1abe aromatic reg~n of the ~pec ~ m on binding ,ofcalciu m ions (fig.2A), w]a~reas no su,oh confoTrnadona] change was found ha the case of the ,dicoumaro'l-Ludueed protein, lu contrast, no ~iff.erenee ;m the pep-
~dde b o n d xe.gion o f the 'spectra were f o u n d t o aCCOln-
p.any ealgmm-bhading by neither protein ,(fig. 2B).
3,b~ J
~
220
-
q
Vo]gme 32, number 2
FEBS LETTERS
4. Disedssio~
Earlier Lnvestigations have shown that the properties o f normal and dicoumaroMnduced prothrombin k~ she absence o f calcium ions are very similar, al~laouo~ not identical. The abnon'nal protein has a somewhat lower electropholetic mobility than normal p r o t h r o m b ~ I t , 1 ] ], and the two proteins can also be disth~maished by quantitative immunoprecipiration 15, 2]. It has been suggested that these differences are due to a ~=~Jnorstructural d~fference, e.g. in d]suhqde pai~ng, Ln a prosthetic group or in conformation. Attempts to reveal a conformational difference b y titration o f phenolic ~ o u p s and by detemfination of fluorescence emission spectra fai]ed kn that identical rosa]is were obtained for both proteins 15]. Neither did the conformafional probes used in the present investigation, i.e. O ~ and CD analyses, produce evidence o f auy difference m the c o n f e r m a t i e n of oNclam-free, non,hal and abnorma] prothrombin. The inference o f these resuits is that the strueturM difference between the two proteins, of whazever kind it m a y be, must be so small that neither o f the latter methods is capable of detecting ~t. A result pertinent to flais conc]usion ~ that recent experiments, where pepfide maps were prepared f r o m NH2-termina] fragments, which constitute appro~dmately one t,hird o f the intact prothrombins, revealed clear differences between the prothrornbins. On the other hand the Cterminal fragments had identical e]ectrophoretic mad irnm~nochengeal properties [11]. Furthermore, N d s e s m e n and Suttie have estab'li~hed that activation o f normal and abnormal prothrombin by trypsin gives rise to the same amounts o f thromb,'m !2], ~ d i c a t i n g that the major varts o f the two molecu]es are identical. When c~deium is added, normal and diconmaroMnduced Nothlombin behave~ differently. The normal prolh~ombia molecule brads three to four cMcium ion~ with l'Agh affinity, wh2~le ~he abnormal pxote~ri binds a~ most on~ calcium ion with comparabN bindins strength I3, 4]. Simultaneously, a change occurs in lhe antigenicstructureof normal pr,othzombin but n o t ~ ,that o f the abnormal protein 13, 5]. This ~ s been interpreted as reflecting a conformafional c~hange induced b y t_he binding ,of ealemm, a conclusion .raft_her ~ p p o r t e d b y the anomalous shape ,of the Scatchard pl0rl:s .obtained Ln equilibrium ~'alysis experiments 13], 'Our cixcular diclaroism data eo~roborrate these results.
J~ne t973
The CD ~pectra ob~dned with and wiLhout Ca ~ in@kate float ,flais ion induces a conformafional transition ~ nor. real p r o t ~ o m b i n but n o t 5n the abnormal protein. The change is expressed only ",anthe aromatic region o f the spectrum and is therefore presumably o n b, z local chin, ge in ~he nei~shbourhood o f some aromatic amhuo ae~_d. This is ~ accoxdance wiLh earlier resuhs, which indicate that the catcium-b,ar.d~g groups o f high afFmiW reside only m the iNq-12-termma! p~rt o f the mo]ezule [11]. However, the experiments do not permit any conclusion about the exact na#ure o f the conformations] change. In conclusion, all tradable da~a indicate that the main functiona] difference between norma~ and dO. cournaroMnduced prothrombin is in their calciumbinding and ~ the properties i,'duced b y this ion. Normal protrarombin binds an opfimN a m o u n t o f calcium ions, which causes ~ e protein to assume a conformation that allows its physiolo~ca! aciPvation ~o thrombin to ~ake p!-~ce. Abnormal prothrombin, however, lacks the ability to bind a comparable a m o u n t o f calcium and @~refore carmot be activated in the normal way. He ¢¢ever, under physioaogical conditions the calcium level is such that the active conformation o f n o r t o n p r o t h r o m b i a is presumably a~]ways maintained in the circulating blood
~Aeknowledgements This irtvestigalion was supported by ~ a m s from the Swedish Naiural Science Research Council (Project No. 3055-]) and ~JaeSwedish Medical Research Counc~ (Project No. B72-iJX-581-08A),
References ~ ] J. Sten.qo an~ P.O. Garao% J. BioL Chem. 247 (~972) 816I). 21 G.L. Ne;~stuen mad J.%V.Sultie, J. BioL Clhern. 247 (1972) 8176. ~3] J. SlenEo anti P,O, Gm~,iot,Biuch~rn. Biophy~ Res, Cornmun. 50 (1973) 98. 4961. 15] J. S*enlqo, 3. Bi~t. Chem, 247 (1972) $a67. ~6] KJ , Dor_~ing~n, M.H. Zaflengo and C. Ta;ffol~ Pxoc. Nail. Ac,ad. SoL U.S. 58 {1967) 996. 345
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17] ]a..E. Calhou, A. Kxalezyerad,Jx. and E. HzTaex, ]3ioehemistry 7 ,O96g) 3958. [8] J.S. l~agwall and 1 ~ . Sehe~aga, ~B~o~hemis~ry~ (:t969) 1860,
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JuneT973
1'9] N. Gxeen~eld, B. Davidson ~md G.D. ~Fasm~xl, Bioche,m~sLry 8 .(2[967) 163~. [ 10] N..Gxeen~e]d and G.D. ~Fasman, B~oc~h,emistry 8 (.:196'9) 4t,08. ].11] J. Ste,nflo, to be l~ubl~shefl.