Activation of goose prothrombin by thrombin

A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988

Zofia B a n a ś - G r u s z k a , B o g u s ł a w a B r e t s z n a j d e r , T a de u s z K ra j ew s ki

A C T I V A T IO N OF. G O OS E P R O T H R O M B I N BY T H R O MB I N

Prothrombin isolated from goose sodium citrate plasma was activated in a system containing goose thrombin and calcium ions. Polyacrylamide gel electrophoresis showed two intermediates of molecular weight of 21 000 and 57 000 in the digest. Ser was found to be the N-terminal a- mino acid residue for intermediate form 1.

INTRODUCTION P r o t h r o m b i n is c o n v e r t e d in to t h r o mb i n b y a c t i o n of f act or Xa. It c an be a ls o a c t i v a t e d b y d i r ec t a ct i on of t h r o mb i n w h i c h sp lit s A r g 156 - S e r 157 b o n d in p r o t h r o m b i n r e l e a s in g f ra gm en t 1 an d i n t e r m e di a t e f or m 1 [8-10]. T he p r e se n ce of a n on a ct i ve i n -t e r m e d i a-t e f or m 1 is a v e r y i mp o rt a nt f ac to r of t he m e c h a n i s m r e -g u l a t i n -g t h r o m b i n c o n c e n t r a t i o n in p l a s m a s in ce th is fo rm is not se n si t i v e to t h ro m b i n action. In t e r m e di a t e f or m 1 doe s no t c o n -ta in a v i t a m i n K d e pe n d e n t fr ag m en t w i t h b i nd i n g sit es for p h o s -p h o l i -pi d s a nd c a l c i u m ions. It cannot, th ere fo re , be a ct i va t ed in p h o s p h o l i p i d - d e p e n d e n t system.

In t his p a pe r w e e x a m i n e d g o os e p r o t h r o m b i n a c t i v a t i on p r o -c es s u s i n g t h ro m b in a n d c h a r a c t e r i z e d i n te r m e d i a te pr oduc ts.

MATERIALS AND METHODS

P r o t h r o m b i n w as i s o l at e d f r om f re sh go o se s o d i u m c i tr a t e p l a -sma (9 : 1) a c c or d i n g to th e m e t h o d of E s n o u f , L l o y d an d J e s t y [4]. E l e c t r o p h o r e t i c a l l y h o m o g e n e o u s p r o t h r o m -b in p r e p a r a t i o n s w e r e a d d i t i o n a l l y p u r i f i e d u s i n g the m e t h o d of

B e n a r o u s , L a b i e a nd J o s s o [3]. T he go ose t h r o m b i n p r e p a r a t i o n s u s e d to a c ti v a t e p r o t h r o m b i n w e r e o b ta i ne d a c c o r d i n g to F e n t o n et al. m e t h o d [5]. P r o t h r o m b i n a c t i -v a t i o n p r o c e s s in t he p r e s e n c e of h o m o l o g e n e o u s t h r o m b in a n d c a l -c i u m ion s w a s p e r f o r m e d b y G r a n t and S u t t i e m e -th o d [6]. P r o t h r o m b i n s a mp l es (200 yg) w e r e i n c ub a t e d w i t h t h r o m -b i n (10 yg) in t he p r e s e n c e of c a l c i u m ions (0.01 M) for 0, 1, 5, 10, 30 a nd 60 min. at 37°C. T he r e a c t i o n w as s t op p e d b y a dd in g s o d i u m d o d e c y l s u l p h a t e (SDS) to a fi n al c o n c e n t r a t i o n of 1%, and h e a t i n g to 7 0° C (wat er bath) for 5-10 min. I d e n t i f i c a t i o n of N- - t er m i n a l am i n o a ci d w a s c a r r i e d ou t by the> m e t h o d of G r o s

a nd L a b o u e s s e [7].

P r o t h r o m b i n a c t i v a t i o n p r o d u c t s (f ra gm ent 1 a nd i nt e r m e di a t e f or m 1) w e r e e x a m i n e d b y S DS p o l y a c r y l a m i d e g el e l e c tr o p h o r e s i s

[11].

RESULTS AND DISCUSSION

• G ee s e p r o t h r o m b i n a nd t h r o m b i n p r e p a r a t i o n s u s e d in e x p e r im e n t a p p e a r e d t o be e l e c t r o p h q r e t i c a l l y h o m o l o g e n e o u s si n ce d u ri n g th e e l e c t r o p h o r e s i s in 7. 5% p o l y a c r y l a m i d e gel' t h ey m i g r a t e d as a s i n g l e b o n d (not shown). It w a s f o u n d tha t p r o t h r o m b i n a c t i v a t i o n p r o c e s s i n a s y s t e m c o n t a i n i n g t h r o m b i n a n d c a l c i u m ions c a u -ses t he a p p e a r a n c e of i n t e r m e d i a t e d e g r a d a t i o n p r o d u c t s a l re a dy a f te r 10 min. S D S p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s s h o w ed the p r e s e n c e of t wo p r o t e i n f r a g m e n t s of m o l e c u l a r w e i g h t s 21 000 a nd 57 000 (Tab. 1, Fig. 1). C h r o m a t o g r a p h i c s e p a r a t i o n of d a n s y l d e r i -v a t i -v e s s h o w e d t ha t s er i ne is t he t e r m i n a l r e s i du e for i n t er m ed i a-T a b l e 1 Molecular weights of prpthrombin and prothrombin activation products

Masa cząsteczkowa protrorabiny i produktów aktywacji protrombiny

Protein Mol. weight Incubation time (min)

0 1 5 10 30 60

'Prothrombin (goose) 79 000 + + + + t

-Intermediate form 1 57 000 - - + + + +

Fig. 1. Densitometer scans of stained polyacrylamide gel electrophoresis of the time course of the activation of goose prothrombin by thrombin (incubation time 0, 1, 5, 10, 30 and 60 min.), Int 1 - intermediate form 1, F 1 - fragment

1, P - prothrombin

Rys. 1. Densytogramy rozdziałów elektroforetycznych protrombiny i produktów aktywacji trombinowej (czas inkubacji 0, 1, 5, 10, 30, 60 min), Int 1 - forma

:

9?

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_{os .}

t

_{<$Z> it}

Fig. 2. Bidimensional thin layer chr o-matography of goose - intermediate form

1 after the dansyXation procedure Rys. 2. Dwukierunkowa chromatografia cienkowarstwowa gęsiej formy poś

red-niej X po procesie dansyXacji

te 1 (Fig 2). M o l e c u l a r w e i g ht s of f r a gm e nt 1 a nd i n t e r m e -d i at e f or m 1 s i mi l ar to c o r -r e s p o n d i n g m o l e c u l a -r w e i g h t s of h um a n a c t i v at e d f ra g m en t s and se r in e as a c o m m o n N - te r m i n a l am in o ac i d r es i d u e in i n t e r m e -d i at e s f or m I s ee m to su gg es t t ha t in ca s e of av i a n p r o t h r o m -b i n a c ti v a t i o n -b y thro m-bin , the sa me k in d of b o n d A r 9i56 - S e r 157 is split. Ou r ea rl ie r i n v e s t i g a t io n s on g o o s e p r o t -h r o m b i n a c t i v a t i o n p r o c e s s by f ac t or Xa [l, 2] as w e l l as the p r e s e n t 'o bse rva ti on s i n d i ca t ed th e a n a l o g y b e t w e e n a vi an an d m a m m a l i a n a c t i v a t i o n pr oce ss es . REFERENCES [ X] B a n a s-G r u s z k a Z., K r a j e w s k i T., B r e t

-s z n a j d e r B. (X985), Acta Bioch. Pol., 32(4), 28X-284.

[ 2 ] B a n a s-G r u s z k a Z., K r a j e w s k i T . , S t e f a n - c z y k B. (X982), I . R . C .S.'Medical Science, 10, 832-833.

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[ 8 ] M a g n u s s o n S., P e t e r s e n T. E. , S o t t r u p-J e n- s e n L., C l a e y s H. (1975), Proteases and biological control, eds E. Reich, D. B. Rifkin, E. Shaw, Cold Spring Harbor, New York 123-149. [ 9 ] M a g n u s s o n S., S o t t r u p-J e n s e n L . , P e t e r s e n

T. E . , D u d e k-W o j c i e c h o w s k a G., C l a e y s H.

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[10] S e e g e r s H. W. , H a s s o u n a H. I., H e w e t t-E m-

m e 1 t D . , W a 1 z D. J. (1975), Seminars in Thrombosis and He-

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[11] W e b e r K., O s b o r n M. (1969), J. Biol. C hem ., 244, 4406- -4412.

Department of Biochemistry University of Łódź

Zofia Banaś-Gruszka, Bogusława Bretsznajder, Tadeusz Krajewski

AKTYWACJA GĘSIEJ PROTROMBINY ZA POMOCĄ TROMBINY

Ze świeżej plazmy cytrynianowej gęsi wyizolowano protrombinę, którą podda-wano aktywacji w układzie homologicznym zawierającym trombinę i jony wapniowe. Za pomocą elektroforazy w 7,5% żelu poliakryloamidowym wykazano obecność w hy- drolizia fragmentu 1 i formy pośredniej 1, odpowiednio o masach cząsteczkowych 21 000 i 57 000. Analiza końcowych reszt aminokwasów wykazała obecność seryny jako N-terminalnego aminokwasu dla formy pośredniej 1.