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 2 , 1983
M aria Adamaka, Roman Gondko
AMINO ACID COMPOSITION OF HEMOCYANIN OF THE CRAYFISH ORCONECTES LTMOSUS
Amino a c i d c o m p o sitio n o f hemocyanin o f O rc o n e c tes lim o- s u s was s t u d i e d . A h ig h c o n te n t o f g lu ta m a te and a s p a r t a t e was fou n d . No d i f f e r e n c e s were r e v e a le d in th e amino a c i d c o m p o sitio n o f h em ocyanins o f th e t h r e e c r a y f i s h s p e c i e s o c c u r in g in Poland v i z . A sta c u s a s t a c u s , A sta c u s l e p t o d a c - t y l u s and O rc o n ec tes l im o s u s .
In tr o d u c tio n
In th e group o f r e s p i r a t o r y p r o t e i n s , c a p a b l e o f r e v e r s i b l e oxygen b in d in g and t r a n s p o r t in g oxygen in a e r o b ic o rg a n ism s, a s p e c i a l r o l e b e lo n g s t o th e c o p p er p r o t e in hem ocyanin.
Hemocyanin, a g i a n t p r o t e in m olec u le o f hemolymph o f a r t h r o -p o d s and m o llu s c s may c o n s t i t u t e u-p t o 90% o f hemolym-ph -p r o t e i n s and o c c u r s in d i f f e r e n t a g g r e g a tio n s t a t e s . The p r o te in d i s s o -c i a t e s in a l k a l i n e m ed ia. D i v a le n t c a t i o n s a f f e c t s p e c i f i c a l l y i t s s t r u c t u r e and f u n c tio n [ 1 , 4 ] ,
H em ocyanins a r e p r e s e n t in two a n im al t y p e s i n h a b it in g b oth t e r r e s t r i a l environm ent ( e . g . a rth ro p o d P o r c e l io l e v i s ) and a s f r e s h ( e . g . a rth r o p o d A s ta c u s a s t a c u s ) a s m arine w a t e r s . In s p i t e o f d i f f e r e n c e s in l i v i n g c o n d i ti o n s , hem ocyanins f u n c tio n in th e same way in a l l th e s e a n im a ls . As f a r a s th e oxygen concen-t r a concen-t i o n i s c onc erned concen-th e w a concen-ter medium i s l e s s a d v a n concen-ta g e o u s concen-th an t e r r e s t r i a l . One l i t e r o f d i s t i l l e d w a te r c o n ta i n s 10 ml o f oxy-gen a t a te m p e ra tu re o f 0°C and t h i s v a lu e d e c r e a s e s w ith in
-c r e a s i n g tem p e ra tu re and s a l i n i t y . At a te m p er a tu re o f 2 0 °C , one l i t e r o f m arin e w a te r c o n t a in s on ly 5 ml o f oxyg en .
I t i s assum ed g e n e r a l l y t h a t th e f u n c t i o n a l and s t r u c t u r a l v a r i a b i l i t y o f hem ocyanins r e s u l t s from a d a p t a t io n o f one mole-c u le to d i f f e r e n t e mole-c o l o g i mole-c a l f a c t o r s d ete rm in in g l i v i n g c o n d i-t i o n s o f in d i v i d u a l a nim al s p e c i e s .
P o lis h f r e s h w a t e rs a r e in h a b it e d by th r e e c r a y f i s h s p e c i e s . Two o f them a re n a t i v e : a ) A s ta c u s a s t a c u s ( L .) and b) A sta c u s l e - p t o d a c t y l u s ( E s c h .) . The t h i r d one, O rc o n e ctes lim o su s ( R a f . ) ( Cambarus a f f i n i s S a y ) was b rou g h t in th e b eg in n in g o f t h i s cen-tu r y from N orth A merica (P e n n s y l v a n ia ) . T h is s p e c i e s was i n t r o -duced in Europe a f t e r th e c r a y f i s h "p l a g u e " epid em ic t h a t e x t e r -m in a ted th e two n a ti v e s p e c i e s .
About 100 i n d i v i d u a l s o f 0 rc o n ec te3 lim o s u s were in tro d u c e d to P olan d by Max von dem Borne from th e D elaw are r i v e r (P e n n s y l-v a n i a ) and s e t t l e d a sm a ll pond in Baranowek co n n e c ted w ith th e M yila r i v e r , a r i g h t a f f l u e n t o f O dra. A n e x t, h ig h e r am ounts o f th e se a n im a ls was b rou g h t in 1911 and s e t t l e d in th e b a s in o f Wda and B rd a r i v e r s . T h is s p e c i e s a s r e s i s t a n t t o th e p la g u e were to s u b s t i t u t e f o r th e n a t i v e s p e c i e s [ 6 ] , Due t o i t s o r i - f i n . O r c o n e c tes lim o su s in c a l l e d commonly " American c r a y f i s h 11.
With r e s p e c t to th e n a t i v e s p e c i e s , Am erican c r a y f i s h i s c h a r a c te r iz e d by a s m a l le r s i z e and a much h ig h e r m o b i li t y . Now i t h a s sp r e a d ou t in to th e whole t e r r i t o r y o f Poland and d i s l o -d g es th e o th e r , more goo-d tem pere-d s p e c i e s .
One can s u s p e c t t h a t w h ile th e European s p e c i e s o r i g i n from a common a n c e s t o r , i t seem s l e s s p r o b a b le f o r O rc o n e c te s lim o - s u s . I t h a s been h y D O t h e s i z e d t h a t th e f r e s h - w a te r c r a y f i s h
s p e c i e s can be d e riv e d from a m arine f o r e f a t h e r .
The aim o f t h i s p a p e r was to compare th e amino a c i d com posi-t i o n o f hemocyanin o f posi-th e posi-two European s p e c i e s and o f O rc o n ec posi-tes lim o su s a s p e c i e s so d i s t a n t from th e s ta n d p o in t o f th e o r i g i n a l g e o g r a p h ic a l d i s t r i b u t i o n . E l u c id a t io n f i r s t o f th e amino a c id c o m p o sitio n and then o f th e amino a c id sequen ce i s in d i s p e n s a b l e f o r th e u n d e r sta n d in g o f f u n c t io n a l p r o p e r t i e s o f any p ro -t e i n .
M a te r ia l and methods
Hemolymph ta ke n from m a le s was k e p t f o r 12 h a t 4°C f o r c l o -t -t i n g . The c l o -t was d i s c a r d e d and -th e serum was f i l t e r e d th roug h a 0 .6 4 jam m il l i p o r e f i l t e r and u l t r a c e n t r i f u g e d a t 100 000 x g f o r 8 h i n a Beckman c e n t r i f u g e . Hemocyanin form ed a b lu e s e d i -ment which was then washed tw ic e and d i s s o lv e d in a sm a ll amount o f w a te r. A f t e r 24 h d i a l y s i s a g a i n s t d i s t i l l e d w a te r, th e p ro -t e i n was c h a r a c -te r i z e d on -th e b a s i s o f a b s o r p -t io n s p e c -t r a and c o p p er c o n te n t .
Hemocyanin was p r e c i p i t a t e d from th e s o l u ti o n w ith 5 v o lu -mes o f c o ld a c e to n e . P r o t e in a l i q u o t s were s u b je c te d t o a c id h y d r o l y s is w ith 3 N p - t o lu e n e s u l f o n i c a c i d and
2% 3 -/2 -a m in o -
e t h y l /i n d o le [ 7 ] . H y d r o ly sis was run a t 110 °C f o r 2 2 , 48 and 72 h, r e s p e c t i v e l y . Amino a c i d s in th e h y d r o ly s a t e were d e term in ed a c -c o rd in g t o S t e i n a nd M o o r e [1 0 ] w ith a J e o l 6AH a u to m a tic a n a l y z e r .R e s u lt s and d i s c u s s i o n
Amino a c id c o m p o sitio n o f hemocyanin o f O rc o n ec tes lim o s u s i s shown in T a b . 1 .
T a b l e 1 Amino a c i d c o m p o sitio n He - O rco n ec tes lim o su s
S k ła d aminokwasowy He - O rco n ec tes lim o su s
AMHHOKKCJiOTHuft cocTaB reMOijHaHHHa p axa - O rcon ectes lim osu s
Amino 22 h 48 h 72 h
%
Amino a c i d
a c i d jiM/mg jiM/mg pM/mg X w eigh t r e s id u e
80 000 MW 1 2 3 4 5 6 7 Trp 0 .0 6 7 5 ± 0 .0 0 9 6 0 .0 6 7 5 1 .0 6 5 .4 0 L ys 0 .3 9 5 2 —0 .0 2 9 1 0 .4 1 3 4 ± 0 .0 0 7 6 ± 0 .0 3 8 10 .3 6 2 6 0 .3 9 0 4 6 .1 4 3 1 .2 3
T a b le 1 (c o n t d .) 1 2 3 4 5 6 7 H is 0 .4 1 8 4 ± 0 .0 3 2 2 ± 0 .0 0 7 60 .4 1 31 ± 0 .0 4 2 70 .3 9 1 6 0 .4 0 7 7 6 .4 1 3 2 .6 2 Arg 0 .2 8 0 0 ± 0 .0 1 5 7 0 .3 0 41 ±0 .0 0 5 0 ± 0 .0 3 2 80 .3 0 81 0 .2 9 7 4 4 .6 7 2 3 .7 9 Asp 0 .8 7 9 7 ± 0 .1 0 3 2 ± 0 .0 2 1 30 .9 4 5 9 0 .8 5 9 7 ± 0 .0 9 1 8 0 .8 9 5 1 1 4 .0 7 7 1 .6 1 Thr 0 .3 1 3 2 ± 0 .0 1 1 2 ± 0 .0 2 7 80 .3 0 4 2 ± 0 .1 1 9 70 .2 6 8 7 0 .3 4 0 0 5 .3 4 2 7 .2 0 S e r 0 .3 5 6 9 ± 0 .0 3 9 7 ± 0 .0 1 8 50 .3 4 5 2 ± 0 .0 1 2 50 .2 7 21 0 .3 8 0 0 5 .9 7 3 0 .4 0 Glu 0 .6 2 6 3 ± 0 .0 5 3 0 ± 0 .0 0 8 10 .6 4 0 4 ± 0 .0 6 2 10 .6 1 21 0 .6 2 6 3 9 .8 5 5 0 .1 0 Pro 0 .3 1 2 6 ± 0 .0 2 2 7 0 .3 1 6 6 ±0 .0 0 3 0 ± 0 .0 3 0 90 .3 0 3 9 0 .3 1 1 0 4 .8 9 2 4 .8 8 Gly .0 .4 3 3 2 - 0 .0 3 1 7 0 .4 3 1 2 ± 0 .0 1 8 4 ± 0 .0 5 2 60 .4 1 1 0 0 .4 2 5 1 6 .6 8 3 4.01 A la 0 .3 9 7 6 ± 0 .0 3 4 2 ± 0 .0 0 4 90 .4 2 31 ± 0 .0 4 5 00 .4 1 61 0 .4 1 2 3 6 .4 8 3 2 .9 8 Cys 0 .0 1 3 2 ± 0 .0 0 2 3 0 .0 1 3 0 ± 0 .0 0 2 2 ± 0 .0 0 0 50 .0 0 1 0 0 .0 0 9 1 0 .1 4 0 .7 3 Val 0 .3 2 2 3 ± 0 .0 2 1 4 ± 0 .0 0 9 20 .3 7 6 5 ± 0 .0 4 0 90 .4 0 7 5 0 .3 6 8 8 5 .8 0 2 9 .5 0 Met 0 .0 3 1 5 ±0 .0 0 1 6 ± 0 .0 0 2 40 .0 2 8 3 ± 0 .0 0 0 80 .0 0 1 7 0 .0 2 0 5 0 .3 2 1 .6 4 l i e 0 .2 1 4 0 ± 0 .0 2 0 5 ± 0 .0 0 2 70 .2 7 1 0 ± 0 .0 2 9 10 .3 0 2 0 0 .2 6 2 3 4 .1 2 2 0 .9 8 Leu 0 .4 8 7 9 ± 0 .0 4 0 9 0 .5 2 5 6 ± 0 .0 0 1 7 ± 0 .0 5 4 80 .5 5 5 9 0 .5 2 3 1 8 .2 2 4 1 .8 5 T yr 0 .2 2 8 2 ± 0 .0 0 9 8 ± 0 .0 1 6 70 .2 5 8 5 0 .2 4 5 4 ± 0 .0 2 5 0 0 .2 4 4 0 3 .8 4 1 9 .5 2 Fhe 0 .3 4 8 3 ± 0 .0 2 5 6 ± 0 .0 1 5 90 .3 9 8 3 .0 .3 9 3 6 ± 0 .0 4 0 6 0 .3 8 0 1 5 .9 8 30.4 1
The d a ta r e p r e s e n t mean v a l u e s from 3 d e te r m in a tio n s e x p r e s -sed a s p m o les o f amino a c i d p e r mg p r o t e i n , a s w e ll a s th e p e r c e n t ab un d an ce. The c o n te n t o f s e r i n e and th r e o n in e was c a l c u l a -t e d by e x -t r a p o l a -t i o n -t o z e ro -tim e . T ryp toph an d oe s n o t s u f f e r d e s t r u c t io n u n d er th e ab ove h y d r o l y s is c o n d i ti o n s [ 7 ] .
A m i n o a c i d c o m p o s i t i o n o f H e t h r e e c r a y f i s h s p e c i e s S k ł a d s m i n o k w a s o w y H e t r z e c h g a t u n k ó w r a k ó w A m h h o k h c j i o t h ł i 3 c o c T a B r e M o u a a H a H a T p e x b ü h o b p a K O B Am in o a c id A st a c u s a st a c u s (9 ) A st a c u s le p to d a c ty lu s (5 ) O rc o n e c te s ü im o su s p M /m g am in o a c id r e si d u e 8 0 0 0 0 M V am in o a c id r e si d u e 8 0 0 0 0 M W fi M /m g am in o a c id r e si d u e 8 0 0 0 0 M W T rp 0 .0 7 3 1 1 .1 0 5 .8 5 0 .0 6 7 5 1 .0 6 5 .4 0 0 .0 6 7 5 1 .0 6 5 .4 0 L y s 0 .3 6 9 9 5 .5 5 2 9 .5 9 0 .3 7 7 0 5 .9 0 3 0 .1 6 0 .3 9 0 4 6 .1 4 3 1 .2 3 H is 0 .4 1 4 4 6 .2 1 3 3 .1 5 0 .4 1 9 3 6 .5 6 3 3 .5 4 0 .4 0 7 7 6 .4 1 3 2 .6 2 A rg 0 .3 1 4 9 4 .7 2 2 5 .1 9 0 .2 9 6 3 4 .6 4 2 3 .7 0 0 .2 9 7 4 4 .6 7 2 3 .7 9 As p 1 .0 5 2 2 1 5 .7 8 8 4 .1 8 0 .9 1 3 9 1 4 .3 0 7 3 .1 1 0 .8 9 5 1 1 4 .0 7 7 1 .6 1 T h r 0 .3 9 0 0 5 .8 5 3 1 .2 0 0 .3 4 0 0 5 .3 2 2 7 .2 0 0 .3 4 0 0 5 .3 4 2 7 .2 0 S e r 0 .3 6 0 0 5 .4 0 2 8 .8 0 0 .3 6 0 0 5 .6 4 2 8 .8 0 0 .3 8 0 0 5 .9 7 3 0 .4 0 G lu 0 .6 6 6 0 9 .9 9 5 3 .2 8 0 .6 2 9 3 9 .8 5 5 0 .3 4 0 .6 2 6 3 9 .8 5 5 0 .1 0 P ro 0 .3 3 0 2 4 .9 5 2 6 .4 2 0 .3 1 2 5 4 .8 9 2 5 .0 0 0 .3 1 1 0 4 .8 9 2 4 .8 8 G ly 0 .4 2 5 8 6 .3 9 3 4 .0 6 0 .4 2 4 0 6 .6 4 3 3 .9 2 0 .4 2 5 1 6 .6 8 3 4 .0 1 A la 0 .4 0 7 9 6 .1 2 3 2 .6 3 0 .4 1 7 4 6 .5 3 3 3 .3 9 0 .4 1 2 3 6 .4 8 3 2 .9 8 C y s 0 .0 1 6 4 0 .2 5 1 .3 1 0 .0 2 1 8 0 .3 4 1 .7 4 0 .0 0 9 1 0 .1 4 0 .7 3 M et 0 .0 5 3 1 0 .8 0 4 .2 5 0 .0 4 4 3 0 .6 9 3 .5 4 0 .0 2 0 5 0 .3 2 1 .6 4 V al 0 .3 7 0 7 5 .5 6 2 9 .6 6 0 .4 0 0 6 6 .2 7 3 2 .0 5 0 .3 6 8 8 5 .8 0 2 9 .5 0 H e 0 .2 5 9 8 3 .9 0 2 0 .7 8 0 .2 5 4 4 3 .9 8 2 0 .3 5 0 .2 6 2 3 4 .1 2 2 0 .9 8 L eu 0 .5 0 2 2 7 .5 3 4 0 .1 8 0 .4 8 4 5 7 .5 8 3 8 .7 6 0 .5 2 3 1 8 .2 2 4 1 .8 5 T y r 0 .2 5 5 5 3 .8 3 2 0 .4 4 0 .2 3 3 3 3 .6 5 1 8 .6 6 0 .2 4 4 0 3 .8 4 1 9 .5 2 P h e 0 .4 0 5 9 6 .0 9 3 2 .4 7 0 .3 9 2 0 6 .1 4 3 1 .3 6 0 .3 8 0 1 5 .9 8 3 0 .4 1
F ig . 1 . P e r c e n t c o n te n t o f am in o a c id s fo r h e m o c y a n in o f th re e c r a y fi sh sp e c ie s P ro c e n to w a z a w a rt o ść am in o k w as ó w w h e m o c y ja n in ie tr z e c h g at u n k ó w ra k ó w ri p o u e H T H h i« a M H H O K H C J iO T H H ft c o c ia B re M O u n a H H H a ip e x b h ą o b pa K O B
T l 1 1 1 I I 1 1 1 H U 1 1 1 1 1 1 I I I l l l l i - Ł T
th r e e c r a y f i s h sr>ec i e s i s g iv e n in T ab . 2 . The d a ta a r e mean v a lu e s f o r 3 h y d r o l y s i s t im e s .
R e s u l ts o f T ab. 2 a r e d em on strated g r a p h ic a l l y in F i g . 1 . No s i g n i f i c a n t d i f f e r e n c e s in amino a c i d c om p o sitio n were found between hem ocyanins o f th e th r e e s p e c i e s s tu d ie d . A c id ic amino a c i d s v i z . g lu ta m a te and a s p a r a t a t e were d om in atin g in a l l c a s e s (a b o u t
25%).
The n e x t m ost abu nd ant amino a c i d s were: g ly -c in e , a l a n i n e , le u -c i n e and p h e n y la la n in e . S u l f u r amino a c i d s were p r e s e n t in s m a l le s t am ounts. The amino a c i d c o m p o sitio n o f c r a y f i s h hem ocyanins d o es n o t d i f f e r t h e r e f o r e .f r o m t h a t s e p o s te d h it h e r t o f o r o th e r an im a l s p e c i e s [ 2 , 3 , 8 , 1 1 ].T ab le 2 show s, t o o , th e number o f amino a c i d r e s id u e s p e r s m a l l e s t f u n c ti o n a l su b u n it o f hemocyanin (5 S ) o f e st im a te d mo-l e c u mo-l a r w eigh t 80 000 D. T h is su b u n it i s form ed by 509 amino a c i d r e s i d u e s .
The s i m i l a r i t y o f amino a c id c o m p o sitio n o f th e th r e e hemo-c y a n in s d em o n strated in t h i s stu d y s u g g e s t s t h a t hem ohemo-cyanins o f th e s p e c i e s s t u d ie d o r i g i n a t e from a common p r o t e i n a n c e s t o r .
REFERENCES
[1 ] B a n n i s t e r J . V ., S tr u c t u r e and Fu n c tion o f Haemo- c y a n in , B e rlin -H eid elb e rg -N e w York (1 9 7 7 ).
[ 2 ] B a y e r E. , F i e d l e r H ., Ann. Chem. 6 3 3 , 14 9-1 54 (1 9 6 2 ). [ 3 ] G h l r e t t i - M a g a l d i A. , N u z z a l e C. , G h i r e t t i F . , B io c h e m istry 5_, 1943-1951 (1 9 6 6 ). [ 4 ] G o n d k o R. , A l e k s i u k S . , A cta U niv. L o d z ., F o l i a biochem . b io p h y s . 1 , 9 3 -9 9 (1 9 8 1 ) . [ 5 ] G o n d k o R . , M i c h a l a k W., I n v e r te b r a t e Oxygen B in d in g P r o t e i n s . S tr u c t u r e , A c tiv e S i t e , and F u n c tio n , ed . J . L a m y , J . L a m y , New Y ork, B a se l (1 9 8 1 ).
[ 6 ] K o s s a k o w s k i J . , R ak i, Warszawa (1 9 6 6 ).
[ 7 ] L i u T - Y ., C h a n g Y. H ., J . B i o l . Chem. 2 4 6 , 2 8 42 - -2 8 4 8 (1 9 7 1 ).
[ 8 ] M o r i n ,g -C l a e s s o n J . , A rk iv . Kemi 10 , 1 -8 (1 9 5 6 ). [ 9 ] R o g a l a A. , G o n d k o R ., Comp. Biochem.
[ 1 0 ] S t e i n W. H ., M o o r e S . , J . B i o l . Chem. 2 1 1 , 3 15 -3 26 (1 9 5 4 ).
[11 ] W a x m a n L . , J . B i o l . Chem. 2 5 0 , 37 96 -3 80 6 (1 9 7 5 ). L a b o ra to r y o f B i o l o g i c a l S c ie n c e s
U n iv e r s it y o f Łódź
M aria Adamska, Roman Gondko
SKŁAD AMIN0KWAS0WY He RAKA PREGOWANEGO (AMERYKAŃSKIEGO) (ORCONECTES LIMOSUS)
Oznaczono s k ła d aminokwasowy h em ocyjan iny ra k a pręgow anego. W w ym ienionej Hc stw ie rd z o n o z n a c zn ą z a w a rto ść kwasu g lu ta m in o -wego i a sp a ra g in o w e g o (2 5% ).
N ie stw ierd z o n o r ó ż n ic w s k ł a d z ie aminokwasowym h em o c y ja n i- ny tr z e c h gatunków raków ż y ją c y c h w P o ls c e : A sta c u s a s t a c u s , A- s t a c u s l e p t o d a c t y lu s i O rc o n e ctes l im o s u s .
Mapa Aj;aMCKa, PoMaH T0HąK0
COCTAB AMHHOKHCJIOTHUK rEMOIJHAHHHA PAKA ORCONECTES LIMOSUS
HccJie^oBSuica aMHHOKHCjioTHuii co cT aB reM o i;HaHHH a p a n a O rconec-t e s l im o s u s . Hamjm BUcoKoe coaepacaHiie r - a y r a M a ia u a c n a p T a ia .
He oÓHapyKHJiH pasJiHuaS b aMHHOKHCJiOTHOM c o c ia B e r e M0u n a H H H0B T p e x b h u o b paKOB »HBymax b riojitmes A sta c u s a s t a c u s , A sta c u s l e p t o d a c -t y l u s a O rc o n e c -te s lim o su s .
