Characterization Acetyl

7/28/2019 Characterization Acetyl

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Biochimica et Biophysica Acta, 657 (1981) 243--256 Els evier/Nor th-Holland Biomedical Press

BBA 69182PURIFICATION AND PROPERTIES OF THE MEMBRANE-BOUNDACETYLCHOLINESTERASE FROM ADULT RAT BRAIN

Z. RA KONC ZAY *, J. MALLOL, H. SCHENK, G. VINCENDON and J.-P. ZANETT ACentre de Neurochimie du CNRS et Institut de Chimie Biologique de la Facultd deMddecine, Universitd Louis Pasteur, 11 rue Humann, 6700 0 Strasbourg (France)(Received July 3rd, 1980)

Key words: Acetylcholinesterase; Af fi ni ty chromatography; (Rat brain)

SummaryThe membrane-bound acetylcholinesterase (acetylcholine acetylhydrolase,EC 3.1.1.7) from adult ra t brain has been purified to homogeneity using

sequential affinity chromatography on Con A-Sepharose and on dimethyl-aminoethylbenzoic acid-Sepharose 4B followed by DEAE-cellulose chromatog-raphy. The yield of the purified enzyme (specific activity: 3068 U/mg protein)is higher than 50%. Polyacrylamide gel electrophoresis in the presence of TritonX-100 gives only one band with acetylcholinesterase activity. With the excep-tion of electrofocusing and pore gradient electrophoresis, where a multipleband pattern was detec ted (which seems to be artefactual), the e nzyme appearsto be homogeneous. Gel filtration and sucrose density gradient centrifugationin the presence of Triton X-100 give only one symmetr ical peak, with a calcu-lated molecular weight of 328 000. Since polyacrylamide gel electrophoresis inthe presence of sodium dodecyl sulfate (SDS) and mercaptoethanol gives onlyone band with a molecular weight of 74 500, a tetrameric structure can be pos-tulated for the membrane-bound acetylcholinesterase from rat brain.

I n t r o du c t i o nSome confusion is present in the literature concern ing the molecular speciesof acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) present inmammalian brain. As reviewed by Rieger and Vigny [1] and Trevor et al. [2]

molecular weights ranging from 60 000 to 650 000 have been described. A com-plex hete rogenei ty has been shown by the use of analytical electrofocusing [ 3 - -* Present address: Centxal Research Laboratory, Medical University, 6 7 2 0 S z e g e d , Hungary.


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2445 ] , e l e c t r o p h o r e s i s [ 4 , 6 - - 1 1 ] , g e l f i l tr a t i o n [ 1 , 4 , 5 , 7 , 1 1 ] , d e n s i t y g r a d i e n t c e n -t r if u g a t i o n [ 1 ] o r a f f i n i ty c h r o m a t o g r a p h y o n i m m o b i l i z e d l e c ti n s [3 ] . H o w -e v er , th i s h e t e r o g e n e i t y w a s a ls o s h o w n t o b e d e p e n d e n t o n t h e m e t h o d u s e d t os o l u b i li z e t h e e n z y m e [ 8 ] . I n t h e c a s e o f l e c t in i n t e r a c t i o n s , c o n c a n a v a l i n Ap r e c i p i t a t e s a ll t h e a c e t y l c h o l i n e s t e r a s e a c t i v i ty o f t h e e n z y m e in r a t b r a i n [ 1 ] ,w h e r e a s c o l u m n s o f t h e Lens culinaris l e c t i n ( w h i c h h a s t h e s a m e s u g a r s p e c i f i c -i t y ) c a n a d s o r b o n l y a p a r t o f it [ 3 ] .

G e l f i lt r a ti o n m e t h o d s s e e m t o g iv e d i f f e r e n t m o l e c u l a r w e i g h ts d e p e n d i n go n t h e m e t h o d o f e n z y m e e x t r a c t io n o r c h r o m a t o g r a p h i c c o n d i ti o n s . I f d iv a-l e n t c a t i o n s a r e s o m e t i m e s u s e d t o a v o i d a gg r e g at io n , s o m e o f th e s o l u b il iz a t io nm e t h o d s u s e E D T A [ 3 - - 5 ] . I n a g r e a t n u m b e r o f st u d ie s , i t is n o t c l e ar i f t h ee n z y m e is tr u e a c e t y l c h o l i n e s t e r a s e o r a c e t y l c h o l i n e s t e r a s e a n d c h o li n e s te r a s e[ 1 , 3 - - 8 ] .R e c e n t s t u d ie s [ 1 , 1 2 - - 1 9 ] h a v e d e m o n s t r a t e d t h a t m o l e c u l a r sp e c ie s w i t hd i f f e r e n t s e d i m e n t a t i o n c o e f f i c i e n t s ar e d e t e c t e d in p r o p o r t i o n s w h i c h ar ec h a r a c t e r i s t ic o f t h e t i ss u e o r o f it s d e g r e e o f m a t u r a t i o n . I n t h e a d u l t r a t b ra i n ,o n l y t w o f o r m s a r e d e t e c t a b l e ( 4 S a n d 1 0 S ), t h e l a t te r b e i n g q u i t e ch a r ac -t e ri st ic o f t h e m e m b r a n e - b o u n d f o r m ( i. e. s o l u b il iz e d o n l y w i th d e t e r g e n t s )[ 1 ] . H o w e v e r n o t h i n g w a s k n o w n a b o u t t h e s t r u c t u re o f t h e p u r i fi e d e n z y m ei n c o n t r a s t w i t h t h e c a s e f o r e l e c t r ic f is h [ 2 0 - - 2 2 ] .

S i n c e it h a s b e e n s u g g e s t e d t h a t a c e t y l c h o l i n e s t e r a s e s h a v e a g re a t a f f i n i t y f o rc o n c a n a v a l i n A [ 1 ] ( a l e c t i n s p e c if i c f o r te r m i n a l g l u c o s e a n d m a n n o s e re s i-d u e s ) a n d s in c e t h e r a t b r a in e n z y m e h a s a s tr o n g a f f i n i t y f o r d i m e t h y l a m i n o -e t h y l b e n z o i c a c i d c o u p l e d t o S e p h a r o s e t h r o u g h a s p a c e r a rm [ 2 3 ] , t h e p u ri fi -c a t i o n o f th i s e n z y m e s e e m e d t o b e q u i t e e a s y . M o r e o v e r , s in c e in ra t b r a i n ah ig h p r o p o r t i o n o f a c e ty l c h o l i n e s t e ra s e h a s th e p r o p e r t i e s o f a m e m b r a n e -b o u n d m o l e c u l e [ 1 ] w e f o c u s e d o u r w o r k o n th is p a r t o f t h e b ra in e n z y m e .T h e a c e t y l c h o l i n e s te r a s e w h i c h is s o lu b l e i n t h e a b s e n c e o f d e t e r g e n t i s t h u se l i m i n a t e d p r i o r t o p u r i f i c a t i o n .

W e p r e s e n t h e r e t h e r e s u l ts o f t h e p u r i f i c a t i o n t o h o m o g e n e i t y o f t h e m e m -b r a n e - b o u n d e n z y m e s o l u b il iz e d in t h e p r e s e n c e o f T r i t o n X - 1 0 0 a n d it s m o l e c -u l a r p r o p e r t i e s .M a t e r i a l s a n d M e t h o d sChemicalsA l l r e a g e n t s w e r e o f a n a l y t i c a l g r a d e . S p e c i a l c h e m i c a l s w e r e o b t a i n e d f r o mt h e f o l l o w i n g s o u r c e s : a c e t y l t h i o c h o l i n e i o d i d e , b u t y r y l t h i o c h o l i n e i o d id e ,~ - m e t h y l - D - m a n n o s i d e , c y t o c h r o m e c , o v a l b u m i n , a l d o l a s e ( E C 4 . 1 . 2 . 1 3 ) , c at a-l a se ( E C 1 . 1 1 . 1 . 6 ) , ~ - g a l a c to s i d a se ( E C 3 . 2 . 1 . 2 3 ) , t h y r o g l o b u l i n , a c e t y l c h o l i n e -s te r a se in h i b i to r : 1 , 5 - b i s - 4 - a l l y l d im e t h y l a m m o n i u m - p h e n y l / p e n t a n 3 - o n e d i b r o -m i d e a n d c h o l i n e st e ra s e i n h i b i t o r t e t r a i s o p r o p y l p y r o p h o s p h o r a m i d e f r o mS i g m a C h e m i c a l s C o . ( S t. L o u i s , U . S . A . ), f l u o r e s c e n t s h e e p a n t i- r a b b it i m m u n o -g l o b u li n s f r o m P a s t e u r P r o d u c t i o n ( P ar is , F r a n c e ) ; 5 , 5 ' - d i t h i o b i s (d i n i t ro b e n z o i ca c i d) f r o m M e r c k ( D a r m s t a d t , F . R . G . ) ; T r i t o n X - 1 0 0 f r o m S e r v a ( H e i d e lb e r g ,F . R . G . ) ; t e t r a e t h y l a m m o n i u m b r o m i d e f r o m F l u k a A G (B u c h s , S w i t ze r la n d ) ,d i e t h y l a m i n o e t h y l ( D E A E ) - c e U u l o s e f r o m W h a t m a n ( S p ri n g fi e ld , U . K . ) c a rr ie ra m p h o l y t e a n d U l t r o d e x f r o m L K B P r o d u c t s ( S t o c k h o l m , S w e d e n ) ; C o n


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245A - S e p h a r o s e , S e p h a r o s e 4 B , B lu e D e x t r a n 2 0 0 0 , p o l y a c r y l a m i d e g r a d ie n t g elP A A 4 / 3 0 , h i gh m o l e c u l a r w e i g h t c a l i b r at i on k i t f r o m P h a r m a c i a F i n e C h e m i -c a ls ( U p p s a l a , S w e d e n ) ; U l t ro g e l A c A ( 2 / 2 ) a n d A c A 3 / 4 f r o m I n d u s t r i e B i o -l o g i q u e F r a n ~ a is e S . A . ( C l i c h y , F r a n c e ) a n d C o o m a s s i e b r i ll a n t b l u e R 2 5 0 f r o mG u r r ( C h a d w e l l H e a t h , E s s e x , U . K . ) .P r e p a r a t i o n o f r a t b r a in a c e t y l c h o l i n e s te r a s eA l l o p e r a t i o n s w e r e c a r ri e d o u t a t 4 C . 6 0 a d u l t r a t b ra i n s w e r e h o m o g e n i z e di n 6 0 0 m l o f s a li ne b u f f e r { 1 2. 5 m M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 2 / 0 . 4 MN a C1 ) in a g la ss h o m o g e n i z e r w i t h T e f l o n p e s t l e a n d t h e s u s p e n s i o n w a s c e n t ri -f u g e d a t 5 3 0 0 0 X g f o r 2 h . T h e s u p e r n a t a n t ( S 1 ) w a s d i s c a r d e d a n d t h e p e l l e t( P 1 ) w a s h o m o g e n i z e d i n 6 0 0 m l o f d is t il l ed w a t e r . T h e s u s p e n s i o n w a s a d d e ds l o w l y t o a s o l u t i o n c o n t a i n i n g 5 . 4 1 o f sa li ne b u f f e r ( 1 3 . 7 5 m M p h o s p h a t e b u f -f e r, p H 7 . 2 / 0 . 4 4 M N a C1 ) c o n t a i n i n g 0 . 5 5 % T r i t o n X - 1 0 0 a n d s t ir re d f o r 3 0r ai n . T h e s u s p e n s i o n w a s t h e n c e n t r i f u g e d a s a b o v e . T h e p e l l e t ( P 2 ) w a s d is -c a r d e d a n d t h e s u p e r n a t a n t ( $ 2 ) w a s u s e d a s s t a rt i n g m a t e r ia l f o r a c e t y l c h o l in e -s t e r a s e p u r i f i c a t i o n .( a) A d s o r p t i o n o f a c e t y l c h o li n e st e ra s e o n t h e C o n A - S e p h a r o s e c o l u m n s .C o n A - S e p h a r o s e w a s w a s h e d b e f o r e a n d a f t e r u s e a s p r e v i o u s l y d e s c r i b e d [ 2 4 ,2 5 ] . T h e s u p e r n a t a n t ( $ 2 ) f ra c t i o n w a s a p p l i e d t o a p r e v i o u s l y e q u i l i b r a t e d( sa l in e b u f f e r c o n t a i n i n g 0 . 5% T r i t o n X - 1 0 0 ) 4 0 X 2 c m C o n A - S e p h a r o s ec o l u m n . T h e f l o w r a t e t h r o u g h t h e c o l u m n w a s k e p t c o n s t a n t w i t h a p e r is ta l ti cp u m p ( 7 0 m l / h ). T h e c o l u m n w a s t h e n w a s h e d w i t h 1 0 c o l u m n v o l. o f b u f f e r .T h e e n z y m e w a s e l u t e d w i t h 6 - - 8 c o l u m n v o l. o f t h e s a m e b u f f e r c o n t a i n i n g0 . 5 M a - m e t h y l - D - m a n n o s i d e .( b ) A f f i n i t y c h r o m a t o g r a p h y o n m - d i m e t h y l a m i n o e t h y l b e n z o ic a c id -S e ph -a r o s e 4 B c o l u m n . D i m e t h y l a m i n o e t h y l b e n z o i c a c i d- S e p h a ro s e 4 B w a s p r e p a r e db y t h e m e t h o d o f M a s s o ul ie a n d B o n [ 2 3 ] . T h e a -m e t h y l - D - m a n n o s i d e e l u e n tf r o m t h e C o n A - S e p h a r o s e c o l u m n w a s a p p l ie d t o a d i m e t h y l a m i n o e t h y l b e n -z o i c a c i d - S e p h a r o s e 4 B c o l u m n ( 4 0 X 2 c m ) p r e v i o u s l y e q u i l i b r a t e d i n s a li n e /T r i t o n X - 1 0 0 b u f f e r a n d w a s h e d w i t h 1 0 c o l u m n v ol . o f t h e sa m e b u ff e r .B o u n d a c e t y l c h o l i n e s t e r a s e w a s e l u t e d w i t h 1 c o l u m n v o l . o f t h e w a s h i n g b u f -f e r c o n t a i n in g 0 .2 M t e t r a e t h y l a m m o n i u m b r o m i d e . A f l o w r a te o f 7 0 m l / h w a sk e p t c o n s t a n t d u r i n g c h r o m a t o g r a p h y . T h i s f ra c t i o n w a s d i a l y z e d a g a i ns t l a rg ev o l u m e s o f 1 2 . 5 m M p h o s p h a t e b u f f e r , p H 7 .2 , c o n t a in i n g 0 . 5% T r it o n X - 1 0 0 ,f o r 2 4 h , w i t h t w o c h a n g e s t o r e m o v e N aC 1, a - m e t h y l - D - m a n n o s i d e a n d t e t r a -e t h y l a m m o n i u m b r o m i d e .( c ) D E A E - c e l l u l o s e c h r o m a t o g r a p h y . T h e d i al y se d t e t r a e t h y l a m m o n i u mb r o m i d e e l u a t e w a s a p p l i e d t o a 1 5 X 1 c m D E A E - c e l l u lo s e c o l u m n p r e v i o u s lye q u i l ib r a t e d w i t h 1 2 . 5 m M p h o s p h a t e b u f f e r c on t a i n in g 0 .5 % T r i to n X - 1 0 0 .T h e e l u t i o n w a s p e r f o r m e d w i t h a 3 0 0 m l li n e a r g r a d i e n t o f Na C1 ( 0 - - 0 .4 M ).T h e f l o w ra t e ( 1 5 m l / h ) w a s k e p t c o n s t a n t d u r i n g c h r o m a t o g r a p h y .( d ) S e c o n d a f f i n i t y c h r o m a t o g r a p h y o n d i m e t h y l a m i n o e t h y l b e n z o i c a c i d -S e p h a r o s e 4 B c o l u m n . T h e m o s t a c t i ve f r a c t io n s w e r e p o o l e d a n d a p p li e d t o as m a ll ( 1 0 X 1 c m ) c o l u m n e q u i l i b r a t e d a s d e s c r i b e d a b o v e . T h e c o l u m n w a st h e n w a s h e d w i t h 1 0 c o l u m n v o l. o f s a li ne b u f f e r c o n t a i n i n g 0 . 0 5 % T r i t o nX - 1 0 0 a n d e l u t e d w i t h t h e s m a l le s t v o l u m e o f t h e s a m e b u f f e r c o n ta i n i n g 0 .2 Mt e t r a e t h y l a m m o n i u m b r o m i d e . F r a c t i o n s ( 0 .5 m l ) w e r e c o l l e c te d a n d a s s ay e d


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246f o r a c e t y l c h o l in e s t e r a s e a c t iv i ty . A f l o w r a t e o f 1 5 m l / h w a s k e p t c o n s t a n td u r i n g c h r o m a t o g r a p h y . T h e p u r i f ie d e n z y m e w a s d ia l y s e d ag a in s t 0 . 0 5 %T r i t o n X - 1 0 0 a n d s t o r e d a t - - 2 0 C .

Enzyme and protein determinationT h e a c t i v it y o f a c e t y l c h o l i n e s t e r a s e w a s d e t e r m i n e d a t 3 7 C , a t p H 7 . 2, b yt h e s p e c t r o p h o t o m e t r i c m e t h o d o f E l l m a n e t a l. [ 2 6 ] , u si n g a c e t y l t h i o c h o l i n ea s s u b s t r a te , b y c o n t i n u o u s r e c o r d i n g o f t h e d i f f e r e n c e o f a b s o r b a n c e o b t a i n e di n t h e p r e s e n c e a n d a b s e n c e o f t h e a c e t y l c h o l i n e s t e r a s e i n h i b i t o r 1 , 5 -b i s- 4 -a l ly l -d i m e t h y l a m m o n i u m - p h e n y l / p e n t a n 3 - on e d ib r o m i d e ( 1 1 0 -s M ) . 1 u n i t ( U ) o fe n z y m e a c t iv i ty is d e f i n e d a s t h e a m o u n t o f e n z y m e t h a t h y d r o l y s e s 1 p m o la c e t y l t h i o c h o l i n e / m i n a t 3 7 C . C h o l i n e s te r a s e w a s d e t e r m i n e d i n s im i la r c o n d i -t i o n s u s in g b u t y r y l t h i o c h o l i n e a s s u b s t r a t e a n d t e t r a i s o p r o p y l p y r o p h o s p h o r -a m i d e ( 1 1 0 - s M ) a s i n h i b i t o r .Gel filtrationG e l f i l tr a t i o n w a s p e r f o r m e d a t 4 C e it h e r o n U l t ro g e l A c A 2 / 2 , U l t r o g e lA c A 3 / 4 o r S e p h a d e x G - 2 0 0 c o l u m n s ( 9 0 1 c m ) i n s a li ne b u f f e r c o n t a i n in g0 . 0 5 o r 0 .5 % T r i t o n X - 1 0 0 . T h e f l o w r a te w a s 4 m l / h . E n z y m e s a m p l e s ( 1 .5 m l )c o n t a i n in g t h e m o l e c u l a r w e i g h t m a r k e r s ( e a c h 1 m g / m l ) w e r e a lw a y s u s e d .F r a c t i o n s o f 0 .5 m l w e r e r e c o v e r e d .T h e f o l l o w i n g c o m p o u n d s w e r e u s e d a s s t an d a r ds o f m o l e c u l a r w e ig h t ( M r)a n d S t o k e s ' r a d i u s ( R e ) : c y t o c h r o m e c (M r, 1 2 4 0 0 ; R e , 1 . 7 4 n m ) , o v a l b u m i n( M r , 4 5 0 0 0 ; R e , 3 . 0 5 n m ) , a l d o l a s e ( M r , 1 5 8 0 0 0 ; R e , 4 . 8 1 n m ) , f l u o r e s c e n tI g G ( M r , 1 6 0 0 0 0 ; R e , 5 . 2 2 n m ) , c a t a l a s e ( M r , 2 5 0 0 0 0 ; R e , 5 . 2 2 n m ) , f l- g al ac to -s i d as e ( M r , 5 4 0 0 0 0 ; R e , 8 . 2 n m ) , t h y r o g l o b u l i n ( M r , 7 5 0 0 0 0 ; R e , 8 .5 n m ) a n dB l u e D e x t r a n 2 0 0 0 . T h e p o s i ti o n s o f t h e m a r k e rs w e r e d e t e r m i n e d b y t h ea b s o r b a n c e ( 6 1 0 n m f o r B l u e D e x t r a n , 5 2 7 n m f o r c y t o c h r o m e c , 2 8 0 n m f o rp r o t e i n s ) o r f lu o r e s c e n c e ( f l u o r e s c e n t I g G ) o r e n z y m e a c t i v i t y f o r f l- g al ac to -s i d as e [ 2 8 ] a n d c a t a l a s e [ 2 9 ] .Sucrose density gradient centrifugationT h e s e d i m e n t a t i o n c o e f f i c ie n t o f t h e m e m b r a n e - b o u n d a c e t y lc h o l in e s t e ra s ew a s e s t i m a t e d i n 5 - - 2 0 % ( w / v ) s u c r o s e g r a d i e n t in sa li n e b u f f e r c o n t a i n i n g 0 . 5 %( w / v ) T r i t o n X - 1 0 0 u s i n g f l -g a la c t os id a s e ( 1 6 S ) , c a t a l a s e ( 1 1 . 3 S ) a n d f l u o -r e s c e n t I gG ( 6 .9 S ), a s s e d i m e n t a t i o n m a r k e r s . C e n t r if u g a t i o n w a s p e r f o r m e d a t4 C i n a S p i n c o L 2 - 6 5 u l t r a c e n t r i f u g e w i t h a S W 6 5 r o t o r ( 8 - - 9 h a t 2 3 0 0 0 0 g ) . 3 5 f r a c t i o n s o f 2 0 0 p l w e r e r e c o v e r e d f r o m t h e g r a d i e n t. T h e s iz e o f t h es a m p l e w a s 2 0 0 p l.Polyacrylamide gel electrophoresisD i sc e l e c t r o p h o r e s i s i n 5 % p o l y a c r y l a m i d e c y l in d r ic a l g e ls w a s p e r f o r m e da c c o r d i n g t o D a vi s [ 3 0 ] , e x c e p t t h a t 0 . 1% T r i t o n X - 1 0 0 w a s a d d e d t o t h e g elsa n d b u f f e r s ( T r is - g ly c i n e , p H 8 . 9 , f o r u p p e r r e s e r v o i r b u f f e r a n d T r is -H C 1 , p H8 . 1 , f o r l o w e r r e s e r v o i r b u f f e r ) .P o r e g r a d i e n t e l e c t r o p h o r e s i s [ 3 1 ] w a s p e r f o r m e d in t h e a b s e n c e o f d e t e r-g e n t o n g r a d i e n t p o l y a c r y l a m i d e g e ls ( P A A 4 / 3 0 f r o m P h a r m a c i a ) . G e l s la b sw e r e p r e - r u n f o r 2 0 m i n a t 7 0 V i n a 0 . 0 9 M T r i s / 0 . 8 M b o r a t e b u f f e r ( p H 8 . 4 )


5/14

247c o n t a i n i n g 2 . 5 m M E D T A . E l e c t r o p h o r e s i s w a s th e n c a r ri e d o u t f o r 1 6 h a t1 5 0 V .

F o r g r a d i e n t p o l y a c r y l a m i d e g el e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f S D S , ge ls la b s w e r e p r e - ru n f o r 6 0 m i n a t 7 0 V in 0 . 0 4 M T r is / 0 . 0 2 M s o d i u m a c e t a t e( p H 7 . 4 ) b u f f e r c o n t a i n in g 2 m M E D T A a n d 0 . 2 % S D S . 1 2 % a c r y l a m i d e ge ls l a b s w e r e a l s o r u n i n i d e n t i c a l c o n d i t i o n s .Elec t ro focus ingA n a l y t ic a l e l e c t r o f o c u s i n g w a s c a rr ie d o u t a t 4 C o n 5 % p o l y a c r y l a m i d e g e ls la b s ( 1 2 5 X 1 2 5 X 0 . 5 m m ) c o n t a i n i n g 0 . 0 5 % T r i t o n X - 1 0 0 . G e l s c o n t a i n in ga m p h o l y t e s ( p H r a ng e 3 - -1 0 ) w e r e p o l y m e r i z e d b y a m m o n i u m p e r su l fa t e . F o l -l o w i n g p o l y m e r i z a t i o n , g e ls w e r e p r e - e l e c t r o f o c u s e d a t 2 0 0 V f o r 6 0 m i n .S a m p l e s w e r e a p p l i e d in t h e b a s ic r e g io n o f th e p H g r a d i e n t ( a r o u n d p H 8 . 5 )a n d t h e n s u b m i t t e d t o e l e c t r o f o c u s i n g f o r 3 h a t 3 0 0 V w i t h c o o l i n g a t 4 C .

T h e p r e p a r a t i v e f l a t- b e d e l e c t r o f o c u s i n g s y s t e m w a s a ls o u s e d o n U l t r o d e xp l a te s ( 5 5 X 1 1 0 X 5 m m ) i n th e p r e s e n c e o f 0 . 0 5 % T r i t o n X - 1 0 0 . P l a te s w e r ep r e - ru n a n d e l e c t r o f o c u s i n g w a s p e r f o r m e d a s d e s c r i b e d a b o v e .Staining af ter polya crylam ide gel e lectrophoresis and electro focusingP r o t e i n s w e r e s t a i n e d o v e r n i g h t w i t h C o o m a s s i e b r il li a n t b l u e R 2 5 0 ( 0 .1 % ,w / v , in m e t h a n o l / a c e t i c a c i d / w a t e r : 4 : 1 : 5 , v / v ) a n d d e s t a i n e d i n t h e s a m em i x t u r e d e v o i d o f d y e . A c e t y l c h o l i n e s t e r a s e a c t iv i t y w a s d e t e c t e d o n t h e g el se i th e r w i th t h e ~ - n a p h t h y l a c e t a t e m e t h o d [ 3 2 ] o r t h e c o p p e r - t h io c h o l i n em e t h o d [ 3 3 ] . B l a n ks w e r e p e r f o r m e d b y p r e in c u b a t i o n ( 3 0 m i n ) a nd in c u b a -t i o n in t h e p re s e n c e o f 1 - 1 0 - S M 1 , 5 - b is - 4 - a l l y ld i m e t h y l a m m o n i u m - p h e n y l /p e n t a n 3 - o n e d i b r o m i d e .Resu l t s

Pu rif icat ion o f ace ty lcholinesteraseW h e n a 1 0 % ( w / v ) h o m o g e n a t e o f r a t br a in in p h o s p h a t e s a l i ne b u f f e r w a sc e n t r i f u g e d a t 5 3 0 0 0 X g f o r 2 h , o n l y 8 % o f t h e a c e t y l c h o l i n e s t e r a s e a c t i v i t yw a s o b t a i n e d in t h e s o l u b l e f r a c t io n S 1 (T a b l e I ). B y t r e a t m e n t o f t h e p e l l e tT A B L E IF L O W S H E E T O F T H E P R O C E D U R E O F T H E E X T R A C T I O N O F R A T B R A I N A C E T Y L C H O L I N E S -T E R A S EF r a c t i o n s are def ined in Mater ial s a n d M e t h o d sF r a c t i o n T ota l V o lume A c t iv i t y T o t a l A c e ty l -

prote in (ml) (U/m l) ac t ivi ty chol ines te rase(mg) (U) solubi l ized

(% of total)H o m o g e n a t e * 1179 134 1.54 206.8Sl ( soluble in sa l ine) 286 120 0.119 14.3 8S2 (soluble in sal ine c o n t a i n i n g

T r i ton X - lO 0) 559 1155 0 .138 159 .0 90P2 ( Insoluble res idue) 119 210 0.018 3.9 2* S t a r t i n g f r o m 12 rat brains.


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7/14

2 4 9

w i t h T r i t o n X - 1 0 0 ( 0 . 5 % i n p h o s p h a t e s a l i n e b u f f e r ) a n d c e n t r i f u g a t i o n ,a p p r o x . 9 8 % o f t h e t o t a l a c e t y l c h o l i n e s t e r a s e a c t i v i ty f r o m t h e p e l l e t ( P 1 )r e m a i n e d i n t h e s u p e r n a t a n t f r a c t i o n ( $ 2 ) ( T a b l e I ).A s u m m a r y o f a t y p i c a l p u r i f i c a t i o n o f T r i t o n - s o l u b i li z e d e n z y m e is g iv e n i n

T a b l e I I. A s c a n b e s e e n , a c e t y l c h o l i n e s t e r a s e w a s q u a n t i t a t i v e l y a d s o r b e d b y aC o n A - S e p h a ro s e 4 B c o l u m n a n d m o r e t h a n 8 0 % o f th e e n z y m e ca n b e e l u t e df r o m t h e c o l u m n w i t h a - m e t h y l - D - m a n n o s i d e . I t s h o u l d b e n o t e d t h a t d e s o r p -t i o n o f a c e t y l c h o l i n e s t e r a s e f r o m C o n A - S e p h a r o s e c o l u m n s is q u i t e d i f f i c u l ta n d t h e e n z y m e i s e l u t e d w i t h a t a il in g p e a k . I n c re a s in g t h e v o l u m e o f th ea - m e t h y l - D - m a n n o s i d e e l u a n t w o u l d g iv e a 1 0 0 % r e c o v e r y o f t h e e n z y m e . T h es e c o n d a f f i n i t y c o l u m n is v e r y e f f i c ie n t , s i n c e d u r i n g th i s p u r i f i c a t i o n s t e p , t h ee n r i c h m e n t is a b o u t 2 5 0 - f o ld ( T a b l e I I) . S u c h e n r i c h m e n t is h o w e v e r m u c hl o w e r ( a b o u t 4 0 - ti m e s ) w h e n $ 2 w a s d i r e c tl y p a s se d t h r o u g h t h e s a m e c o l u m n ,p r o b a b l y d u e t o i n c re a s in g n o n s p e c i f i c a d s o r p t i o n . T h e D E A E - c e l l u l o s e s t e pw a s i n t r o d u c e d t o e l im i n a t e t h e m a t e ri a l w h i c h c o u l d ha v e b e e n a d s o r b e d no n -

m

- I - - ! -

4

A B C A B C DF i g . 1 . 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 o f t h e p u r i f i e d a c e t y l c h o l l n e s t e r a s e ( 1 0 / a g ) i n 5 % a c r y l a m i d eg e ls c o n t a i n i n g T r i t o n X - 1 0 0 . A : p r o t e i n s t a i n w i t h C o o m a s s i e h r i l l a n t b l u e R 2 5 0 . B : a c e t y l c h o l i n e s t e r a s es t a i n w i t h t h e c o p p e r t h i o c h o l i n e m e t h o d [ 3 3 ] . C : s a m e as B e x c e p t t h a t 1 , 5 - b i s - 4 - a l l y l d i m e t h y l a m -m o n i u m - p h e n y l / p e n t a n 3 - o n e d i b r o m i d e ( 1 1 0 - 5 M ) w a s a d d e d t o t h e i n c u b a t i o n m i x t u r e .F i g . 2 . ( A ) 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 x o p h o r e s i s o f t h e p u r i f i e d a c e t y l c h o l i n e s t e r a s e ( 1 0 ~ g ) i n th ep r e s e n c e o f 2 - m e r c a p t o e t h a n o l o n a p o l y a c r y l a m i d e g r a d i e n t . E l e c t r o p h o r e t i c c o n d i t i o n s ax e g i v e n i nM a t e r i a l a n d M e t h o d s . M r w a s d e t e r m i n e d b y u s i n g t h e f o l l o w i n g p r o t e i n s a s s t a n d a r d s ( B ) : f e r r i t i n( 1 8 5 0 0 ) ; l a c t a te d e h y d r o g e n a s e ( 3 6 0 0 0 ) ; c a t a l a s e ( 6 0 0 0 0 ) ; a l b u m i n ( 6 7 0 0 0 ) ; f e r r i t in ( 2 2 0 0 0 0 ) a n dt h y r o g i o b u l i n ( 3 3 0 0 0 0 ) . ( C ) P o r e g r a d i e n t e l e c t r o p h o r e s is w i t h o u t d e t e r g e n t o f t h e p u r i f i e d m e m b r a n e -b o u n d a c e t y l c h o l i n e s t e r a s e ( 5 ~ g ) . O n l y a s m a l l p r o p o r t i o n o f e n z y m e p e n e t r a t e t h e g e l N o t e t h e t a l l t n g so f t h e m a j o r b a n d s . M r v a l u e s w e r e d e t e r m i n e d u s i n g t h e f o l l o w i n g p r o t e i n s a s s t a n d a r d : ( D ) a l b u m i n( 6 7 0 0 0 ) ; l a c t a t e d e h y d x o g e n a s e ( 1 4 0 0 0 0 ) ; c a t a l a s e ( 2 3 2 0 0 0 ) ; f e r t i t i n ( 4 4 0 0 0 0 ) ; t h y r o g i o b u l l n ( 6 6 9 0 0 0 ) .


8/14

2 5 0

T A B L E I I IM O L E C U L A R P R O P E R T I E S O F T H E P U R I F I E D M E M B R A N E - B O U N D A C E T Y L C H O L I N E S T E R A S EF R O M R A T B R A I N

V a l u e s S . D . N u m b e r o fexperiments

M o l e c u l a r w e i g h t o f t h e m o n o m e r( S D S - p o l y a c r y l a m i d e g e l electrophoresis)

S t o k e s ' r a d i u s ( g e l f i l t r a t i o n )Sedimentation coefficientC a l c u l a t e d m o l e c u l a r w e i g h tIsoelectric point (major band)

7 4 5 0 0 3 0 0 0 ( 4 )7 . 1 5 0 . 3 5 n m * ( 1 5 )8 . 0 5 0 . 3 5 n m * * ( 1 5 )9 . 7 5 0 . 2 S ( 4 )

2 9 4 0 0 0 " 2 0 4 0 03 2 8 0 0 0 * * 2 0 1 0 0

5 . 2 1 0 . 0 2 ( 6 )* Calculated according to Siegel and Monty [ 3 4 ] .

* * Calculated according to Ackers [ 3 5 ] .

specifically on the two affi nit y columns. E luti on by a linear NaC1 gradient pro-duced a symmetrical peak of activity eluted between 0.07 and 0.15 M sodiumchloride. The last step of affinity chroma togra phy was introduced to concen-t/ate the purified enzyme and to reduce the concentration of Triton X-100.The specific activity of the purified enzyme was found to be 3068 U/mg pro-tein which represents a 17 550-fold purification . The overall yield of the prepa-ration (about 50%) should be increased if control aliquots at various stages aretaken into account.It should be noted that the acetylcholinesterase activity decreases duringstorage at 4C. During the 5 days o f the prepara tion, the enzymat ic activit y in$2 decreases by 25%. With the purified enzyme, virtually all the activity is lostif it is stored for 1 mo nt h at 4C. The activity is however very stable if theenz yme is stored frozen at --20C.Pu r i t y o f t h e Tr i to n - so lu b le a ce ty l ch o l in es t e ra sePolya cryl amid e gel electrophoresis in 5% acrylamid e, in the presence o f0.05% Triton X-100, of the purified acetylcholinesterase gave only one slowlymigrating protein band (Fig. 1) with enzyme activity. No other protein bandswere detected. Polya cryla mide gel electrophoresis in th e presence of 5 mMmer cap toe tha nol and 0.2% SDS in a gradient of acrylamid e (Fig. 2) gave only asingle band with a molecula r weight around 74 500 + 3000 (Table III). Whenthe gels are overloaded and stained with Coomassie brilliant blue R250, twoother bands of lower Mr could be detected as traces (2--5% of the total area ofthe densitometric tracing}.Gel fi l tra t io n a n d su cro se g ra d ien t ce n t r i fu g a t io n o f a ce ty l ch o lin es t e ra seGel filtrat ion of the purified acetylcholine sterase in the presence of 0.05%Triton X-100 (w/v) allowed the determination of its apparent molecularweight. A mean value of 487 000 + 21 000 was found for the purified enzyme.The e nzy me migrates as a symme trical peak an d shoulders or multiplepeak pat terns were never detected. The Stokes' radius was calculated from thegel filtration parameters by two different equations [34,35]. With the method


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251of Siegel and M on ty [34] a radius of 7.15 + 0.35 n m was fo un d which differssubstantially from that found using the equation of Ackers [35], 8.05 0.35nm (Table III). Since in ou r condi tion s (--log Kd) 1/2 [34] was no t fou nd to beproportional to the Stokes' radii of globular marker proteins, and since wefoun d consistent results with the me tho d of Ackers [35] (for Ultrogel AcA 3/4the effective pore radius was 23.3 + 1.0 nm) we believe tha t the value Re, 8.05 -+0.35 nm is more probable.A symmetrical peak with sedimentation constant s = 9.75 + 0.2 S (Table III)was also observed both for $2 enzym e and the purified acetylcholinesterase bycentrifuga tion in sucrose dens ity gradients in the presence of detergent.Electro focusing of acetylcholinesteraseElectrofocusing of purified acetylcholinesterase gave different patternsdepending on the system used. Preparative electrofocusing on Ultrodex platesgave only one peak of activity at pH 5.2. In the presence of 2-merca ptoethanol,two peaks of activi ty (Fig. 3) at pH 5.2--5.3 and 6.4, respectively, were det ect ed.The picture obtained with analytical electrofocusing is more complex either inthe presence or absence of reducing agents (Fig. 4). In both cases, a major pro-tein band with enzymatic activity is detected at pH 5.21 and minor bands atpH 5.12 and 4.95. In the absence of 2-mercaptoethanol, two other weaklystained bands are detected (pH 4.85 and 5.38, respectively). In the presence of

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pH"7

4A B C D E

Fig. 3 . I soe lec t r ic focusing of the pur i f ied ace tylchol i nes te rase (ACHE ) on Ul t ro dex pla tes in t h e p r e s e n c eof me r c a p toe th a no l . F r a c t i ons w e r e a s sa ye d f o r e nz yme a c t i v i t y by t he me thod o f E11man e t al . [ 26 ] : , enz yme ac t ivi ty (a rbi t ra ry uni t s ) : o . . . . . . o pH.Fig. 4 . Drawing of the pa t te rn obt a ined by e lec txofocusing of pur i f ied ace tylcho l ines te rase on 5% acryl -amid ge ls . (A) 5 mm f rac t ions of the ge l were cut and ace tylchol ines te ras e ac t ivi ty was meas ured by t h emet hod of E l lman e t a l . [2 6] . (B) The enzy me fou nd in the dashed zone in A was subjec ted to re-e lectxo-focusing under the same cond i t ions . (C) The ge l was run in the absence of m e r c a p t o e t h a n o l a n d s ta inedf o r p r o t e ins ( Cooma ss i e b ri ] l a n t b lue R250) . ( D ) T he ge l w a s r un i n t he p r e se nc e o f me r c a p toe th a no! a ndsta ined for ace ty lchol ines te rase ac t ivi ty [3 2] . (E) The ge l was run i n t h e pr e se nc e o f me r c a p toe tha no l a nds t a i n e d f o r p r o t e i n s (Coomassie br i l lant b lue R2 50) .


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2522-mercaptoethanol, these two bands disappeared, whereas a protein band withacetylcholinesterase activity is detected at pH 6.35. It should be emphasizedthat diffuse protein and enzyme stain is always present between pH 4.65 and5.75.

We have tested the possibility that the multiple band pattern could be due toartefacts. The major band (pH 5.21) was isolated after analytical electrofocus-ing (Fig. 4a) and submitted to the same procedure in identical conditions. Asshown in Fig. 4b, this band gives rise to a diffuse patt ern , suggesting arte fact sdue to electrofocusing.Pore gradient electrophoresis on polyacrylamide gradient in the absence ofdetergentThe picture obtai ned was seen in Fig. 2 with a prepar ation of purified acetyl-cholinesterase stored at --20C. Only one band with a molecular weight of420 000 can be detected within the gel. When the gels are overloaded (10--20pg) t wo min or bands with molecul ar weight of 520 000 and 720 000, respec-tively, are also present. However, most of the protein was precipitated at thestart of the gel. With preparations stored at 4C for 1 month (which lost theiractivity), a multiple band pattern was detected with the purified acetylcholine-sterase. Analysis of the electrophoretic mobility of the various bands (data notshown) indicated th at all of them could be multiples of the faster-migratingco mp on en t (Mr, 74 700): 150 000 (X2); 22 00 00 (X3); 3700 00 (X5); 44 00 00(X6); 560 000 (X8); 730 000 (XlO) and 1 000 000 (X 15).D i s c u s s i o n

Method of purification of the membrane-bound acetylcholinesteraseAs suggested in the Introduction of this paper, contradictory views exist inthe literature concerning the solubilization of rat brain acetylcholinesterase.Chan et al. [4] and Wenthold et al. [5] claimed tha t a sucrose/EDTA mixtu re isable to solubilize ' mos t' of the enzyme from rat brain. It is clear however tha tonly 20% of the enzyme can be solubilized by this method including part ofthe 10% which is soluble in water or othe r aqueou s buffers of high or low ionicstrength (Ref. 1 and ou r own data). The other 10% are extra cted after stirringovernight at 4C. Since it has been demonstrated that, in the head of house-flyor in mouse brain, incubation in sucrose/EDTA mixtures at room temperatureor 37C is more efficient [36,37 or 11], it is possible tha t this solubilization isdue to mild proteolysis.Preliminary experiments have shown that extraction by water or bufferedsaline (0.4 M NaC1) yields only abo ut 10% of the tota l ace tylcholinesterasefound in adult rat brain. Repeated extractions with the same media do notsolubilize more enzyme. We considered thus that the remaining enzyme has thecharacteristics of a membrane-bound molecule.Purity of the enzymeUsing the procedure described in Materials and Methods, the membrane-bou nd acetylchol inesterase can be purified with in a week in very high yield.The specific activity of our purified enzyme (3067 U/mg protein) represents


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253more than 17 500-fold purif icat ion and is very similar to activities of brainacetylcholinesterase reporte d in the literature: 4500 U/mg protein [5] for thesucrose/EDTA soluble enzyme and 4250 U/mg for the total bovine caudatenucleus enzym e [10]. The differences observed for these purified enzyme s arepossibly due to difficulties in the d eterm inat ion o f very small amou nt of pro-tein, mainly in the presence of Triton X-100, as observed by Ruess et al. [10],and also to the different conditions used for enzymatic determinations [5,10].No further comparison of the purity of the enzyme can be achieved sinceelectrophoreti c photogra phs are not given by others [4,5,10]. Assuming tha tthere are no c onta mina nts with th e same charge and molecular weight for theprotein and the same molecular weight for the subunits, we can estimate fromthe scan of the SDS-polyacrylamide gels stained with Coomassie brilliant blueR250 at least a 95--98% pur ity for our en zyme. Our purified preparations arecomple tely devoid of nonspecific esterase activities on t he basis of the action ofinhibitors. In fact, during the preparation, the low quantity of cholinesterasepresent in the $2 fraction is adsorbed on the Con A-Sepharose column but notadsorbed on the di met hyl ami noe thy l benzoic acid-Sepharose 4B column. Sincethis column is used twice during the preparation and since cholinesterase is notdetectable in the purified preparat ion, we have good reason to believe tha t it isnot a contaminant.M o l e c u l a r p r o p e r t i e s o f t h e m e m b r a n e - b o u n d a c e t yl c h o li n e st e r a se(a ) Mo lecu la r we ig h t a n d o l ig o mer ic s t ru c tu re . As mentioned above, gelfiltration gave only one peak with an apparent molecular weight of 487 000 +21 000 which is in agreement with the results of Rieger and Vigny [1] but incontradiction with results showing a profile with multiple peaks [4,5,7,11]. Inour working conditions, the recoveries of enzymatic activities from the gelfiltration columns are quantitative and, thus, it cannot be postulated that somemolecular forms are lost on the column. The possibility that they are lostduring th e preparation of the enzy me (50% yield) can be excluded since onlyone symmetrical peak is obtained from the $2 fraction which contained all themembrane-bound acetylcholinesterase.

The presence of a unique peak cou ld be an artefact due to t he intera ctions ofTrit on X-100 with the enzym es as suggested [38,39] for the acetylcholine-sterase of eryt hroc yte membranes. Our a tte mpts to eliminate Triton X-100 byprolonged dialysis or chromatography on Biobeads SX2 [40] were unsuccess-ful due to e nzyme precipitati on. Our observations confirm the results of Riegerand Vigny [1], who observed multi ple and high molecular weight aggregateswhen the Triton-soluble en zy me from rat brain was submit ted to gel filtration(or sucrose gradient centrifugation) in the absence of Triton X-100. It is thusmore likely that artefacts are produce d by t he eli mination of Triton X-100.An artificial increase of Stokes' radius due to interactions of Triton X-100with the enzyme, suggested by others [1], is possible, but we have not as yet anexperimenta l proo f tha t such a pheno men on occurs, since the enzym e precipi-tates as soon as the detergent was eliminated (see above). An increase of Sto-kes' radius has been observed [1] for a soluble 10 S form of acetylcholine-sterase when this enzy me is chrom atogr aphe d in the presence of Triton X-100(from 7.4 t o 8.5 n m). It is no t clear however if this soluble 10 S form is identi-


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254cal to the 10 S membrane-b ound fo rm and fu rthe r studies are necessary toclarify this point. However, the value determined here {8.05 + 0.35 nm) is inthe same range as the previous one.

The s value of the purified membrane- bound acetylcholinesterase determinedhere (9.75 + 0.2 S) agrees well with the da ta given in Ref. 1 either for t hesoluble 'hard to solubilize' form either in the presence or in the absence ofdetergent or for the membran e-bound 'hard to solubilize' form analysed in thepresence of detergent (about 10 S).The calculation of the molecular weight of the purified acetylcholinesteraseis dubious since uncertainty exists for the determinations of Stokes' radii, sedi-men tat ion coefficients and partial specific volumes in the presence of deter-gents. However if we assume (i) that Stokes' radii and sedi ment atio n coeffi-cients determined in the presence or in the absence of detergents are not dra-mati cal ly diff eren t [1,14] and (ii) th at the partial specific volume (v-) is similarto those of the m arker proteins and thus to those o f the various forms of acetyl-cholinesterase found in various tissues and species [14] and to the 'hard tosolubilize' 10 S form of the rat brain acetylcholinesterase (~= 0.76) [1], cal-culations of the molecular weight from the Stokes' radius and sedimentationcoe ffi cient give 294 000 and 328 000, respect ively, according to Siegel andMo nty [34] or to Ackers [35]. These results as well as Stokes' radius and sedi-mentation coefficient are very close to those found for the globular G4 formfrom bovine supe rior cervical ganglia [14] . From our calculated values a tetra-meric structure of a mo no me r with M r abo ut 75 000 has thus to be expected.

(b) " G ly co p r o te in n a tu re o f t h e m em b ra n e -b o u n d a ce ty l ch o lin es te ra se . Asshown before, the membra ne-boun d acetylcholinesterase binds completely toCon A-Sepharose columns. The specificity of this lectin for terminal gluco- andmanno-pyrannosid e [41], also demon strat ed by the specific elution of theenz yme by these sugars, indicates tha t ace tylcholineste rase is a glycoprote inand that the membrane-bound enzyme appears to be homogeneous with regardto the affinity for the lectin. These results are also in agreement with thealmost complete coprecipitation of concanavalin A and rat brain acetylcholine-sterase foun d by others [1] but differ substantially from the heterogene ity ob-served by Gurd [3] with the mem brane-b ound en zyme prepared by the sucrose/EDTA extraction method.( c ) H e t e r o g e n e i t y o f t h e m e m b r a n e - b o u n d a c e ty l c ho l in e s te r a s e: t h e r o le o far te fac ts . As mentioned in the Introduction of this paper, a very complexhete rogeneity of acetylcholinesterase has been shown using electrophoresis orelectrofocusing together with density gradient centrifugation. It has not beenpossible to correlate these various kinds of heterogeneity [18]. Only two of themetho ds used by us to test the molecular properties of the pure membrane-bou nd acetylcholines terase, i.e., pore gradient electrophoresis and isoelectricfocusing give evidence o f hete rogen eity.The method of pore gradient electrophoresis was introduced in an attemptto measure the apparent molecular weight of the purified membrane-boundacetylcholinesterase in the absence of detergent [31], since Triton X-100 (anuncharged detergent) does not penetrate the gel. The main result of this techni-que was that with fresh enzyme preparations, precipitation occurs as soon asthe enzyme penetrates the polyacrylamide gel. Only a small proportion of the


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255e n z y m e m i g r a t e d w i t h t a il in g , g i vin g a m a j o r b a n d w i t h an a p p a r e n t M r o f4 2 0 0 0 0 a n d t w o m i n o r b a n d s . T h e s e r e s u l ts a r e v e r y s im i la r t o th o s e o f R u e s se t a l. [ 1 0 ] f o r t h e b o v i n e c a u d a t e n u c l e u s e n z y m e a n d o f G r o s s m a n a n d L ie f -l a n d e r [ 4 2 ] f o r t h e c a l f e r y t h r o c y t e a c e t y lc h o l in e s te r a s e , w h e n t h e s e e n z y m e sw e r e s u b m i t t e d t o 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 in t h e a b s e n c e o f d e te r -g e n t a n d h a v e t o b e i n t e r p r e t e d a s a r t e f a c t s d u e t o th i s i n a p p r o p r i a t e t e c h n i-q u e .H o w e v e r , t h e r e s u l t s o b t a i n e d w i t h o l d e n z y m e p r e p a r a t io n s a r e p a r t ic u l a r lyi n t e r e s ti n g , s i n c e n o p r e c i p i t a t i o n o c c u r s a t t h e s t a r t o f t h e g e l a n d a d e f i n i t em u l t ip l e b a n d p a t t e r n is o b s e r v e d w i t h M r r a n g i ng f r o m 7 5 0 0 0 t o se v e ra l m i l-l io n s . T h i s h e t e r o g e n e i t y i s c l e a r ly n o t d u e t o p r o t e o l y s i s s i n c e t h e s e o l d p r e p -a r a t i o n s , i n 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 i n t h e p r e s e n c e o f 2- m e r -c a p t o e t h a n o l , g iv e o n l y o n e b a n d i d e n t i c a l t o t h a t o b t a i n e d w i t h fr e s h p r e p a -r a t i o n s . I d e n t i c a l r e s u lt s w e r e o b t a i n e d f o r t h e e r y t h r o c y t e m e m b r a n e a c e t y l-c h o l i n e s t e r a s e b y N i d a y e t al . [ 4 3 ] , a n d o u r i n t e r p r e t a t i o n s a r e v e r y s im i l ar :d u r i n g s t o r a g e f o r a l o n g p e r i o d , d e n a t u r a t i o n p r o c e s s e s o c c u r , in v o l v i n g r e d is -t r ib u t i o n o f th e o l ig o m e r ic s t r u c t u re s o f t h e m e m b r a n e - b o u n d e n z y m e . A s o b -s e r v ed b y o t h e r s [ 3 9 ] , c h a n g e s in s e d i m e n t a t i o n p a t t e r n s a r e a l so d e t e c t e d i ns im i la r c o n d i t i o n s w i t h a c e t y l c h o li n e s te r a s e f r o m e r y t h r o c y t e s m e m b r a n e s .

T h e r e s u lt s o f e le c t r o f o c u s i n g o f t h e p u r if i e d a c e t y l c h o l i n e s t e r a s e c o u l di n d ic a t e a c o m p l e x h e t e r o g e n e i t y o f t h e m e m b r a n e - b o u n d e n z y m e . H o w e v e r ,in c o n t r a s t w i t h w h a t h a s b e e n o b s e r v e d b y o t h e r s f o r th e s u c r o s e / E D T Ae n z y m e [ 5 ] , r e r u n n i n g o f t h e m a j o r b a n d ( p H 5 . 2 1 ) g iv es i n o u r re s u l t s a d if -f u s e a n d b r o a d p e a k o f a c t iv i t y . T h is m e a n s th a t t h e m u l t i p le b a n d p a t t e r n o b -t a i n e d is a n a r t e f a c t d u e t o t h e e l e c t r o f o c u s i n g s t e p i t se l f , p o s s i b l y b y in t e r a c -t io n s b e t w e e n th e e n z y m e a n d a m p h o l y t e s [ 4 4 ] . T h e d e m o n s t r a t i o n o f o t h e r s[ 3 9 , 4 5 ] t h a t a c e t y lc h o l in e s t e ra s e i so l at e d f r o m h u m a n e r y t h r o c y t e m e m b r a n e sw i t h T r i t o n X - 1 0 0 is a s s o c i a t e d w i t h l ip i ds ( m a i n l y p h o s p h a t i d y l s e r i n e ) h a s a ls ot o b e t a k e n i n to a c c o u n t i n th e i n te r p r e t a ti o n o f t h e h e te r o g e n e i t y o b t a i n e dw i t h e l e c tr o f o c u si n g . T h e e f f e c t o f m e r c a p t o e t h a n o l a n d t h e a p p e a ra n c e o f ap r o t e i n b a n d w i t h a c e t y l c h o l i n e s t e r a s e a c t i v i ty a t p H 6 . 3 m i g h t i n d i c a te t h a t a na c t u a l h e t e r o g e n e i t y o f th e e n z y m e e x i s ts . I t a p p e a r s h o w e v e r ( in p r e p a r a t i o n )t h a t t h is b a n d c o r r e s p o n d s t o a n i n c o m p l e t e m o d i f i c a t i o n o f t h e o l ig o m e r i cs t ru c t u r e o f t h e m e m b r a n e - b o u n d e n z y m e d u e t o t h e p a rt ia l r e d u c t i o n o f dis ul -f i d e b r i d g e s .

I m m u n o c h e m i c a l s t u d i e s a re n o w i n p r o g r e ss t o o b t a i n m o r e p r e c is i o n s o nt h e s t r u c t u r e a n d p r o p e r t i e s o f th e r a t b r a i n a c e t y l c h o l in e s t e r a s e .A c k n o w l e d g e m e n t s

W e t h a n k D r s . J . M a s s o u l i e , M . V i g n y a n d S . B o n f o r u s e f u l d is c u s s i o n a n dD r . O . K . L a n g l e y f o r r e v i e w i n g th i s m a n u s c r i p t . J . -P . Z . i s a C h ar g ~ d e R e c h e r -c h e a u C N R S .Re f e r e n c e s

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