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Influence of acetylcholine on rebinding of soluble acetylcholinesterase to a synaptosomal fraction from rat brain
BIOCHIMIE, 1980, 62, 671-680.
Influence of acetylcholine on rebinding of soluble acetylcholinesterase to a synaptosomal fraction from rat brain. Claude BURGUN, J'ean-Marie WARTER, Pierre VEIL, G6rard ~GREM~EL (*) and Albert VCAKSJMAN(**) <>. (Re~u le 1-2-1980. Accept~ apr~s r e m a n i e m e n t le 12-5-1980).
Centre de N e u r o c h i m i e da Centre National de la R e c h e r c h e Scientifique, Institut de Chimie Biologique de la Facultd de Mddecine et Clinique Neurologique da C.H.U., 11 rue H u m a n n , 67085 Strasbourg Cedex (France).
Rdsum~.
Summary.
II est montr6 q u e r a c 6 t y l c h o l i n e s t 6 r a s e relargu6e p a r u n e fraction s y n a p t o s o m a l e de cerv e a u de rat se r a t t a c h e & cette fraction apr~s son i n c u b a t i o n en p r e s e n c e de 3 m M d'ac~ty1choline. Ce p h ~ n o m ~ n e est r~versible. Le glutamate, r a s p a r t a t e et le T-aminobutyrate antagonisent l'effet de l'ac6tylcholine. Le Lubrol WX et Ie Triton X 100 solubilisent plus difficilement les prot6ines totales et l'ac6tylcholinest6rase
A c e t y l c h o l i n e s t e r a s e (Acetylcholine acety1h y d r o l a s e E.C. 3.1.1.7.) r e l e a s e d from a rat b r a i n s y n a p t o s o m a l fraction w a s s h o w n to rebind u p o n incubation in the p r e s e n c e of 3 m M acetylcholine. This action w a s s h o w n to be reversible. Glutamate, a s p a r t a t e a n d y-aminob u t y r a t e a n t a g o n i s e this eifech Solubilization of both bulk protein a n d a c e t y l c h o l i n e s t e r a s e b y Lubrol W'X a n d Triton X 100 after acetylcholine i n c u b a t i o n of s y n a p t o s o m a l fractions is m u c h lower t h a n in the n o n i n c u b a t e d preparation. Local production of protons due to the hydrolysis of acetylcholine b y the e n z y m e could partially explain the reassociation of the e n z y m e . W e s u g g e s t that the o b s e r v e d p h e n o m e n o n m a y p l a y some p h y s i o l o g i c a l role in the function of acetylcholinesterase.
des fractions aprbs incubation en pr6sence d'ac6tylcholine. La production locale de protons lors de l ' h y d r o l y s e de l'ac~tylcholine p a r l ' e n z y m e pourrait expliquer partiellement la r6association de l'ensyme. Le p h 6 n o m ~ n e observ6 pourrait jouer un r61e p h y s i o l o g i q u e clans la fonction de l'acStylcholinest6rase. Mots-cl6s : ACh ; ac6tyleholinest6rase ; liberation ; fixation ; synaptosomes.
Key-words: ACh ; Acetylcholinesterase ; release ; binding ; synaptosomes.
Introduction.
to exist in a broad spectrum of living systems [1, 4, 5, 6, 7] and were found to be either predominantly soluble or membrane bound.
The existence of different molecular forms of acetylcholinesterase is now supported by strong evidence [1, 2, 3]. These forms have been shown
Abbreviations : Acetylcholinesterase Acetylcholine Acetylthiocholine Tetraisopropylpyrophosphoramide Diisopropylfluorophosphate
: : : : :
ACHE. ACh. ATCh. ISOMPA. DFP.
(*) Charg~ de Recherche h I'INSERM. (") Maitre de Recherche au CNRS. <> To whom all correspondence should be addressed.
Aggregation between some of the forms has been suggested to be a possible mechanism relating soluble to bound enzyme. Although much emphasis has been placed on the molecular structure and the metabolic aspects of acetylcholinesterase, little is known about the membrane dynamics in which this enzyme system is involved, and the possible role of this dynamic in neurohumoral transmission. In a previous study where rat liver mitochondrial membranes were used as a model system, strong evidence was produced that some
672
C. Bur.qun and coll.
e n z y m e s a n d p r o t e i n s w e r e a b l e to b e r e l e a s e d and rebound to the inner mitochondrial membrane in response to changes in the external environment. This phenomenon, which might have iml~ortant functional and structural implications was shown to be reversible, specific, and dependent on the lipid composition of the membrane [8, 9 ] . I n o r d e r t o r e a c h a b e t t e r u n d e r s t a n d i n g of such molecular processes involved at the syna, p t i c l e v e l , i t s e e m e d o f i n t e r e s t to u s to e x a mine whether protein movements comparable to those detected in the mitochondria could be observed in synaptosomal preparations. The enzyme chosen as model was acetylcholinesterase (Acetylcholine Acetylhydrolase 3.1.1.17.). In the present article, release and rebinding of acetylcholinesterase to synaptosomal preparations is reported under defined experimental conditions.
Materials and Methods. All p r o d u c t s w e r e o f t h e p u r e s t q u a l i t y a v a i l a b l e . Sucrose (Analar) was obtained from BDH chemicals L d t (Poole Engl,and). Fieoll 400 w a s f r o m P h a r m a c i a F i n e C h e m i c a l s ( S w e d e n ) . S o d i u m a s p a r t a t e a n d glut a m a t e w e r e p u r c h a s e d f r o m E. M e r c k AG ( D a r m s t a d t ) . 7 - a m i n o b u t y r i c acid, a c e t y l e h o l i n e c h l o r i d e , a e e t y l t h i o c h o l i n e iodide, c h o l i n e c h l o r i d e , a s w e l l a s b o v i n e s e r u m a l b u m i n e a n d T r i t o n X 100 w e r e f r o m S i g m a C h e m i c a l CO, S t - L o u i s MO. M i n e r a l s a l t s , b a s e s a n d acids were obtained from Prolabo, Rh6ne-Poulenc. Lubrol WX was purchased from Imperial Chemical Industries. Labelled products were from Amersham France. All s o l u t i o n s w e r e p r e p a r e d in 0.32 M s u c r o s e a d j u s t e d to p H 6.4 w i t h HC1 o r N a O H , d e p e n d i n g o n t h e effector u s e d . All c e n t r i f u g a t i o n s w e r e p e r f o r m e d in a S p i n e o m o d e l L 50 c e n t r i f u g e , a n d all m a n i p u l a t i o n s w e r e p e r f o r m e d a t 4°C u n l e s s o t h e r w i s e m e n t i o n e d .
or 0.32 M s u c r o s e c o n t a i n i n g t h e s t u d i e d effectors. All f u r t h e r a d d i t i o n s a r e i n d i c a t e d in t h e legend,s o f figures. T h e t o t a l v o l u m e w a s a d j u s t e d to 2 m l a t a p H of 6.4. A f t e r i n c u b a t i o n , t h e p r e p a r a t i o n s w e r e c e n t r i f u ged a t 106,000 g f o r 60 w i n i n a R 40 r o t o r . S u p e r n a t e n t s w e r e r e c o v e r e d . P e l l e t s w e r e s u s p e n d e d in 4.5 m l w a t e r a n d h o m o g e n i z e d f o r 30 s in a g l a s s - t e f l o n h o m o g e n i z e r ( T h o m a s ) a t 880 r p m . F o r d e t e r g e n t t r e a t m e n t , 500 p.1 of a s o l u t i o n o f d e t e r g e n t ( L u b r o l W X o r T r i t o n X 1,00,) at d i f f e r e n t c o n c e n trations, were a d d e d to e i t h e r c r u d e or p u r i f i e d s y n a p tosomal fractions. The mixtures were incubated for 15 m i n a t 37°C in r o t a r y s h a k i n g b a t h , and' c e n t r i f u g e d f o r 60 m i n a t 105,000 g in a R 40 t y p e r o t o r . S u p e r n a tants were recovered. The pellets were suspended by h o m o g e n i z a t i o n a s a b o v e a n d b r o u g h t to a final v o l u m e of 5 m l w i t h w a t e r . E n z y m a t i c a n d p r o t e i n m e a s u r e m e n t s w e r e p e r f o r m e d on t h e d i f f e r e n t f r a c t i o n s . I n t h e a b o v e a s s a y s , t h e i n c u b a t i o n in s u c r o s e corr e s p o n d s to a r e l e a s e e x p e r i m e n t , w h e r e a s t h e i n c u b a t i o n i n t h e p r e s e n c e of a c e t y l c h o l i n e w h i c h a l w a y s f o l l o w s a s u c r o s e i n c u b a t i o n c o r r e s p o n d s to a r e b i n ding experiment. I n t h e c a s e of r e v e r s i b i l i t y e x p e r i m e n t s , t h r e e c r u d e s y n a p t o s o m a l f r a c t i o n s w e r e p r e p a r e d . T h e first f r a c t i o n w a s i n c u b a t e d in 0.32 M s u c r o s e , 1.5 m l A C h (acet y t e h o l i n e ) a n d 3.0 m M A C h w e r e a d d e d r e s p e c t i v e l y to t h e 2 n d a n d 3rd f r a c t i o n . T h e s e f r a c t i o n s w e r e t h e n c e n t r i f u g e d f o r 1 h a t 105,0~)0 g in a R 40 r o t o r . T h e petlets were discarded and the supernatants -- namely : s u c r o s e , A C h 1.5 m M a n d A C h 3.0 m M w e r e d i a l y z e d at 4 ° C a g a i n s t 4 × 2 l i t e r s 0.3.2 M s u c r o s e f o r 24 h. I d e n t i c a l f r e s h l y p r e p a r e d p e l l e t s (2D m g p r o t e i n / m l ) n a m e l y s u c r o s e , A C h 1.5 m M a n d A C h 3.0 m M w e r e i n c u b a t e d e i t h e r in 0.32 M s u c r o s e f o r c o n t r o l , or in presence of their diaIyzed corresponding supernatant, o r in t h e p r e s e n c e of t h e a b o v e m e n t i o n e d , s u p e r n a t a n t s in w h i c h t h e i n i t i a l A C h c o n c e n t r a t i o n w a s r e s t o r e d . P r o t e i n c o n c e n t r a t i o n in i n c u b a t i o n m i x t u r e w a s b r o u g h t to 20 m g p e r m l . In~cubations w e r e p e r f o r m e d a t 37°C f o r 5 m i n . T h e n , t h e t h r e e p r e p a r a t i o n s w e r e c e n t r i f u g e d a t 10'5,000 g in a R 40 r o t o r f o r 1 h a t 4°C. T h e s u p e r n a t a n t s w e r e collected a n d t h e p e l l e t s were r e s u s p e n d e d a n d h o m o g e n i z e d in 4.5 m l ice-cooled water ; protein and AChE (acetylcholine esterase) determinations were performed on each fraction.
Preparation of crude and purified synaptosomaI fractions.
Labelling of AChE with synaptosomal fraction.
T h e m e t h o d f o r t h e p r e p a r a t i o n of b o t h c r u d e a n d purified synaptosomal fractions was essentially derived from the technique described by Morgan and coll. [10].
A C h E f r o m r a t b r a i n w a s p u r i f i e d a c c o r d i n g to t h e m e t h o d of M a s s o u l i 6 a n d B o n [11]. T h e p u r i f i e d e n z y m e w a s l a b e l l e d f o l l o w i n g t h e t e c h n i q u e of B o l t o n a n d H u n t e r [12]. 1 ~g of e n z y m e d i s s o l v e d i n 100 ~1 0.1 M p h o s p h a t e b u f f e r a t p H 8 w a s t r e a t e d d u r i n g 15 m i n at 0°C, in t h e p r e s e n c e of 1 m C i of B o l t o n a n d H u n t e r reagent.
Adult albino Wistar rats were killed by decapitat i o n ; t h e b r a i n s removed~ a n d placed' i n ice-cold 0.32 M sucrose. The original method was applied except that E D T A - p o t a s s i u m p h o s p h a t e w a s o m i t t e d in t h e h o m o genization medium anti the crude synaptosomal fraction was washed twice before use. For the purified synaptosomal fraction, EDTA- potassium phosphate was also omitted from the preparation.
Incubation of fractions and detergent treatments. S y n a p t o s o m a l f r a c t i o n s c o r r e s p o n d i n g to 20 m g of p r o t e i n s were i n c u b a t e d f o r 5 m i n a t 37°C in a r o t a r y s h a k i n g b a t h in t h e p r e s e n c e of e i t h e r 0.32 M s u c r o s e ,
BIOCH1MIE, 1980, 62, n ° 10.
l~st and incubation
with
T h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of 0.5 m l 0.2 M g l y e i n e in 0.1 M p h o s p h a t e b u f f e r a t p H 8. Labelled enzyme was separated from excess reagent on a G 25 S e p h a d e x c o l u m n e q u i l i b r a t e d w i t h 50 m M p h o s p h a t e b u f f e r at p H 7.5. F r a c t i o n s o f 5.00 ~l w e r e collected. 20 ~l of t h e m o s t active f r a c t i o n c o n t a i n i n g 1 ~g l a b e l l e d e n z y m e a n d c o r r e s p o n , d i n g to a b o u t 355.10~ c p m w a s a d d e d to 1 ml of s y n a p t o s o m a l s ~ s p e n s i o n c o n t a i n i n g 2'0 m g of p r o t e i n in 0.32 M s u c r o s e . F i n a l v o l u m e w a s a d j u s t e d to 2 m l w i t h 0.32 M s u c r o s e .
Rebinding of acetylcholinesterase to synaptosomes. I n c u b a t i o n s were p e r f o r m e d in the presence or absence of 2 mM ACh u n d e r conditions described above.
Labelling of acetylcholinesterase with FSH] diisopropylfluorophosphate. The method applied was basically t h a t of Bellhorn and coll. [13]. A s u p e r n a t a n t fraction obtained after incubating a s y u a p t o s o m a l fraction (2,0 mg p r o t e i n / m l ) in the presence of 0.32 M sucrose for 5 rain at 37°C was isolated, concentrated 5-fold on an Amicon PM 30' filter, centrifuged 30 rain a t 1D0:,000 g and a d j u s t e d to final 10 raM, pH 7.6 by a d d i t i o n of concentrated p h o s p h a t e buffer. a) An aliquot (500 /xl) of this fraction was incubated for 30 min at room t e m p e r a t u r e in the presence of 0.2 M b u t y r y l c h o l i n e , in order to protect the active site of AChE ; it was t h e n incubated for 30 rain at room t e m p e r a t u r e in the presence of lt0-6 M u n l a b e l l e d DFP. After being passed, on a G 25 Sephadex c o l u m n , and eluted w i t h 0.1 M p h o s p h a t e buffer at pH 7.6, the protein fraction was reincubated for 30 m i n at room t e m p e r a t u r e in presence of 5 ~Ci [3H] DFP (10-6 M). The final preparation, was dialyze~ f o r 24 h at 4°C against 3 × 2 l, lfi mM p h o s p h a t e buffer at pH 7. b) In parallel a n o t h e r 500 B1 aliquot of the initial p r e p a r a t i o n was i n c u b a t e d directly in the presence of 5 ~Ci [3H] DFP (10-6 M) and dialyzed as above. Control of enzyme activity showed t h a t b u t y r y l c h o l i n e completely protected the enzyme site, and t h a t a f t e r its e l i m i n a t i o n and t h a t of un.labelle(~ DFP, AChE activity was preserved. The addition of [aH] DFP then totally inhibited the enzyme. In the absence of butyrylcholine, cold DFP inhibits 90 per cent of AChE activity. Labelling was of 10,400 c p m / m g p r o t e i n for a) a n d 1'3,000 c p m / m g protein f o r b).
673
p h o t o m e t e r . The concentration of ATCh was 1 mM. During the m e a s u r e m e n t s of the AChE activity, the ¢ exogenously introduced >> ACh in the euvette never exceeded 0.18 mM and was generally a r o u n d 0.075 raM. Under these conditions, it w a s d e m o n s t r a t e d t h a t t h i s small a m o u n t of ACh did not interfere w i t h the ACkE activity m e a s u r e d by the ATCh method, p r o b a b l y because u n d e r our working con.ditions, this ACh was already h y d r o l y z e d at the t i m e AChE was as.sayed. F u r t h e r m o r e , AChE activity was not different w h e n m e a s u r e d on the j u s t mentioned' s u p e r u a t a n t fluid f r o m which the exogenous ACh had been previously dialyzed. The m e t h o d of Lowry and coll. [16] was used for protein d e t e r m i n a t i o n w i t h s e r u m a l b u m i n as s t a n dard.
Results. I n f l u e n c e of acetylcholine on the partition o[ acetylcholinesterase a c t i v i t y associated to di[[erent subcellular fractions in a sucrose gradient. S u b f r a c t i o n a t i o n of a c r u d e s y n a p t o s o m a l fraction on a s u c r o s e g r a d i e n t r e v e a l e d that AChE activity partitionned differently between myelin, synapt0somal and mitochondrial fractions when c e n t r i f u g a t i o n w a s p e r f o r m e d on a 0.32 M s u c r o s e i n c u b a t e d c r u d e f r a c t i o n or in a f r a c t i o n i n c u b a -
TABLE I.
I n f l u e n c e of acetyteholine on the partition o[ acetytcholinesterase a c t i v i t y associated to d i f f e r e n t subcellular [factions, in a sucrose gradient. sucrose
My. Sy. Mit.
3 mM ACh
activity
(per cent)
activity
{per cent)
4.16 1.93 0.28
65.3 30.3 4.4
1.13 3.52 1.14
29.3 53.4 17.3
sucrose
3 mM ACh
specific activity 0.108 0.060 0. 010
0.073 0.096 0. 037
The three fractions were obtained by r e c e n t r i f u g a t i o n of a crude s y n a p t o s o m a l f r a c t i o n on a 0.8 M/1.0 M/1.2 M sucrose gradient. The so-called Myelin fraetinn contains soluble AChE and myelin. My. : Myelin ; Sy. : Synaptosomes ; Mit. : M,itochondria. Activity is expressed' as, t~moles ATCh h y d r o l y z e d per rain per fraction. Specific activity is expressed as ~moles ATCh h y d r o l y z e d per m i n / m g protein.
Enzymatic assays and protein determinations. Aeetylcholinesterase aetlvity w a s determined~ according to the method, of E l l m a n and coll. [14], the m e a s u r e m e n t s were p e r f o r m e d at 30°C in the presence of p h o s p h a t e buffer 0.1 M, pH 7.6, and 1 mM of 5,5rdithiobis-(2-nitrobenzoie acid) and 10-5 M ISOMPA to i n h i b i t non-specific esterases [15]. Readings were perf o r m e d at 412 n.m in a Beckman Aeta III type speetro-
BIOCH1MIE, 1980, 62, n ° 10.
t e d in t h e p r e s e n c e o f 3 m M A C h ( t a b l e I). It c a n b e s e e n t h a t i n t h e p r e s e n c e of A,Ch, 55 p e r c e n t of t h e A C h E p r e v i o u s l y p r e s e n t i n t h e m y e l i n f r a c t i o n (My), w h i c h also c o r r e s p o n d e d to t h e p o o l of s o l u b i l i z e d e n z y m e , s h i f t e d t o w a r d s b o t h s y n a p t o s o m a l - r i c h f r a c t i o n s : i.e. p u r e s y n a p t o s o m e s (Sy) a n d m i t o c h o n d r i a (Mi).
C. B u r g u n and coll.
674
Influence of acetylcholine and acetglthiocholine on release and binding of acetylcholinesterase by a crude synaptosomal fraction. Table II s u m m a r i z e s the i n f l u e n c e of sucrose or sucrose s u p p l e m e n t e d w i t h N,aC1 or KC1, acetate, c h o l i n e and a c e t y l c h o l i n e on the solubilization of a c e t y l c h o l i n e s t e r a s e after i n c u b a t i o n of a c r u d e s y n a p t o s o m a l f r a c t i o n , as d e s c r i b e d in Methods. As can be seen, u n d e r these c o n d i t i o n s , only acet y l e h o l i n e has a m a r k e d i n h i b i t o r y effect on the release of a e e t y l c h o l i n e s t e r a s e . F i g u r e 1 s h o w s r e b i n d i n g of r e l e a s e d a c e t y l c h o l i n e s t e r a s e as a f u n c t i o n of a c e t y l c h o l i n e or a c e t y l t h i o c h o l i n e c o n c e n t r a t i o n . R e b i n d i n g of a c e t y l c h o l i n e s t e r a s e levels off at 3 mM a c e t y l c h o l i n e .
ting 12~I labelled AChE w i t h a s y n a p t o s o m a l fraction in the p r e s e n c e of a c e t y l c h o l i n e . Results are s u m m a r i z e d in table IV. In the p r e s e n c e of 0.32 M s u c r o s e alone, 78 p e r cent of the labelled e n z y m e
t0AS o.,o
TABLE II.
Influence of acetylcholine and other substances on release and binding of acetylcholinesterase on a crude sllnaptosomal fraction.
Sucrose (0.32 M) Acetate Choline Acetate -~- choline Acetylcholine NaCI KC1
Specific activity of soluble acetylcholinesterase ,~moles per mg protein • per mill
Per cent of soluble acetylcholinesterase
0.22 0.23 0.26 0.31 0.08 0.30 0.32
10.5 :5.2 4.5 :5.2 0.8 r'6.6 7.0
AJll substances were used at a concentration of 20 mM, except aeetylcholine which was 3 mM.
Rebinding o[ acetylcholinesterase to a purified synaptosomal fraction in absence and presence of exogenous acetylcholine. I n c u b a t i o n of a p u r i f i e d s y n a p t o s o m a l f r a c t i o n in the p r e s e n c e or absence of 3 mM a c e t y l c h o l i n e was p e r f o r m e d u n d e r the above d e s c r i b e d conditions. A t y p i c a l set of results is s u m m a r i z e d in table III. T h e y c l e a r l y s h o w that f o r such a prep a r a t i o n , a c e t y l c h o l i n e s t e r a s e is r e l e a s e d f r o m the m e m b r a n e f r a c t i o n in 0.32 M sucrose and b o u n d to that f r a c t i o n w h e n 3 mM a c e t y l c h o l i n e is present.
Rebinding of le5I labelled acetylcholinesterase to a crude synaptosomal fraction in presence of acelylcholine. R e b i n d i n g of A C h E to s y n a p t o s o m a l m e m b r a n e s can be d e m o n s t r a t e d u n a m b i g u o u s l y by i n c u b a -
BIOCHIMIE, 1980, 62, n ° 10.
w i
mM
Ach •
or
ATCh o
FIG. 1. - - B i n d i n g influence of acetglcholine (ACh) or acetltithiocholine (ATCh) on the acet!tlcholinesterase solubilized from a crude sllnaptosomal fraction. The activity of the enzyme is expressed in ixmol of aeetylth~iocholine hydTolyzed per min per 20 mg synaptosomal protein.
w a s f o u n d in the s u p e r n a t a n t f r a c t i o n and 22 p e r cent w a s associated to the s y n a p t o s o m a l pellet, w h e r e a s in t h e p r e s e n c e of 2 mM ACh the situation w a s r e v e r s e d , w i t h 76 p e r cent r a d i o a c t i v e e n z y m e b o u n d to the s y n a p t o s o m a l fraction. T h e ratio of r a d i o a c t i v e AChE to e n z y m e activ i t y r e m a i n i n g in s u p e r n a t a n t after r e b i n d i n g is i d e n t i c a l f o r s u c r o s e treated or ACh t r e a t e d synaptosomes, thus c o n f i r m i n g that both labelled protein a n d a c t i v i t y r e b i n d to the same e x t e n d to the s y n a p t o s o m a l m e m b r a n e . In v i t r o reversibility of the binding phenomenon. I n c u b a t i o n of a c r u d e s y n a p t o s o m a l f r a c t i o n in the p r e s e n c e of e i t h e r 1.5 mM a c e t y l c h o l i n e or 3 mM a e e t y l c h o l i n e results in b i n d i n g of the soluble a c e t y l c h o l i n e s t e r a s e to the m e m b r a n e f r a c t i o n as seen above. R e i n c u b a t i o n of such a f r a c t i o n in the p r e s e n c e of e i t h e r 0.32 M sucrose or the s o l u b i l i z e d fractions o b t a i n e d a f t e r sucrose or sucrose + acetyl-
Rebinding of acetylcholinesterase to synaplosomes. c h o l i n e t r e a t m e n t and d i a l y z e d against 0.32 M sucrose, results in the f u r t h e r release of acetylcholinesterase. This suggests that the r 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 can be released once r e b o u n d to the m e m b r a n e fraction, f o l l o w i n g i n c u b a t i o n in the p r e s e n c e of 1.5 mM or 3.0 mM a c e t y l c h o l i n e (table VA).
675
Moreover, if i n c u b a t i o n o c c u r s in the p r e s e n c e of s y n a p t o s o m e s aged for 36 h at 4°C no b i n d i n g of A~ChE o c c u r s even at ACh c o n c e n t r a t i o n s up to 5 mM (see figure 3). T h i s is c o n t r a r y to the results obtained with a freshly prepared synaptosomal f r a c t i o n w h i c h strongly r e b i n d s ACHE, as s h o w n above.
TABLE HI.
Influence of acetylcholine on release and binding of bulk protein and acetylcholinesterase on a purified synaptosomal fraction. Specific activity of acetyleholinesterase ~moles per mg protein per min Fraction
Synaptosomes Fluid
Lubrol supernatant
Residual pellet
Total acetylcholinesterase activity
Sucrose -~- acetylcholine
2.5 10 -s (0.15 per cent)
7.2 10 -3 (3.2 per cent)
28.9 10 -3 (96.6 per cent)
2.60
Sucrose
23.9 I0 -a ( 2 . 4 per cent)
8.1 10 -3 (6.7 per cent)
30.8 10 -3 (90.9 per cent)
2.58
Per cent of total protein Fraction
Synaptosomes
Fluid
Lubrol supernatant
Residual pellet
Total recovery
Sucrose -~-acetylcholine
1.6 per cent
11.6 per cent
86.8 per cent
100 per cent
Sucrose
2 . 6 per cent
21.4 per cent
76 per cent
92.2 per cent
After incubation either in sucrose alone or in sucro.se plus acetyleholine, the synaptosomal fraction was separated from the fluid fraet.ion and treated with 0.16 mg Lubrol per mg protein ; a pel]et and a supernatant were then separated (105,0100 g, 1 h). Percentage of AChE in each fraction is given.
F u r t h e r m o r e , w h e n i n c u b a t e d in the p r e s e n c e of the d i a l y z e d s u p e r n a t a n t to w h i c h t h e i r original c o n c e n t r a t i o n of a c e t y l c h o l i n e w a s restored, c r u d e s y n a p t o s o m a l f r a c t i o n s s h o w e d no release of a c e t y l c h o l i n e s t e r a s e (table VB). H o w e v e r , w h e n i n c u b a t e d in the p r e s e n c e of 0.32 M sucrose alone, these f r a c t i o n s w h e t h e r p r e i n c u b a t e d in sucrose or in the p r e s e n c e of 1.5. mM or 3.0 mM a c e t y l c h o l i n e (table V'C) s h o w e d release of a c e l y l c h o l i n e s t e r a s e activity.
Influence of aging of the sgnaptosomaI fraction on the rebinding of ACHE. The above results could be due to the p r e c i p i t a tion of ~ C h E in the course of A,Ch h y d r o l y s i s . Tllis p o s s i b i l i t y was r u l e d out by the fact that in the absence of s y u a p t o s o m a l m a t e r i a l t h e r e is no delectable, p e l l e l a b l e activity.
BIOCHIM1E, 1980, 62, n ° 10.
Influence of pH on rebinding of AChE to synaptosomal fraction. Little is k n o w n about the b u f f e r i n g c a p a c i t y of the s y n a p t i c cleft m i c r o e n v i r o n m e n l . Our first e x p e r i m e n t s w e r e t h e r e f o r e p e r f o r m e d in sucrose at pH 6.4. T h e y thus i n d i c a t e a p o t e n t i a l beh a v i o u r of this system. I n f l u e n c e of pH w a s c o m p a r e d in two d i f f e r e n t situations : a) the final pH w a s i m p o s e d by 10 mM v a r i o u s buffers at different pH. b) the final pH w a s the result of h y d r o l y s i s of different initial c o n c e n t r a t i o n s of ACh. It can be seen that at equal final pH, m u c h m o r e AChE a c t i v i t y r e b i n d s in the p r e s e n c e of ACh than in its absence (figure 2).
676
C. B u r g u n a n d coll.
Thus, at a final p H of 6.0, 85 p e r cent of the AChE a c t i v i t y ffisappears f r o m the i n c u b a t i o n med i u m in the p r e s e n c e of ACh a n d only 15 p e r cent in its absence, suggesting a d i f f e r e n t i a l effect of both p H (or buffer type) and ACh on the rebinding of .~ChE to a s y n a p t o s o m a l fraction. Rebinding of half of the soluble AChE o c c u r s at p H 6.7 in the p r e s e n c e of ACh and at pH 5.35 in its absence.
Results s h o w that only 9 p e r c e n t of labelled e n z y m e is to be f o u n d in s y n a p t o s o m a l p e l l e t (table VI), in 0.32 M sucrose alone against 47 p e r
% released
AChE
100-
% released
AChE
100. 50-
50. 0 ACh (raM)
01
Final ]~ pH in supernatant
Fro. 2. - - Influence of final pH on AChE rebinding to synaptosomes in presence of ACh (•) and in buffered solutions at various pH (D). The activity is expressed as per cent of activity in supernatan1 at p,H 7.
F u r t h e r m o r e , in buffered m e d i a at pH 7, rebinding o c c u r s as s h o w n in figure 3. At 2 mM ACh, the d i f f e r e n t buffers do not lead to the same extent of r e b i n d i n g . R e b i n d i n g is 45 p e r cent of total a c t i v i t y in 1,0 mM Tris buffer, 30 p e r cent in 10 mM p h o s p h a t e buffer and 20 p e r cent in 1~) mM Hepes buffer. At 5 mM ACh h o w e v e r , r e b i n d i n g is of 55 p e r cent regardless of the nature of the buffer used. These results suggest a d i s t i n c t i n f l u e n c e of ACh, ions and p H on r e b i n d i n g of ACHE. I n f l u e n c e of e n z y m e inhibition on e n z y m e rebinding. T h e above results show that both ACh a n d pH influence AChE r e b i n d i n g to s y n a p t o s o m a l fraction. To distinguish these t w o effects, AChE was i n h i b i t e d by b l o c k i n g of its active site w i t h aH labelled D FP. R e a s s o c i a t i o n of labelled, i n h i b i t e d e n z y m e was then m e a s u r e d in the p r e s e n c e and absence of ACh. BIOCHIMIE, 1980, 62, n ° 10.
FIn. 3. - - Influence of various 10 mM buffers in the presence of increasing ACh concentrations on rebinding of AChE to synaplosomes. The activity is expressed as per cent of activity in sucrose supernatant. Control shows no rebinding to aged synaptosomes. • • aged synaptosomes. © © sucrose • • >> + PO, 10 mM -k "k >> + tris PH 7 • • >) + Hepes
/IGLUTAMATE
y_
/
•
/
~. o.1 -~ .-c_
I.u .¢
2 =
/
-
/:" // /..
OG
.
.'WASPAR i A T E
.../' '
,/ ~20
150
TIO0
1125 mM
Amino
acid
Fro. 4. - - Antagonistic influence of glutamate, asparrate and 7-aminobutyratc (GABA) to the binding of acetylcholineslerase provoked by 5 mM acetylcholine. Activity of soluble acetyleho]inesterase is expressed in p.mol'of acetylthiocholine hydrolized per min per 20 mg synaplosomal protein.
Rebinding
of acelylcholinesterose
c e n t in the p r e s e n c e of 3 mM ACh, t h e last figure is to be c o m p a r e d to 76 p e r c e n t a c t i v i t y in the case of l a b e l l e d n o n - i n h i b i t e d e n z y m e .
I n f l u e n c e of acetylcholine on the solubilisation of acetylcholinesterase by Lubrol W X and Triton X 100.
R e b i n d i n g of 1~5I labelled acetylcholinesterase to s y n a p t o s o m a l fraction in the p r e s e n c e of acetylcholine.
100-
Radioactivity cpm Ct0-:*) Supernatant
Pellet
Total
279.5
76.5
356
.° Sucrose
ACh 2 mM
90
277
B
367
Supernatant AChE activity
"/protein
50.
oe i i
i l
b. "/activity r
sucrose
ACh 2 mM
132
498
2.1
38
383
2.4
Binding of 1251 labelled AChE : a) radioactivity associated to supernatant and pellet ; b) enzymatie aetivity and ratio of radioaetivity/prorein o r / e n z y m a t i c activity. Radioactivity is expressed in 1,0-3 epm. Enzymatic activity is expres:sed in nmoles of ATCh hydrolyzed p e r ran. Protein is expressed in rag.
62,
n °
10.
2150 mg
!ubrol / mg protein B
-t
50
0
O
J
m9
t r i t o n / rng protein
FiG. 5.
Antagonist influence of glutamate, asportate and 7-aminobutyrate to the action of aeetylcholine.
1980,
I
P,
A.-
If t h e o b s e r v e d r e l e a s e a n d b i n d i n g of a e e t y l c h o l i n e s t e r a s e w e r e to p l a y s o m e p h y s i o l o g i c a l r o l e in t h e m o d u l a t i o n of n e r v o u s e v e n t s , t h e n o n e w o u l d e x p e c t a g e n t s s u c h as 7 - a m i n o b u t y r a t e , g l u t a m a t e o r a s p a r t a t e to h a v e s o m e p o s s i b l e inf l u e n c e o n t h i s r e l e a s e a n d b i n d i n g . I n t h e following experiments crude synaptosomal fractions w e r e i n c u b a t e d in t h e p r e s e n c e of 5 m M a e e t y l c h o l i n e a n d i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r g l u t a m a t e , a s p a r t a t e o r , t - a m i n o b u t y r a t e . All t h r e e c o m p o u n d s w e r e i n h i b i t o r y t o w a r d s t h e a c t i o n of a c e t y l e h o l i n e on t h e r e l e a s e of a e e t y l e h o l i n e s t e r a s e (figure 4).
1.125
,°°{
Tire f a c t t h a t t h e r a t i o of r a d i o a c t i v i t y u p o n e n z y m e a c t i v i t y i n c r e a s e s 5.6-fotd in the s u p e r n a t a n t f r a c t i o n in t h e p r e s e n c e of A~ih s t r o n g l y s u g g e s t s t h a t i n h i b i t e d e n z y m e r e a s s o e i a t e s less w e l l to m e m b r a n e f r a c t i o n t h a n a c t i v e e n z y m e .
BIOCHIMIE,
677
T h e q u e s t i o n as to w h e t h e r in the p r e s e n c e of a c e t y l c h o l i n e t h e r e l e a s e d a c e t y l c h o l i n e s t e r a s e is
TABLE IV.
A
to s y n a p t o s o m e s .
Solubilization by Lubrol WX of bound acetylcholineaterasc and bulk protein in the presence or absence of 5 raM accttllcholine. ---
in 0.32 M sucrose alone ; in 0.82 M sucrose supplemented with 5 mM aeetyleholine.
B. - - Solubilization by Triton X 100 of bound acetylcholinesterase and bulk protein in the presence or absence of 5 mM acetylcholine.
in 0.32 M sucrose alone ; in 0.82 M sucrose supplemented with 5 mM aeetyleholine. Activity is expressed as percentage of activity in pellet after action of detergent as compared to the activity in pellet without detergent (A typical set of ---
CUI'Ves),
s u p e r f i c i a l l y b o u n d to t h e m e m b r a n e o r is m o r e embedded was approached by treating the memb r a n e s w i t h d e t e r g e n t s . R e s u l t s of t h e i n f l u e n c e
C. B u r g u n a n d coll.
678
of L u b r o l W X a n d T r i t o n X 100 a r e s h o w n i n f i g u r e s ~A, 5B. S o . l u b i l i z a t i o n of b o u n d a c e t y l cholinesterase and protein was compared in crude s y n a p t o s o m e s t r e a t e d w i t h e i t h e r 0.32 M s u c r o s e alone or supplemented with 5 mM acetylcholine.
In addition, the hypotonic shock which occurs during these experiments showed a di,fferential p r o t e i n r e l e a s e i n t h e c o n t r o l a s s a y s (in t h e p r e s e n c e o r a b s e n c e of a c e t y l c h o l i n e ) . TABLE VI.
W h e r e a s L u b r o l W X at 0.5 m g / m g of p r o t e i n s o l u b i l i z e d u p to 90 p e r c e n t of t h e b o u n d a c e t y l cholinesterase in a sucrose-treated preparation, o n l y 40 p e r c e n t of t h e e n z y m e w a s e x t r a c t e d when synaptosomes were pretreated with 5 mM a c e t y l c h o l i n e ( f i g u r e 5A). C o m p a r a b l e s o l u b i l i z a -
R e b i n d i n g of [ZH]DFP labelled AChE to synaplosomes. Radioactivily cpm (10-:~1 Supernatant
TABLE V. In supernatants AChE activity (nmoles ATCh hydrolyzed/min~ _ in fraction
1. Sucrose
2. ACh 1.5 mM 3. ACh 3 mM
Before
reineubation of dialyzed supernatants with their corresponding pellets 41 50 13 50,5 9 42
2. ACh 1.5 mM 3. ACh 3 mM
Before
AIter
reincubation o! dialyzed supernatants with their corresponding pellets and their initial ACh concentration 13 5 9 3
Controls AChE activity (amoles ATCh hydrolyzed/min) in fraction
ACh 2 mM
223.4
Total
40
449
195
418
Supernatant
Sucrose
ACh
AChE activity
"/protein
"/activity
84
1231
4.9
8
2031
27.9
a) D i s t r i b u t i o n of [aH] labelled ACRE in soluble and s y n a p t o s o m a l fraction, w h e n s y n a p t o s o m e s were incub a t e d in sucrose a l o n e or sucrose plus 3 mM ACh. b) AChE activity, r a t e of r a d i o a c t i v i t y / p r o t e i n or e n z y m a t i c activity. Activity is expressed in, nmoles ATCh h y d r o l y z e d per min/fraetion.
[00
In sucrose 0.32 M
Sucrose ACh 1 . 5 rnM
51 50,5
ACh 3 mM
43
R~versibility of AChE b i n d i n g to s y n a p t o s o m a l membranes. A. Released AChE w h e n sucrose dialyzed s u p e r n a t a n t s are r e i n e u b a t e d w i t h t h e i r corresponding synaptosomes ; 1, sucrose a l o n e ; 2 a n d 3, with 1.5 mM a n d 3 mM ACh, respectively. B. AChE r e b o u n d wl~et~ dialyzed s u p e r n a t a n t s are reineubated with their corresponding synaptosomes in the presence of 1.5 mM (2) a n d 3 mM (3) ACh. C. Released AChE w h e n s y n a p t o s o m c s are r e i n c u b a t e d in sucrose alone.
t i o n i n b o t h s a m p l e s w a s o n l y o b t a i n e d at c o n c e n t r a t i o n s o.f L u b r o l a b o v e 2 m g p e r m g of p r o t e i n . I n a simil,ar m a n n e r , i n s u c r o s e - t r e a t e d s y n a p t o s o m e s 42 p e r c e n l of t h e t o t a l , p r o t e i n w e r e e x t r a c t e d at 0.5 m g L u b r o l p e r m g of p r o t e i n , w h e r e a s i n a c e t y l c h o l i n e - t r e a t e d p r e p a r a t i o n s o n l y 25 p e r c e n t of t h e t o t a l p r o t e i n w e r e e x t r a c t e d (~igure 5A).
BIOCHIM1E, 1980, 62, n ° 10.
408.7
AIter
in supernatants AChE activity (nmoles ATCh hydrolyzed/mio~ iu traction
Sucrose
Pellet
110
120 mM ACh
FIG. 6. - - Solubilization of acetylcholinesterase by
0.5 m 9 Lubrol WX per log protein in the presence ot increasing concentrations o f acetfflcholine.
Activity is, expressed as p e r cent of activity in pellet a f t e r action of A C h / a c t i v i t y in pellet in the absence of ACh.
T r i t o n X 10,0 ( u p to 4 p e r c e n t v / v ) s o l u b i l i z e d n o m o r e t h a n 17 p e r c e n t a c e t y l c h o l i n e s t e r a s e i n acetylcholine-treated synaptosomes a n d u p to
Rebinding
of acetylcholinesterase
to s y n a p t o s o m e s .
679
90 p e r cent in sucrose-treated particles (figure 5B). S i m i l a r l y d i f f e r e n t i a l proteins solubilization was also observed. In the presence or absence of acetylcholine, 50 per cent a n d 90' per cent of total protein were extracted, respectively (figure 5~B).
cleft is the major n a t u r a l source protons. This would also fit the r e b o u n d at m u c h h i g h e r pH directly hydrolyzed, t h a n w h e n was due to global pH change.
The effects of 0,.5 mg Lubrol W X / m g p r o t e i n on the s o l u b i l i z a t i o n of acetylcholinesteras.e .as a f u n c t i o n of a c e t y l c h o l i n e c o n c e n t r a t i o n is summarized in figure 6. The results o b t a i n e d w i t h inc r e a s i n g a c e t y l c h o l i n e c o n c e n t r a t i o n s show that the solubilization of the enzyme is only possible at a c o n c e n t r a t i o n of acetylcholine i n f e r i o r to 10 mM.
Although c o n f i g u r a t i o n a l m o d i f i c a t i o n of AChE as a c o n s e q u e n c e of DFP i n t e r a c t i o n could be one of the reasons for lesser r e b i n d i n g of the enzyme to the synaptosomes, this h y p o t h e s i s does not seem to allow an easy i n t e r p r e t a t i o n of the pH results.
Conclusions.
The above results show that in a p u r i f i e d synaptosome p r e p a r a t i o n , acetylcholinesterase is released from the m e m b r a n e s . This is reversed, as clearly d e m o n s t r a t e d b y r e b i n d i n g of 125 I labelled ACHE, at a threshold c o n c e n t r a t i o n of 3 mM acetylcholine. The action of acetylcholine is in t u r n a n t a g o n i z e d b y glutamate, aspartate a n d ,t-aminobutyrate. Release a n d b i n d i n g of the enzyme seems to be associated w i t h the p r e s e n c e or absence of acetylcholine in the e x t e r n a l m e d i u m . As both a c e t y l c h o l i n e and ~,,-aminobutyrate conc e n t r a t i o n s are at levels possibly e n c o u n t e r e d in the n e r v o u s system [17~0], the observed p h e n o m e n o n could be of some consequence to the functions of acetylcholinesterase. W h e t h e r it could have a m o d u l a t o r y i n f l u e n c e on the s y n a p t o s o m a l acetylcholinesterase activity by e m b e d d i n g the enzyme at threshold levels of acetylcholine has to be examined. The suggestion that the pH changes consecutive to a c e t y l c h o l i n e h y d r o l y s e s could be i n v o l v e d in depolarization of M cells was put f o r w a r d by Podleski a n d C h a n g e u x [21]. In view of our i g n o r a n c e of pH regulation in the s y n a p t i c cleft this is a possibility. Our results p o i n t t o w a r d s both pH a n d A,Gh as h a v i n g an effect on the reassociation of AChF to the s y n a p tic m e m b r a n e . The results obtained w i t h D.FP lead to the c o n c l u s i o n that active enzyme r e b i n d s about 6 times m o r e efficiently, at e q u i v a l e n t final pH, t h a n i n a c t i v e enzyme, suggesting that if protons w e r e i n v o l v e d i n this r e b i n d i n g , it w o u l d r a t h e r be n a s c e n t protons. U n d e r physiological conditions, h y d r o l y s i s of ACh in the s y n a p t i c
BIOCHIMIE, 1980, 62, n ° 10.
of local e x t e r n a l fact that enzyme w h e n ACh was proton increase
In this context, v - a m i n o b u t y r i e acid could of course play a buffering role in our e x p e r i m e n t s . But the fact is that it is p r e s e n t more specifically in the s y n a p t i e cleft a n d as such could thus interfere with the m e c h a n i s m of r e b i n d i n g of ACHE. The m e m b r a n e p r o p e r t i e s are also m o d i f i e d b y acetylcholine as i n d i c a t e d by the accessibility of proteins and aeetyleholinesterase to detergents. This suggest a r e o r g a n i z a t i o n of this m e m b r a n e w i t h higher i n t e r a c t i o n b e t w e e n some of its components. These observations open n e w perspectives for the regulatory f u n c t i o n s of the described release and rebinding phenomenon. As a m a t t e r of fact, one could speculate that i n the course of the s y n a p t i c discharge, w h e n ACh reaches the threshold level of 2 raM, in the synaptic cleft, the (( free >> AChE p r e s e n t in this structure r e b i n d s either to pre- or p o s t - s y n a p t i c memb r a n e s or both ( w h i c h is not yet k n o w n ) , thus c l e a r i n g the cleft and m a M n g it more p r o b a b l e for the released ACh to reach the n o w accessible receptor site. Compounds, like GABA, w o u l d antagonize this, w e t h e r directly or t h r o u g h t h e i r b u f f e r i n g effect, m a k i n g it more difficult for the A.Ch signal to reach its receptor site and thus i n h i b i t i n g the t r a n s m i s s i o n of the influx. F u r t h e r studies w i t h more i n t e g r a t e d in s i t u systems are presently i n progress. Acknowledgements. We wish to acknowledge the s k i l f u l l technical assistance of Mrs E. Bother, N. Cr~mel and R. L. Rendon. Part of this work has been supported by the (>, grant n ° 77.$.100.3 and by the <
REFERENCES. 1. Kremzner, L. T. & Wilson, I. B. (1964) Biochemistry U.S.A., 3, 194)2~-190,5. 2. Massouli~, J. a Rieger, F. (1969) Eur. J. Biochem., 11, 441-455. 46
680
C. B u r g u n
3. Massouli~, J., Rieger, F. ~ Tsuji, S. (1970) Eur. J. Biochem., 14, 430-439. 4. Vigny, M., Di Giamberard4no, L., Courand, J. Y., Rieger, F. ~ Koenig, J. (1976) FEBS Lett., 69, 277280. 5. Jackson, R. L. ,~ Aprison, M. H. (1966) J. Neurochem., 13, 1351-1365. 6. Davis, G. A. ~ Agranoff, B. W. (1968) Nature, 220, 277-280. 7. Hall, Z. W. (1973) J. Neurobiol., 4, 343-361. 8. W a k s m a n , A. ~ Rendon, A. (1974) Biochimie, 56, 907-924. 9. CrSmel, G., Rebel, G., Warter, J.-M., Rendon, A. Waks~nan, A. (1976) Arch. Biochem. Biophys., 173, 255-263. 10. Morgan, I. G., Wolfe, L. S., Mandel, P. ~ Gombos, G. (1971) Biochim. Biophtls. Aeta, 241, 737-751. 11. Massouli6, J. ~ Bon, S. (1976) Eur. J. Biochem., 68, 531-539. 12. Boltoth A. E. ~ Hunter, W. M. (1973) Bioehem. J., 133, 529-539.
BIOCHIMIE, 1980, 62, n ° 10.
a n d coll. 13. Bellhorn, M. B., Blumenfeld, O. O. ~ Gallop, P. M. (1970) Biochem. Bioph!Is. Res. Commun., 39, 267273. 14. Ellman, G. L., Courtney, K. D., Andres, V. J. Featherstone, R. M. (1961) Biochem. Pharmaeol., 7, 88-95. 15. Siakotos, A. N., Filbert, M. ,~ Hestvr, R. (1969) Biochem. Med., 3, 1-12. 16. Lowry, O. G., Rosebrough, N. J., Farr, A. L. Randall, R. J. (1951) J. Biol. Chem., 193, 265-275. 17. F o n n u m , F. • Walberg, F. (197~3) Brain Res., 54, 11512~7. 18. F o n n u m , F. • Walberg, F. (19.73) Brain Res., 62, 577579. 19. Dowdall, M. J., Boyne, A. F. ~ Whittaker, V. P. (1974) Biochem. J., 140, 1-12. 20. Kuffler, S. W. • Yoshikami, D. (1975) J. Physiol., 251, 465-482. 21. Podleski, T. ~ Changeux, J.-P. (1967) Science, 157, 1579-1581.
Influence of acetylcholine on rebinding of soluble acetylcholinesterase to a synaptosomal fraction from rat brain. Claude BURGUN, J'ean-Marie WARTER, Pierre VEIL, G6rard ~GREM~EL (*) and Albert VCAKSJMAN(**) <>. (Re~u le 1-2-1980. Accept~ apr~s r e m a n i e m e n t le 12-5-1980).
Centre de N e u r o c h i m i e da Centre National de la R e c h e r c h e Scientifique, Institut de Chimie Biologique de la Facultd de Mddecine et Clinique Neurologique da C.H.U., 11 rue H u m a n n , 67085 Strasbourg Cedex (France).
Rdsum~.
Summary.
II est montr6 q u e r a c 6 t y l c h o l i n e s t 6 r a s e relargu6e p a r u n e fraction s y n a p t o s o m a l e de cerv e a u de rat se r a t t a c h e & cette fraction apr~s son i n c u b a t i o n en p r e s e n c e de 3 m M d'ac~ty1choline. Ce p h ~ n o m ~ n e est r~versible. Le glutamate, r a s p a r t a t e et le T-aminobutyrate antagonisent l'effet de l'ac6tylcholine. Le Lubrol WX et Ie Triton X 100 solubilisent plus difficilement les prot6ines totales et l'ac6tylcholinest6rase
A c e t y l c h o l i n e s t e r a s e (Acetylcholine acety1h y d r o l a s e E.C. 3.1.1.7.) r e l e a s e d from a rat b r a i n s y n a p t o s o m a l fraction w a s s h o w n to rebind u p o n incubation in the p r e s e n c e of 3 m M acetylcholine. This action w a s s h o w n to be reversible. Glutamate, a s p a r t a t e a n d y-aminob u t y r a t e a n t a g o n i s e this eifech Solubilization of both bulk protein a n d a c e t y l c h o l i n e s t e r a s e b y Lubrol W'X a n d Triton X 100 after acetylcholine i n c u b a t i o n of s y n a p t o s o m a l fractions is m u c h lower t h a n in the n o n i n c u b a t e d preparation. Local production of protons due to the hydrolysis of acetylcholine b y the e n z y m e could partially explain the reassociation of the e n z y m e . W e s u g g e s t that the o b s e r v e d p h e n o m e n o n m a y p l a y some p h y s i o l o g i c a l role in the function of acetylcholinesterase.
des fractions aprbs incubation en pr6sence d'ac6tylcholine. La production locale de protons lors de l ' h y d r o l y s e de l'ac~tylcholine p a r l ' e n z y m e pourrait expliquer partiellement la r6association de l'ensyme. Le p h 6 n o m ~ n e observ6 pourrait jouer un r61e p h y s i o l o g i q u e clans la fonction de l'acStylcholinest6rase. Mots-cl6s : ACh ; ac6tyleholinest6rase ; liberation ; fixation ; synaptosomes.
Key-words: ACh ; Acetylcholinesterase ; release ; binding ; synaptosomes.
Introduction.
to exist in a broad spectrum of living systems [1, 4, 5, 6, 7] and were found to be either predominantly soluble or membrane bound.
The existence of different molecular forms of acetylcholinesterase is now supported by strong evidence [1, 2, 3]. These forms have been shown
Abbreviations : Acetylcholinesterase Acetylcholine Acetylthiocholine Tetraisopropylpyrophosphoramide Diisopropylfluorophosphate
: : : : :
ACHE. ACh. ATCh. ISOMPA. DFP.
(*) Charg~ de Recherche h I'INSERM. (") Maitre de Recherche au CNRS. <> To whom all correspondence should be addressed.
Aggregation between some of the forms has been suggested to be a possible mechanism relating soluble to bound enzyme. Although much emphasis has been placed on the molecular structure and the metabolic aspects of acetylcholinesterase, little is known about the membrane dynamics in which this enzyme system is involved, and the possible role of this dynamic in neurohumoral transmission. In a previous study where rat liver mitochondrial membranes were used as a model system, strong evidence was produced that some
672
C. Bur.qun and coll.
e n z y m e s a n d p r o t e i n s w e r e a b l e to b e r e l e a s e d and rebound to the inner mitochondrial membrane in response to changes in the external environment. This phenomenon, which might have iml~ortant functional and structural implications was shown to be reversible, specific, and dependent on the lipid composition of the membrane [8, 9 ] . I n o r d e r t o r e a c h a b e t t e r u n d e r s t a n d i n g of such molecular processes involved at the syna, p t i c l e v e l , i t s e e m e d o f i n t e r e s t to u s to e x a mine whether protein movements comparable to those detected in the mitochondria could be observed in synaptosomal preparations. The enzyme chosen as model was acetylcholinesterase (Acetylcholine Acetylhydrolase 3.1.1.17.). In the present article, release and rebinding of acetylcholinesterase to synaptosomal preparations is reported under defined experimental conditions.
Materials and Methods. All p r o d u c t s w e r e o f t h e p u r e s t q u a l i t y a v a i l a b l e . Sucrose (Analar) was obtained from BDH chemicals L d t (Poole Engl,and). Fieoll 400 w a s f r o m P h a r m a c i a F i n e C h e m i c a l s ( S w e d e n ) . S o d i u m a s p a r t a t e a n d glut a m a t e w e r e p u r c h a s e d f r o m E. M e r c k AG ( D a r m s t a d t ) . 7 - a m i n o b u t y r i c acid, a c e t y l e h o l i n e c h l o r i d e , a e e t y l t h i o c h o l i n e iodide, c h o l i n e c h l o r i d e , a s w e l l a s b o v i n e s e r u m a l b u m i n e a n d T r i t o n X 100 w e r e f r o m S i g m a C h e m i c a l CO, S t - L o u i s MO. M i n e r a l s a l t s , b a s e s a n d acids were obtained from Prolabo, Rh6ne-Poulenc. Lubrol WX was purchased from Imperial Chemical Industries. Labelled products were from Amersham France. All s o l u t i o n s w e r e p r e p a r e d in 0.32 M s u c r o s e a d j u s t e d to p H 6.4 w i t h HC1 o r N a O H , d e p e n d i n g o n t h e effector u s e d . All c e n t r i f u g a t i o n s w e r e p e r f o r m e d in a S p i n e o m o d e l L 50 c e n t r i f u g e , a n d all m a n i p u l a t i o n s w e r e p e r f o r m e d a t 4°C u n l e s s o t h e r w i s e m e n t i o n e d .
or 0.32 M s u c r o s e c o n t a i n i n g t h e s t u d i e d effectors. All f u r t h e r a d d i t i o n s a r e i n d i c a t e d in t h e legend,s o f figures. T h e t o t a l v o l u m e w a s a d j u s t e d to 2 m l a t a p H of 6.4. A f t e r i n c u b a t i o n , t h e p r e p a r a t i o n s w e r e c e n t r i f u ged a t 106,000 g f o r 60 w i n i n a R 40 r o t o r . S u p e r n a t e n t s w e r e r e c o v e r e d . P e l l e t s w e r e s u s p e n d e d in 4.5 m l w a t e r a n d h o m o g e n i z e d f o r 30 s in a g l a s s - t e f l o n h o m o g e n i z e r ( T h o m a s ) a t 880 r p m . F o r d e t e r g e n t t r e a t m e n t , 500 p.1 of a s o l u t i o n o f d e t e r g e n t ( L u b r o l W X o r T r i t o n X 1,00,) at d i f f e r e n t c o n c e n trations, were a d d e d to e i t h e r c r u d e or p u r i f i e d s y n a p tosomal fractions. The mixtures were incubated for 15 m i n a t 37°C in r o t a r y s h a k i n g b a t h , and' c e n t r i f u g e d f o r 60 m i n a t 105,000 g in a R 40 t y p e r o t o r . S u p e r n a tants were recovered. The pellets were suspended by h o m o g e n i z a t i o n a s a b o v e a n d b r o u g h t to a final v o l u m e of 5 m l w i t h w a t e r . E n z y m a t i c a n d p r o t e i n m e a s u r e m e n t s w e r e p e r f o r m e d on t h e d i f f e r e n t f r a c t i o n s . I n t h e a b o v e a s s a y s , t h e i n c u b a t i o n in s u c r o s e corr e s p o n d s to a r e l e a s e e x p e r i m e n t , w h e r e a s t h e i n c u b a t i o n i n t h e p r e s e n c e of a c e t y l c h o l i n e w h i c h a l w a y s f o l l o w s a s u c r o s e i n c u b a t i o n c o r r e s p o n d s to a r e b i n ding experiment. I n t h e c a s e of r e v e r s i b i l i t y e x p e r i m e n t s , t h r e e c r u d e s y n a p t o s o m a l f r a c t i o n s w e r e p r e p a r e d . T h e first f r a c t i o n w a s i n c u b a t e d in 0.32 M s u c r o s e , 1.5 m l A C h (acet y t e h o l i n e ) a n d 3.0 m M A C h w e r e a d d e d r e s p e c t i v e l y to t h e 2 n d a n d 3rd f r a c t i o n . T h e s e f r a c t i o n s w e r e t h e n c e n t r i f u g e d f o r 1 h a t 105,0~)0 g in a R 40 r o t o r . T h e petlets were discarded and the supernatants -- namely : s u c r o s e , A C h 1.5 m M a n d A C h 3.0 m M w e r e d i a l y z e d at 4 ° C a g a i n s t 4 × 2 l i t e r s 0.3.2 M s u c r o s e f o r 24 h. I d e n t i c a l f r e s h l y p r e p a r e d p e l l e t s (2D m g p r o t e i n / m l ) n a m e l y s u c r o s e , A C h 1.5 m M a n d A C h 3.0 m M w e r e i n c u b a t e d e i t h e r in 0.32 M s u c r o s e f o r c o n t r o l , or in presence of their diaIyzed corresponding supernatant, o r in t h e p r e s e n c e of t h e a b o v e m e n t i o n e d , s u p e r n a t a n t s in w h i c h t h e i n i t i a l A C h c o n c e n t r a t i o n w a s r e s t o r e d . P r o t e i n c o n c e n t r a t i o n in i n c u b a t i o n m i x t u r e w a s b r o u g h t to 20 m g p e r m l . In~cubations w e r e p e r f o r m e d a t 37°C f o r 5 m i n . T h e n , t h e t h r e e p r e p a r a t i o n s w e r e c e n t r i f u g e d a t 10'5,000 g in a R 40 r o t o r f o r 1 h a t 4°C. T h e s u p e r n a t a n t s w e r e collected a n d t h e p e l l e t s were r e s u s p e n d e d a n d h o m o g e n i z e d in 4.5 m l ice-cooled water ; protein and AChE (acetylcholine esterase) determinations were performed on each fraction.
Preparation of crude and purified synaptosomaI fractions.
Labelling of AChE with synaptosomal fraction.
T h e m e t h o d f o r t h e p r e p a r a t i o n of b o t h c r u d e a n d purified synaptosomal fractions was essentially derived from the technique described by Morgan and coll. [10].
A C h E f r o m r a t b r a i n w a s p u r i f i e d a c c o r d i n g to t h e m e t h o d of M a s s o u l i 6 a n d B o n [11]. T h e p u r i f i e d e n z y m e w a s l a b e l l e d f o l l o w i n g t h e t e c h n i q u e of B o l t o n a n d H u n t e r [12]. 1 ~g of e n z y m e d i s s o l v e d i n 100 ~1 0.1 M p h o s p h a t e b u f f e r a t p H 8 w a s t r e a t e d d u r i n g 15 m i n at 0°C, in t h e p r e s e n c e of 1 m C i of B o l t o n a n d H u n t e r reagent.
Adult albino Wistar rats were killed by decapitat i o n ; t h e b r a i n s removed~ a n d placed' i n ice-cold 0.32 M sucrose. The original method was applied except that E D T A - p o t a s s i u m p h o s p h a t e w a s o m i t t e d in t h e h o m o genization medium anti the crude synaptosomal fraction was washed twice before use. For the purified synaptosomal fraction, EDTA- potassium phosphate was also omitted from the preparation.
Incubation of fractions and detergent treatments. S y n a p t o s o m a l f r a c t i o n s c o r r e s p o n d i n g to 20 m g of p r o t e i n s were i n c u b a t e d f o r 5 m i n a t 37°C in a r o t a r y s h a k i n g b a t h in t h e p r e s e n c e of e i t h e r 0.32 M s u c r o s e ,
BIOCH1MIE, 1980, 62, n ° 10.
l~st and incubation
with
T h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of 0.5 m l 0.2 M g l y e i n e in 0.1 M p h o s p h a t e b u f f e r a t p H 8. Labelled enzyme was separated from excess reagent on a G 25 S e p h a d e x c o l u m n e q u i l i b r a t e d w i t h 50 m M p h o s p h a t e b u f f e r at p H 7.5. F r a c t i o n s o f 5.00 ~l w e r e collected. 20 ~l of t h e m o s t active f r a c t i o n c o n t a i n i n g 1 ~g l a b e l l e d e n z y m e a n d c o r r e s p o n , d i n g to a b o u t 355.10~ c p m w a s a d d e d to 1 ml of s y n a p t o s o m a l s ~ s p e n s i o n c o n t a i n i n g 2'0 m g of p r o t e i n in 0.32 M s u c r o s e . F i n a l v o l u m e w a s a d j u s t e d to 2 m l w i t h 0.32 M s u c r o s e .
Rebinding of acetylcholinesterase to synaptosomes. I n c u b a t i o n s were p e r f o r m e d in the presence or absence of 2 mM ACh u n d e r conditions described above.
Labelling of acetylcholinesterase with FSH] diisopropylfluorophosphate. The method applied was basically t h a t of Bellhorn and coll. [13]. A s u p e r n a t a n t fraction obtained after incubating a s y u a p t o s o m a l fraction (2,0 mg p r o t e i n / m l ) in the presence of 0.32 M sucrose for 5 rain at 37°C was isolated, concentrated 5-fold on an Amicon PM 30' filter, centrifuged 30 rain a t 1D0:,000 g and a d j u s t e d to final 10 raM, pH 7.6 by a d d i t i o n of concentrated p h o s p h a t e buffer. a) An aliquot (500 /xl) of this fraction was incubated for 30 min at room t e m p e r a t u r e in the presence of 0.2 M b u t y r y l c h o l i n e , in order to protect the active site of AChE ; it was t h e n incubated for 30 rain at room t e m p e r a t u r e in the presence of lt0-6 M u n l a b e l l e d DFP. After being passed, on a G 25 Sephadex c o l u m n , and eluted w i t h 0.1 M p h o s p h a t e buffer at pH 7.6, the protein fraction was reincubated for 30 m i n at room t e m p e r a t u r e in presence of 5 ~Ci [3H] DFP (10-6 M). The final preparation, was dialyze~ f o r 24 h at 4°C against 3 × 2 l, lfi mM p h o s p h a t e buffer at pH 7. b) In parallel a n o t h e r 500 B1 aliquot of the initial p r e p a r a t i o n was i n c u b a t e d directly in the presence of 5 ~Ci [3H] DFP (10-6 M) and dialyzed as above. Control of enzyme activity showed t h a t b u t y r y l c h o l i n e completely protected the enzyme site, and t h a t a f t e r its e l i m i n a t i o n and t h a t of un.labelle(~ DFP, AChE activity was preserved. The addition of [aH] DFP then totally inhibited the enzyme. In the absence of butyrylcholine, cold DFP inhibits 90 per cent of AChE activity. Labelling was of 10,400 c p m / m g p r o t e i n for a) a n d 1'3,000 c p m / m g protein f o r b).
673
p h o t o m e t e r . The concentration of ATCh was 1 mM. During the m e a s u r e m e n t s of the AChE activity, the ¢ exogenously introduced >> ACh in the euvette never exceeded 0.18 mM and was generally a r o u n d 0.075 raM. Under these conditions, it w a s d e m o n s t r a t e d t h a t t h i s small a m o u n t of ACh did not interfere w i t h the ACkE activity m e a s u r e d by the ATCh method, p r o b a b l y because u n d e r our working con.ditions, this ACh was already h y d r o l y z e d at the t i m e AChE was as.sayed. F u r t h e r m o r e , AChE activity was not different w h e n m e a s u r e d on the j u s t mentioned' s u p e r u a t a n t fluid f r o m which the exogenous ACh had been previously dialyzed. The m e t h o d of Lowry and coll. [16] was used for protein d e t e r m i n a t i o n w i t h s e r u m a l b u m i n as s t a n dard.
Results. I n f l u e n c e of acetylcholine on the partition o[ acetylcholinesterase a c t i v i t y associated to di[[erent subcellular fractions in a sucrose gradient. S u b f r a c t i o n a t i o n of a c r u d e s y n a p t o s o m a l fraction on a s u c r o s e g r a d i e n t r e v e a l e d that AChE activity partitionned differently between myelin, synapt0somal and mitochondrial fractions when c e n t r i f u g a t i o n w a s p e r f o r m e d on a 0.32 M s u c r o s e i n c u b a t e d c r u d e f r a c t i o n or in a f r a c t i o n i n c u b a -
TABLE I.
I n f l u e n c e of acetyteholine on the partition o[ acetytcholinesterase a c t i v i t y associated to d i f f e r e n t subcellular [factions, in a sucrose gradient. sucrose
My. Sy. Mit.
3 mM ACh
activity
(per cent)
activity
{per cent)
4.16 1.93 0.28
65.3 30.3 4.4
1.13 3.52 1.14
29.3 53.4 17.3
sucrose
3 mM ACh
specific activity 0.108 0.060 0. 010
0.073 0.096 0. 037
The three fractions were obtained by r e c e n t r i f u g a t i o n of a crude s y n a p t o s o m a l f r a c t i o n on a 0.8 M/1.0 M/1.2 M sucrose gradient. The so-called Myelin fraetinn contains soluble AChE and myelin. My. : Myelin ; Sy. : Synaptosomes ; Mit. : M,itochondria. Activity is expressed' as, t~moles ATCh h y d r o l y z e d per rain per fraction. Specific activity is expressed as ~moles ATCh h y d r o l y z e d per m i n / m g protein.
Enzymatic assays and protein determinations. Aeetylcholinesterase aetlvity w a s determined~ according to the method, of E l l m a n and coll. [14], the m e a s u r e m e n t s were p e r f o r m e d at 30°C in the presence of p h o s p h a t e buffer 0.1 M, pH 7.6, and 1 mM of 5,5rdithiobis-(2-nitrobenzoie acid) and 10-5 M ISOMPA to i n h i b i t non-specific esterases [15]. Readings were perf o r m e d at 412 n.m in a Beckman Aeta III type speetro-
BIOCH1MIE, 1980, 62, n ° 10.
t e d in t h e p r e s e n c e o f 3 m M A C h ( t a b l e I). It c a n b e s e e n t h a t i n t h e p r e s e n c e of A,Ch, 55 p e r c e n t of t h e A C h E p r e v i o u s l y p r e s e n t i n t h e m y e l i n f r a c t i o n (My), w h i c h also c o r r e s p o n d e d to t h e p o o l of s o l u b i l i z e d e n z y m e , s h i f t e d t o w a r d s b o t h s y n a p t o s o m a l - r i c h f r a c t i o n s : i.e. p u r e s y n a p t o s o m e s (Sy) a n d m i t o c h o n d r i a (Mi).
C. B u r g u n and coll.
674
Influence of acetylcholine and acetglthiocholine on release and binding of acetylcholinesterase by a crude synaptosomal fraction. Table II s u m m a r i z e s the i n f l u e n c e of sucrose or sucrose s u p p l e m e n t e d w i t h N,aC1 or KC1, acetate, c h o l i n e and a c e t y l c h o l i n e on the solubilization of a c e t y l c h o l i n e s t e r a s e after i n c u b a t i o n of a c r u d e s y n a p t o s o m a l f r a c t i o n , as d e s c r i b e d in Methods. As can be seen, u n d e r these c o n d i t i o n s , only acet y l e h o l i n e has a m a r k e d i n h i b i t o r y effect on the release of a e e t y l c h o l i n e s t e r a s e . F i g u r e 1 s h o w s r e b i n d i n g of r e l e a s e d a c e t y l c h o l i n e s t e r a s e as a f u n c t i o n of a c e t y l c h o l i n e or a c e t y l t h i o c h o l i n e c o n c e n t r a t i o n . R e b i n d i n g of a c e t y l c h o l i n e s t e r a s e levels off at 3 mM a c e t y l c h o l i n e .
ting 12~I labelled AChE w i t h a s y n a p t o s o m a l fraction in the p r e s e n c e of a c e t y l c h o l i n e . Results are s u m m a r i z e d in table IV. In the p r e s e n c e of 0.32 M s u c r o s e alone, 78 p e r cent of the labelled e n z y m e
t0AS o.,o
TABLE II.
Influence of acetylcholine and other substances on release and binding of acetylcholinesterase on a crude sllnaptosomal fraction.
Sucrose (0.32 M) Acetate Choline Acetate -~- choline Acetylcholine NaCI KC1
Specific activity of soluble acetylcholinesterase ,~moles per mg protein • per mill
Per cent of soluble acetylcholinesterase
0.22 0.23 0.26 0.31 0.08 0.30 0.32
10.5 :5.2 4.5 :5.2 0.8 r'6.6 7.0
AJll substances were used at a concentration of 20 mM, except aeetylcholine which was 3 mM.
Rebinding o[ acetylcholinesterase to a purified synaptosomal fraction in absence and presence of exogenous acetylcholine. I n c u b a t i o n of a p u r i f i e d s y n a p t o s o m a l f r a c t i o n in the p r e s e n c e or absence of 3 mM a c e t y l c h o l i n e was p e r f o r m e d u n d e r the above d e s c r i b e d conditions. A t y p i c a l set of results is s u m m a r i z e d in table III. T h e y c l e a r l y s h o w that f o r such a prep a r a t i o n , a c e t y l c h o l i n e s t e r a s e is r e l e a s e d f r o m the m e m b r a n e f r a c t i o n in 0.32 M sucrose and b o u n d to that f r a c t i o n w h e n 3 mM a c e t y l c h o l i n e is present.
Rebinding of le5I labelled acetylcholinesterase to a crude synaptosomal fraction in presence of acelylcholine. R e b i n d i n g of A C h E to s y n a p t o s o m a l m e m b r a n e s can be d e m o n s t r a t e d u n a m b i g u o u s l y by i n c u b a -
BIOCHIMIE, 1980, 62, n ° 10.
w i
mM
Ach •
or
ATCh o
FIG. 1. - - B i n d i n g influence of acetglcholine (ACh) or acetltithiocholine (ATCh) on the acet!tlcholinesterase solubilized from a crude sllnaptosomal fraction. The activity of the enzyme is expressed in ixmol of aeetylth~iocholine hydTolyzed per min per 20 mg synaptosomal protein.
w a s f o u n d in the s u p e r n a t a n t f r a c t i o n and 22 p e r cent w a s associated to the s y n a p t o s o m a l pellet, w h e r e a s in t h e p r e s e n c e of 2 mM ACh the situation w a s r e v e r s e d , w i t h 76 p e r cent r a d i o a c t i v e e n z y m e b o u n d to the s y n a p t o s o m a l fraction. T h e ratio of r a d i o a c t i v e AChE to e n z y m e activ i t y r e m a i n i n g in s u p e r n a t a n t after r e b i n d i n g is i d e n t i c a l f o r s u c r o s e treated or ACh t r e a t e d synaptosomes, thus c o n f i r m i n g that both labelled protein a n d a c t i v i t y r e b i n d to the same e x t e n d to the s y n a p t o s o m a l m e m b r a n e . In v i t r o reversibility of the binding phenomenon. I n c u b a t i o n of a c r u d e s y n a p t o s o m a l f r a c t i o n in the p r e s e n c e of e i t h e r 1.5 mM a c e t y l c h o l i n e or 3 mM a e e t y l c h o l i n e results in b i n d i n g of the soluble a c e t y l c h o l i n e s t e r a s e to the m e m b r a n e f r a c t i o n as seen above. R e i n c u b a t i o n of such a f r a c t i o n in the p r e s e n c e of e i t h e r 0.32 M sucrose or the s o l u b i l i z e d fractions o b t a i n e d a f t e r sucrose or sucrose + acetyl-
Rebinding of acetylcholinesterase to synaplosomes. c h o l i n e t r e a t m e n t and d i a l y z e d against 0.32 M sucrose, results in the f u r t h e r release of acetylcholinesterase. This suggests that the r 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 can be released once r e b o u n d to the m e m b r a n e fraction, f o l l o w i n g i n c u b a t i o n in the p r e s e n c e of 1.5 mM or 3.0 mM a c e t y l c h o l i n e (table VA).
675
Moreover, if i n c u b a t i o n o c c u r s in the p r e s e n c e of s y n a p t o s o m e s aged for 36 h at 4°C no b i n d i n g of A~ChE o c c u r s even at ACh c o n c e n t r a t i o n s up to 5 mM (see figure 3). T h i s is c o n t r a r y to the results obtained with a freshly prepared synaptosomal f r a c t i o n w h i c h strongly r e b i n d s ACHE, as s h o w n above.
TABLE HI.
Influence of acetylcholine on release and binding of bulk protein and acetylcholinesterase on a purified synaptosomal fraction. Specific activity of acetyleholinesterase ~moles per mg protein per min Fraction
Synaptosomes Fluid
Lubrol supernatant
Residual pellet
Total acetylcholinesterase activity
Sucrose -~- acetylcholine
2.5 10 -s (0.15 per cent)
7.2 10 -3 (3.2 per cent)
28.9 10 -3 (96.6 per cent)
2.60
Sucrose
23.9 I0 -a ( 2 . 4 per cent)
8.1 10 -3 (6.7 per cent)
30.8 10 -3 (90.9 per cent)
2.58
Per cent of total protein Fraction
Synaptosomes
Fluid
Lubrol supernatant
Residual pellet
Total recovery
Sucrose -~-acetylcholine
1.6 per cent
11.6 per cent
86.8 per cent
100 per cent
Sucrose
2 . 6 per cent
21.4 per cent
76 per cent
92.2 per cent
After incubation either in sucrose alone or in sucro.se plus acetyleholine, the synaptosomal fraction was separated from the fluid fraet.ion and treated with 0.16 mg Lubrol per mg protein ; a pel]et and a supernatant were then separated (105,0100 g, 1 h). Percentage of AChE in each fraction is given.
F u r t h e r m o r e , w h e n i n c u b a t e d in the p r e s e n c e of the d i a l y z e d s u p e r n a t a n t to w h i c h t h e i r original c o n c e n t r a t i o n of a c e t y l c h o l i n e w a s restored, c r u d e s y n a p t o s o m a l f r a c t i o n s s h o w e d no release of a c e t y l c h o l i n e s t e r a s e (table VB). H o w e v e r , w h e n i n c u b a t e d in the p r e s e n c e of 0.32 M sucrose alone, these f r a c t i o n s w h e t h e r p r e i n c u b a t e d in sucrose or in the p r e s e n c e of 1.5. mM or 3.0 mM a c e t y l c h o l i n e (table V'C) s h o w e d release of a c e l y l c h o l i n e s t e r a s e activity.
Influence of aging of the sgnaptosomaI fraction on the rebinding of ACHE. The above results could be due to the p r e c i p i t a tion of ~ C h E in the course of A,Ch h y d r o l y s i s . Tllis p o s s i b i l i t y was r u l e d out by the fact that in the absence of s y u a p t o s o m a l m a t e r i a l t h e r e is no delectable, p e l l e l a b l e activity.
BIOCHIM1E, 1980, 62, n ° 10.
Influence of pH on rebinding of AChE to synaptosomal fraction. Little is k n o w n about the b u f f e r i n g c a p a c i t y of the s y n a p t i c cleft m i c r o e n v i r o n m e n l . Our first e x p e r i m e n t s w e r e t h e r e f o r e p e r f o r m e d in sucrose at pH 6.4. T h e y thus i n d i c a t e a p o t e n t i a l beh a v i o u r of this system. I n f l u e n c e of pH w a s c o m p a r e d in two d i f f e r e n t situations : a) the final pH w a s i m p o s e d by 10 mM v a r i o u s buffers at different pH. b) the final pH w a s the result of h y d r o l y s i s of different initial c o n c e n t r a t i o n s of ACh. It can be seen that at equal final pH, m u c h m o r e AChE a c t i v i t y r e b i n d s in the p r e s e n c e of ACh than in its absence (figure 2).
676
C. B u r g u n a n d coll.
Thus, at a final p H of 6.0, 85 p e r cent of the AChE a c t i v i t y ffisappears f r o m the i n c u b a t i o n med i u m in the p r e s e n c e of ACh a n d only 15 p e r cent in its absence, suggesting a d i f f e r e n t i a l effect of both p H (or buffer type) and ACh on the rebinding of .~ChE to a s y n a p t o s o m a l fraction. Rebinding of half of the soluble AChE o c c u r s at p H 6.7 in the p r e s e n c e of ACh and at pH 5.35 in its absence.
Results s h o w that only 9 p e r c e n t of labelled e n z y m e is to be f o u n d in s y n a p t o s o m a l p e l l e t (table VI), in 0.32 M sucrose alone against 47 p e r
% released
AChE
100-
% released
AChE
100. 50-
50. 0 ACh (raM)
01
Final ]~ pH in supernatant
Fro. 2. - - Influence of final pH on AChE rebinding to synaptosomes in presence of ACh (•) and in buffered solutions at various pH (D). The activity is expressed as per cent of activity in supernatan1 at p,H 7.
F u r t h e r m o r e , in buffered m e d i a at pH 7, rebinding o c c u r s as s h o w n in figure 3. At 2 mM ACh, the d i f f e r e n t buffers do not lead to the same extent of r e b i n d i n g . R e b i n d i n g is 45 p e r cent of total a c t i v i t y in 1,0 mM Tris buffer, 30 p e r cent in 10 mM p h o s p h a t e buffer and 20 p e r cent in 1~) mM Hepes buffer. At 5 mM ACh h o w e v e r , r e b i n d i n g is of 55 p e r cent regardless of the nature of the buffer used. These results suggest a d i s t i n c t i n f l u e n c e of ACh, ions and p H on r e b i n d i n g of ACHE. I n f l u e n c e of e n z y m e inhibition on e n z y m e rebinding. T h e above results show that both ACh a n d pH influence AChE r e b i n d i n g to s y n a p t o s o m a l fraction. To distinguish these t w o effects, AChE was i n h i b i t e d by b l o c k i n g of its active site w i t h aH labelled D FP. R e a s s o c i a t i o n of labelled, i n h i b i t e d e n z y m e was then m e a s u r e d in the p r e s e n c e and absence of ACh. BIOCHIMIE, 1980, 62, n ° 10.
FIn. 3. - - Influence of various 10 mM buffers in the presence of increasing ACh concentrations on rebinding of AChE to synaplosomes. The activity is expressed as per cent of activity in sucrose supernatant. Control shows no rebinding to aged synaptosomes. • • aged synaptosomes. © © sucrose • • >> + PO, 10 mM -k "k >> + tris PH 7 • • >) + Hepes
/IGLUTAMATE
y_
/
•
/
~. o.1 -~ .-c_
I.u .¢
2 =
/
-
/:" // /..
OG
.
.'WASPAR i A T E
.../' '
,/ ~20
150
TIO0
1125 mM
Amino
acid
Fro. 4. - - Antagonistic influence of glutamate, asparrate and 7-aminobutyratc (GABA) to the binding of acetylcholineslerase provoked by 5 mM acetylcholine. Activity of soluble acetyleho]inesterase is expressed in p.mol'of acetylthiocholine hydrolized per min per 20 mg synaplosomal protein.
Rebinding
of acelylcholinesterose
c e n t in the p r e s e n c e of 3 mM ACh, t h e last figure is to be c o m p a r e d to 76 p e r c e n t a c t i v i t y in the case of l a b e l l e d n o n - i n h i b i t e d e n z y m e .
I n f l u e n c e of acetylcholine on the solubilisation of acetylcholinesterase by Lubrol W X and Triton X 100.
R e b i n d i n g of 1~5I labelled acetylcholinesterase to s y n a p t o s o m a l fraction in the p r e s e n c e of acetylcholine.
100-
Radioactivity cpm Ct0-:*) Supernatant
Pellet
Total
279.5
76.5
356
.° Sucrose
ACh 2 mM
90
277
B
367
Supernatant AChE activity
"/protein
50.
oe i i
i l
b. "/activity r
sucrose
ACh 2 mM
132
498
2.1
38
383
2.4
Binding of 1251 labelled AChE : a) radioactivity associated to supernatant and pellet ; b) enzymatie aetivity and ratio of radioaetivity/prorein o r / e n z y m a t i c activity. Radioactivity is expressed in 1,0-3 epm. Enzymatic activity is expres:sed in nmoles of ATCh hydrolyzed p e r ran. Protein is expressed in rag.
62,
n °
10.
2150 mg
!ubrol / mg protein B
-t
50
0
O
J
m9
t r i t o n / rng protein
FiG. 5.
Antagonist influence of glutamate, asportate and 7-aminobutyrate to the action of aeetylcholine.
1980,
I
P,
A.-
If t h e o b s e r v e d r e l e a s e a n d b i n d i n g of a e e t y l c h o l i n e s t e r a s e w e r e to p l a y s o m e p h y s i o l o g i c a l r o l e in t h e m o d u l a t i o n of n e r v o u s e v e n t s , t h e n o n e w o u l d e x p e c t a g e n t s s u c h as 7 - a m i n o b u t y r a t e , g l u t a m a t e o r a s p a r t a t e to h a v e s o m e p o s s i b l e inf l u e n c e o n t h i s r e l e a s e a n d b i n d i n g . I n t h e following experiments crude synaptosomal fractions w e r e i n c u b a t e d in t h e p r e s e n c e of 5 m M a e e t y l c h o l i n e a n d i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r g l u t a m a t e , a s p a r t a t e o r , t - a m i n o b u t y r a t e . All t h r e e c o m p o u n d s w e r e i n h i b i t o r y t o w a r d s t h e a c t i o n of a c e t y l e h o l i n e on t h e r e l e a s e of a e e t y l e h o l i n e s t e r a s e (figure 4).
1.125
,°°{
Tire f a c t t h a t t h e r a t i o of r a d i o a c t i v i t y u p o n e n z y m e a c t i v i t y i n c r e a s e s 5.6-fotd in the s u p e r n a t a n t f r a c t i o n in t h e p r e s e n c e of A~ih s t r o n g l y s u g g e s t s t h a t i n h i b i t e d e n z y m e r e a s s o e i a t e s less w e l l to m e m b r a n e f r a c t i o n t h a n a c t i v e e n z y m e .
BIOCHIMIE,
677
T h e q u e s t i o n as to w h e t h e r in the p r e s e n c e of a c e t y l c h o l i n e t h e r e l e a s e d a c e t y l c h o l i n e s t e r a s e is
TABLE IV.
A
to s y n a p t o s o m e s .
Solubilization by Lubrol WX of bound acetylcholineaterasc and bulk protein in the presence or absence of 5 raM accttllcholine. ---
in 0.32 M sucrose alone ; in 0.82 M sucrose supplemented with 5 mM aeetyleholine.
B. - - Solubilization by Triton X 100 of bound acetylcholinesterase and bulk protein in the presence or absence of 5 mM acetylcholine.
in 0.32 M sucrose alone ; in 0.82 M sucrose supplemented with 5 mM aeetyleholine. Activity is expressed as percentage of activity in pellet after action of detergent as compared to the activity in pellet without detergent (A typical set of ---
CUI'Ves),
s u p e r f i c i a l l y b o u n d to t h e m e m b r a n e o r is m o r e embedded was approached by treating the memb r a n e s w i t h d e t e r g e n t s . R e s u l t s of t h e i n f l u e n c e
C. B u r g u n a n d coll.
678
of L u b r o l W X a n d T r i t o n X 100 a r e s h o w n i n f i g u r e s ~A, 5B. S o . l u b i l i z a t i o n of b o u n d a c e t y l cholinesterase and protein was compared in crude s y n a p t o s o m e s t r e a t e d w i t h e i t h e r 0.32 M s u c r o s e alone or supplemented with 5 mM acetylcholine.
In addition, the hypotonic shock which occurs during these experiments showed a di,fferential p r o t e i n r e l e a s e i n t h e c o n t r o l a s s a y s (in t h e p r e s e n c e o r a b s e n c e of a c e t y l c h o l i n e ) . TABLE VI.
W h e r e a s L u b r o l W X at 0.5 m g / m g of p r o t e i n s o l u b i l i z e d u p to 90 p e r c e n t of t h e b o u n d a c e t y l cholinesterase in a sucrose-treated preparation, o n l y 40 p e r c e n t of t h e e n z y m e w a s e x t r a c t e d when synaptosomes were pretreated with 5 mM a c e t y l c h o l i n e ( f i g u r e 5A). C o m p a r a b l e s o l u b i l i z a -
R e b i n d i n g of [ZH]DFP labelled AChE to synaplosomes. Radioactivily cpm (10-:~1 Supernatant
TABLE V. In supernatants AChE activity (nmoles ATCh hydrolyzed/min~ _ in fraction
1. Sucrose
2. ACh 1.5 mM 3. ACh 3 mM
Before
reineubation of dialyzed supernatants with their corresponding pellets 41 50 13 50,5 9 42
2. ACh 1.5 mM 3. ACh 3 mM
Before
AIter
reincubation o! dialyzed supernatants with their corresponding pellets and their initial ACh concentration 13 5 9 3
Controls AChE activity (amoles ATCh hydrolyzed/min) in fraction
ACh 2 mM
223.4
Total
40
449
195
418
Supernatant
Sucrose
ACh
AChE activity
"/protein
"/activity
84
1231
4.9
8
2031
27.9
a) D i s t r i b u t i o n of [aH] labelled ACRE in soluble and s y n a p t o s o m a l fraction, w h e n s y n a p t o s o m e s were incub a t e d in sucrose a l o n e or sucrose plus 3 mM ACh. b) AChE activity, r a t e of r a d i o a c t i v i t y / p r o t e i n or e n z y m a t i c activity. Activity is expressed in, nmoles ATCh h y d r o l y z e d per min/fraetion.
[00
In sucrose 0.32 M
Sucrose ACh 1 . 5 rnM
51 50,5
ACh 3 mM
43
R~versibility of AChE b i n d i n g to s y n a p t o s o m a l membranes. A. Released AChE w h e n sucrose dialyzed s u p e r n a t a n t s are r e i n e u b a t e d w i t h t h e i r corresponding synaptosomes ; 1, sucrose a l o n e ; 2 a n d 3, with 1.5 mM a n d 3 mM ACh, respectively. B. AChE r e b o u n d wl~et~ dialyzed s u p e r n a t a n t s are reineubated with their corresponding synaptosomes in the presence of 1.5 mM (2) a n d 3 mM (3) ACh. C. Released AChE w h e n s y n a p t o s o m c s are r e i n c u b a t e d in sucrose alone.
t i o n i n b o t h s a m p l e s w a s o n l y o b t a i n e d at c o n c e n t r a t i o n s o.f L u b r o l a b o v e 2 m g p e r m g of p r o t e i n . I n a simil,ar m a n n e r , i n s u c r o s e - t r e a t e d s y n a p t o s o m e s 42 p e r c e n l of t h e t o t a l , p r o t e i n w e r e e x t r a c t e d at 0.5 m g L u b r o l p e r m g of p r o t e i n , w h e r e a s i n a c e t y l c h o l i n e - t r e a t e d p r e p a r a t i o n s o n l y 25 p e r c e n t of t h e t o t a l p r o t e i n w e r e e x t r a c t e d (~igure 5A).
BIOCHIM1E, 1980, 62, n ° 10.
408.7
AIter
in supernatants AChE activity (nmoles ATCh hydrolyzed/mio~ iu traction
Sucrose
Pellet
110
120 mM ACh
FIG. 6. - - Solubilization of acetylcholinesterase by
0.5 m 9 Lubrol WX per log protein in the presence ot increasing concentrations o f acetfflcholine.
Activity is, expressed as p e r cent of activity in pellet a f t e r action of A C h / a c t i v i t y in pellet in the absence of ACh.
T r i t o n X 10,0 ( u p to 4 p e r c e n t v / v ) s o l u b i l i z e d n o m o r e t h a n 17 p e r c e n t a c e t y l c h o l i n e s t e r a s e i n acetylcholine-treated synaptosomes a n d u p to
Rebinding
of acetylcholinesterase
to s y n a p t o s o m e s .
679
90 p e r cent in sucrose-treated particles (figure 5B). S i m i l a r l y d i f f e r e n t i a l proteins solubilization was also observed. In the presence or absence of acetylcholine, 50 per cent a n d 90' per cent of total protein were extracted, respectively (figure 5~B).
cleft is the major n a t u r a l source protons. This would also fit the r e b o u n d at m u c h h i g h e r pH directly hydrolyzed, t h a n w h e n was due to global pH change.
The effects of 0,.5 mg Lubrol W X / m g p r o t e i n on the s o l u b i l i z a t i o n of acetylcholinesteras.e .as a f u n c t i o n of a c e t y l c h o l i n e c o n c e n t r a t i o n is summarized in figure 6. The results o b t a i n e d w i t h inc r e a s i n g a c e t y l c h o l i n e c o n c e n t r a t i o n s show that the solubilization of the enzyme is only possible at a c o n c e n t r a t i o n of acetylcholine i n f e r i o r to 10 mM.
Although c o n f i g u r a t i o n a l m o d i f i c a t i o n of AChE as a c o n s e q u e n c e of DFP i n t e r a c t i o n could be one of the reasons for lesser r e b i n d i n g of the enzyme to the synaptosomes, this h y p o t h e s i s does not seem to allow an easy i n t e r p r e t a t i o n of the pH results.
Conclusions.
The above results show that in a p u r i f i e d synaptosome p r e p a r a t i o n , acetylcholinesterase is released from the m e m b r a n e s . This is reversed, as clearly d e m o n s t r a t e d b y r e b i n d i n g of 125 I labelled ACHE, at a threshold c o n c e n t r a t i o n of 3 mM acetylcholine. The action of acetylcholine is in t u r n a n t a g o n i z e d b y glutamate, aspartate a n d ,t-aminobutyrate. Release a n d b i n d i n g of the enzyme seems to be associated w i t h the p r e s e n c e or absence of acetylcholine in the e x t e r n a l m e d i u m . As both a c e t y l c h o l i n e and ~,,-aminobutyrate conc e n t r a t i o n s are at levels possibly e n c o u n t e r e d in the n e r v o u s system [17~0], the observed p h e n o m e n o n could be of some consequence to the functions of acetylcholinesterase. W h e t h e r it could have a m o d u l a t o r y i n f l u e n c e on the s y n a p t o s o m a l acetylcholinesterase activity by e m b e d d i n g the enzyme at threshold levels of acetylcholine has to be examined. The suggestion that the pH changes consecutive to a c e t y l c h o l i n e h y d r o l y s e s could be i n v o l v e d in depolarization of M cells was put f o r w a r d by Podleski a n d C h a n g e u x [21]. In view of our i g n o r a n c e of pH regulation in the s y n a p t i c cleft this is a possibility. Our results p o i n t t o w a r d s both pH a n d A,Gh as h a v i n g an effect on the reassociation of AChF to the s y n a p tic m e m b r a n e . The results obtained w i t h D.FP lead to the c o n c l u s i o n that active enzyme r e b i n d s about 6 times m o r e efficiently, at e q u i v a l e n t final pH, t h a n i n a c t i v e enzyme, suggesting that if protons w e r e i n v o l v e d i n this r e b i n d i n g , it w o u l d r a t h e r be n a s c e n t protons. U n d e r physiological conditions, h y d r o l y s i s of ACh in the s y n a p t i c
BIOCHIMIE, 1980, 62, n ° 10.
of local e x t e r n a l fact that enzyme w h e n ACh was proton increase
In this context, v - a m i n o b u t y r i e acid could of course play a buffering role in our e x p e r i m e n t s . But the fact is that it is p r e s e n t more specifically in the s y n a p t i e cleft a n d as such could thus interfere with the m e c h a n i s m of r e b i n d i n g of ACHE. The m e m b r a n e p r o p e r t i e s are also m o d i f i e d b y acetylcholine as i n d i c a t e d by the accessibility of proteins and aeetyleholinesterase to detergents. This suggest a r e o r g a n i z a t i o n of this m e m b r a n e w i t h higher i n t e r a c t i o n b e t w e e n some of its components. These observations open n e w perspectives for the regulatory f u n c t i o n s of the described release and rebinding phenomenon. As a m a t t e r of fact, one could speculate that i n the course of the s y n a p t i c discharge, w h e n ACh reaches the threshold level of 2 raM, in the synaptic cleft, the (( free >> AChE p r e s e n t in this structure r e b i n d s either to pre- or p o s t - s y n a p t i c memb r a n e s or both ( w h i c h is not yet k n o w n ) , thus c l e a r i n g the cleft and m a M n g it more p r o b a b l e for the released ACh to reach the n o w accessible receptor site. Compounds, like GABA, w o u l d antagonize this, w e t h e r directly or t h r o u g h t h e i r b u f f e r i n g effect, m a k i n g it more difficult for the A.Ch signal to reach its receptor site and thus i n h i b i t i n g the t r a n s m i s s i o n of the influx. F u r t h e r studies w i t h more i n t e g r a t e d in s i t u systems are presently i n progress. Acknowledgements. We wish to acknowledge the s k i l f u l l technical assistance of Mrs E. Bother, N. Cr~mel and R. L. Rendon. Part of this work has been supported by the (>, grant n ° 77.$.100.3 and by the <
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