Henry G. Mautner and Eva Bartels- Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs...
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Proceedings o f th e National Academy o f Sciences Vol. 6 7 , N o . 1 , p p . 74-78, September 1970 Interactions o f p-Nitrobenzene Diazonium Fluoroborate a n d Analogs with the Active Sites o f Acetylcholine-Receptor a n d -Esterase* Henry G . Mautner a n d E v a Bartels DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY, TUFTS UNIVERSITY SCHOOL OF MEDICINE, BOSTON, MASSACHUSETTS 02111, AND DEPARTMENT OF NEUROLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, YORK, YORK 10032 Communicated b y David Nachmansohn, June 4 , 1970 Abstract. p-Nitrobenzene diazonium fluoroborate (NDF) i s a potent i n - hibitor o f t h e carbamylcholine-induced depolarization o f t h e electroplax a n d o f acetylcholinesterase. I t probably forms covalent bonds with t h e acetylcholine- receptor a n d -esterase a t th e active site o f t h e proteins. I t s inhibitory strength a t least t h e same a s that o f trimethylammonium diazonium fluoroborate (TDF). T e p-acetoxy analog, with i t s weaker electron-withdrawing group, i s about t e n times weaker a s a n inhibitor than t h e trimethylammonium o r p - nitro analogs, both o f which have strong electron-withdrawing groups. After treatment o f t h e electroplax preparation with dithiothreitol, NDF remains a n irreversible receptor-inhibitor, while TD F becomes a potent reversible receptor- activator. T D F i s self-inhibitory: applied before reduction, i t n o longer d e - polarizes. Although t h e first observations o n TD F suggested that t h e compound labels both proteins b y virtue o f t h e steric complementary o f i t s trimethylammonium group t o a negative subsite i n t h e proteins, t h e present study indicates that i t i s t h e positively charged diazonium group that reacts with t h e active sites o f t h e proteins t o form a covalent bond with a n appropriate amino-acid residue. Introduction. A considerable body o f evidence h a s accumulated i n favor o f t h e view that acetylcholine induces a conformational change o f t h e receptor protein i n excitable membranes, thereby triggering o f f a series o f reactions resulting i n increased i o n permeability.1 2 Much effort h a s been expended o n isolating t h e biopolymer o n which acetylcholine acts during activity.3'4 work would b e aided b y highly specific reagents suitable f o r labeling o f t h e mac- romolecules i n question b y formation o f covalent bonds with groups a t o r near t h e active site. I n recent years, several such "active-site directed" labeling reagents have been reported.5-8 This present study i s a n extension o f t h e work b y Changeux e t al.8 with t h e acetylcholine receptor a n d o f Wofsy a n d Michaeli9 with acetylcholine esterase o n on e o f these reagents, p-trimethylammonium ben- zene diazonium fluoroborate (TDF). T h e latter compound, which h a d been used a s a n affinity-labeling reagent f o r anti-p-azophenyl trimethylammonium antibodies, 1 0 i s a n irreversible inhibitor o f t h e acetylcholine-receptor site o f th e electroplax a t t h e level o f t h e junctions. 7 4
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P r o c e e d i n g s o f t h e N a t i o n a l A c a d e m y o f S c i e n c e sV o l . 6 7 , N o . 1 , p p . 7 4 - 7 8 , S e p t e m b e r 1 9 7 0
I n t e r a c t i o n s o f p - N i t r o b e n z e n e Diazonium F l u o r o b o r a t eand A n a l o g s w i t h t h e A c t i v e S i t e s o f A c e t y l c h o l i n e - R e c e p t o r
and - E s t e r a s e *
Henry G . Mautner and E v a B a r t e l s
DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY, TUFTS UNIVERSITY SCHOOL OF MEDICINE,
BOSTON, MASSACHUSETTS 02111, AND DEPARTMENT OF NEUROLOGY, COLLEGE OF PHYSICIANS
AND SURGEONS, COLUMBIA UNIVERSITY, NE W YORK, NE W YORK 10032
C o m m u n i c a t e d b y D a v i d N a c h m a n s o h n , J u n e 4 ,1 9 7 0
A b s t r a c t . p - N i t r o b e n z e n e d i a z o n i u m f l u o r o b o r a t e ( N D F ) i s a p o t e n t i n -
h i b i t o r o f t h e c a r b a m y l c h o l i n e - i n d u c e d d e p o l a r i z a t i o n o f t h e e l e c t r o p l a x a n d o fa c e t y l c h o l i n e s t e r a s e . I t p r o b a b l y f o r m s c o v a l e n t b o n d s w i t h t h e a c e t y l c h o l i n e -r e c e p t o r a n d - e s t e r a s e a t t h e a c t i v e s i t e o f t h e p r o t e i n s . I t s i n h i b i t o r y s t r e n g t h
i s a t l e a s t t h e s a m e a s t h a t o f t r i m e t h y l a m m o n i u m d i a z o n i u m f l u o r o b o r a t e( T D F ) . T h e p - a c e t o x y a n a l o g , w i t h i t s w e a k e r e l e c t r o n - w i t h d r a w i n g g r o u p ,
i s a b o u t t e n t i m e s w e a k e r a s a n i n h i b i t o r t h a n t h e t r i m e t h y l a m m o n i u m o r p -
n i t r o a n a l o g s , b o t h o f w h i c h h a v e s t r o n g e l e c t r o n - w i t h d r a w i n g g r o u p s . A f t e r
t r e a t m e n t o f t h e e l e c t r o p l a x p r e p a r a t i o n w i t h d i t h i o t h r e i t o l , NDF r e m a i n s a n
i r r e v e r s i b l e r e c e p t o r - i n h i b i t o r , w h i l e TD F b e c o m e s a p o t e n t r e v e r s i b l e r e c e p t o r -a c t i v a t o r . TD F i s s e l f - i n h i b i t o r y : a p p l i e d b e f o r e r e d u c t i o n , i t n o l o n g e r d e -p o l a r i z e s .
A l t h o u g h t h e f i r s t o b s e r v a t i o n s o n TD F s u g g e s t e d t h a t t h e c o m p o u n d l a b e l sb o t h p r o t e i n s b y v i r t u e o f t h e s t e r i c c o m p l e m e n t a r y o f i t s t r i m e t h y l a m m o n i u m
g r o u p t o a n e g a t i v e s u b s i t e i n t h e p r o t e i n s , t h e p r e s e n t s t u d y i n d i c a t e s t h a t i t i s
t h e p o s i t i v e l y c h a r g e d d i a z o n i u m g r o u p t h a t r e a c t s w i t h t h e a c t i v e s i t e s o f t h e
p r o t e i n s t o f o r m a c o v a l e n t b o n d w i t h a n a p p r o p r i a t e a m i n o - a c i d r e s i d u e .
I n t r o d u c t i o n . A c o n s i d e r a b l e b o d y o f e v i d e n c e h a s a c c u m u l a t e d i n f a v o r o f
t h e v i e w t h a t a c e t y l c h o l i n e i n d u c e s a c o n f o r m a t i o n a l c h a n g e o f t h e r e c e p t o rp r o t e i n i n e x c i t a b l e m e m b r a n e s , t h e r e b y t r i g g e r i n g o f f a s e r i e s o f r e a c t i o n sr e s u l t i n g i n i n c r e a s e d i o n p e r m e a b i l i t y . 1 2 Much e f f o r t h a s b e e n e x p e n d e d o n
i s o l a t i n g t h e b i o p o l y m e r o n w h i c h a c e t y l c h o l i n e a c t s d u r i n g a c t i v i t y . 3 ' 4 S u c h
w o r k w o u l d b e a i d e d b y h i g h l y s p e c i f i c r e a g e n t s s u i t a b l e f o r l a b e l i n g o f t h e m a c -
r o m o l e c u l e s i n q u e s t i o n b y f o r m a t i o n o f c o v a l e n t b o n d s w i t h g r o u p s a t o r n e a r
t h e a c t i v e s i t e . I n r e c e n t y e a r s , s e v e r a l s u c h " a c t i v e - s i t e d i r e c t e d " l a b e l i n gr e a g e n t s h a v e b e e n r e p o r t e d . 5 - 8 T h i s p r e s e n t s t u d y i s a n e x t e n s i o n o f t h e w o r k
b y C h a n g e u x e t a l . 8 w i t h t h e a c e t y l c h o l i n e r e c e p t o r a n d o f W o f s y a n d M i c h a e l i 9w i t h a c e t y l c h o l i n e e s t e r a s e o n o n e o f t h e s e r e a g e n t s , p - t r i m e t h y l a m m o n i u m b e n -
z e n e d i a z o n i u m f l u o r o b o r a t e ( T D F ) .T h e l a t t e r c o m p o u n d , w h i c h h a d b e e n u s e d a s a n a f f i n i t y - l a b e l i n g r e a g e n t f o r
a n t i - p - a z o p h e n y l t r i m e t h y l a m m o n i u m a n t i b o d i e s , 1 0 i s a n i r r e v e r s i b l e i n h i b i t o r o ft h e a c e t y l c h o l i n e - r e c e p t o r s i t e o f t h e e l e c t r o p l a x a t t h e l e v e l o f t h e j u n c t i o n s .
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V o i _ . 6 7 , 1 9 7 0 ACETYLCHOLINE RECEPTOR AND ESTERASE 7 5
TDF, b e i n g a d i a z o n i u m s a l t , r e a d i l y f o r m s c o v a l e n t b o n d s w i t h s u i t a b l e a m i n o
a c i d r e s i d u e s . The p o t e n t r e c e p t o r i n h i b i t o r s d - t u b o c u r a r i n e , d i m e t h y l d -
t u b o c u r a r i n e , a n d f l a x e d i l p r o t e c t a g a i n s t t h e i r r e v e r s i b l e i n h i b i t i o n b y TDF,e v e n w h e n t e n t i m e s l a r g e r c o n c e n t r a t i o n s o f TD F a r e u s e d . 8
I n t h e p r e s e n t s t u d y we h a v e t e s t e d TDF, i t s d i m e t h y l a n a l o g ( D D F ) , i t sp - n i t r o a n a l o g ( N D F ) , a n d i t s p - a c e t o x y a n a l o g ( A D F ) . T h e p o t e n c i e s o f t h e s ec o m p o u n d s i n a f f e c t i n g t h e m o n o c e l l u l a r e l e c t r o p l a x p r e p a r a t i o n w e r e c o m p a r e d
a n d t h e i r s t a b i l i t i e s i n s o l u t i o n w e r e d e t e r m i n e d . T h e r e l a t i v e a b i l i t i e s o f T D F la n d NDF t o i n h i b i t a c e t y l c h o l i n e s t e r a s e w e r e a l s o s t u d i e d .
M e t h o d . A l l c o m p o u n d s w e r e s y n t h e s i z e d a n d p u r i f i e d b y m e t h o d s d e s c r i b e d i l lt h e l i t e r a t u r e ( T D F , 1 ' D D F , 1 2 N D F , 1 3 A D F 1 3 ) .
B e c a u s e t h e e l e c t r o p l a x e x p e ri m e n t s w e r e c a r r i e d o u t i n pH 6 e e l R i n g e r ' s s o l u t i o n ,s t u d i e s o f t h e d e c o m p o s i t i o n o f t h e a b o v e c o m p o u n d s w e r e c a r r i e d o u t i n t h a t s o l v e n tu s i n g 1 m g / 1 0 0 m l s o l u t i o n o f TD F a n d i t s a n a l o g s . R e a c t i o n r a t e s w e r e f o l l o w e d b yo b s e r v i n g t h e d i s a p p e a r a n c e o f u l t r a v i o l e t e x t i n c t i o n a t 2 5 3 n m f o r TDF, 3 7 8 n m f o rDDF, 2 6 0 n m f o r NDF, a n d 2 6 9 n m f o r ADF. T h e c o m p o u n d s w e r e p l a c e d i n t h e r m o -r e g u l a t e d ( 2 5 . 0 0 C ) , s t o p p e r e d q u a r t z c u v e t t e s . A C a r y m o d e l 1 5 r e c o r d i n g s p e c t r o -p h o t o m e t e r was u s e d . P l o t s o f l o g A a g a i n s t t i m e y i e l d e d s t r a i g h t l i n e s , f r o m t h e s l o p e so f w h i c h t h e f i r s t - o r d e r r a t e c o n s t a n t s o f d e c o m p o s i t i o n w e r e o b t a i n e d ( s e e T a b l e 1 ) .
S i n g l e c e l l s f r o m t h e e l e c t r i c o r g a n o f t h e e l e c t r i c e e l w e r e d i s s e c t e d a n d m o u n t e d i n al u c i t e c h a m b e r a c c o r d i n g t o t h e p r o c e d u r e d e v e l o p e d b y S c h o f f e n i e l s . 1 4 T h e m e t h o d o fr e c o r d i n g t h e r e s t i n g p o t e n t i a l w a s t h e s a m e a s t h a t d e s c r i b e d p r e v i o u s l y . " 5 T h e c o m -p o s i t i o n o f t h e e e l R i n g e r ' s s o l u t i o n w a s N a C l 1 6 0 , K C l 5 , C a C l 2 2 , M g C l 2 2 , N a H 2 P O 4 0 . 3 .a n d N a 2 H P O 4 1 . 2 mM. T h e i n c u b a t i o n pH w a s 7 . 0 , t h e t e m p e r a t u r e 2 5 0 C . T h e c o m -p o u n d s w e r e d i s s o l v e d i n R i n g e r ' s s o l u t i o n a t pH 6 . 0 , w i t h a p h o s p h a t e b u f f e r c o n c e n t r a -
t i o n o f 5 0 mM i m m e d i a t e l y b e f o r e u s e . I n s o m e e x p e r i m e n t s 1 mM L - h i s t i d i n e w a sa d d e d a f t e r t h e a p p l i c a t i o n o f TD F a n d NDF, t o q u e n c h u n r e a c t e d m o l e c u l e s , b u t n od i f f e r e n c e i n t h e r e s u l t s w a s o b s e r v e d w h e n . t h e - c e l l w a s w a s h e d i n R i n g e r ' s o n l y .
R e s u l t s . F i g u r e 1 s h o w s d o s e : - r e s p o n s e c u r v e s f o r c a r b a m y l c h o l i n e w i t h a n dw i t h o u t p r e t r e a t m e n t w i t h 0 . 0 1 mM TDF a n d NDF o r 0 . 1 mM AD F f o r 2 0
m i n . I t c a n b e s e e n t ha t TDF a n d , I E R i n h i b i t t h e c a r b a m y l c h o l i n e r e s p o n s e
t o a n a l m o s t i d e n t i c a l e x t e n t " w h i M e ' A D F i s t e n t i m e s w e a k e r . I f a c e l l i s i n -c u b a t e d w i t h 0 . 0 5 m r n M TDF o r NDF f o r 2 0 m i n , t h e r e s p o n s e t o 0 . 0 5 mM c a r -b a m y l c h o l i n e i s a b o l i s h e d - a n d 1 mM c a r b a m y l c h o l i n e c a u s e s o n l y 5 - 1 0 mV
d e p o l a r i z a t i o n . NDF a l s o i n h i b i t s t h e d e p o l a r i z a t i o n s p r o d u c e d b y t e t r a -
m e t h y l a m m o n i u m a n d p h e n y l t r i m e t h y l a m m o n i u m . T h e o b s e r v a t i o n s o fC h a n g e u x e t a l . 8 h a v e s u g g e s t e d t h a t p h e n y l t r i m e t h y l a m m o n i u m a n d c u r a r e
c o m p e t e w i t h TD F f o r t h e s a m e b i n d i n g s i t e s o n t h e r e c e p t o r . S i m i l a r r e s u l t sh a v e now b e e n o b t a i n e d w i t h NDF. H e x a m e t h o n i u m , c u r a r e , a n d t o a l e s s e rd e g r e e t e t r a m e t h y l a m m o n i u m p r o t e c t e d t h e b i n d i n g s i t e s a g a i n s t t h e a c t i o n o f
F I G . 1 . - D o s e - r e s p o n s e c u r v e s f o r c a r b a m Y l - E - E oc h o l i n e b e f o r e a n d a f t e r e x p o s u r e o f t h e c e l l t o m Vp - t r i m e t h y l a m m o n i u m b e n z e n e d i a z o n i u m f l u o - 6 0r o b o r a t e ( T D F ) a n d i t s p - n i t r o ( N D F ) a n d p - 5 0
a c e t o x y ( A D F ) a n a l o g s . Tw o d o s e - r e s p o n s e 4 0
c u r v e s o f c a r b a m y l c h o l i n e w e r e o b t a i n e d o n a 3 0,o *-
s i n g l e c e l l ,o n e
b e f o r ea n d o n e a f t e r
a p p l i c a t i o no f
2 0e i t h e r 0 . 0 1 mM T D F , 0 . 0 1 mM NDF, o r 0 . 1 mM l o l ' -ADF f o r 2 0 m i n . 0 , E l , A c o n t r o l s , X NDF, 0 i-,T D F , A A D F , * 0 . 0 5 mM ND F o r TDF. E o = 5 - *i n i t i a l r e s t i n g p o t e n t i a l , E = s t e a d y s t a t e p o t e n - c a r b , 1 0 - 5 Mt i a l i n p r e s e n c e o f c a r b a m y l c h o l i n e .
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7 6 BIOCHEMISTRY: MA UTNER AND BARTELS P u o c . N . A . S .
NDF. A s s h o w n i n F i g u r e 2 B , t h e r e i p o n s 4 t o 0 . 0 5 mM c a r b a m y i c h o l i n e w a s
o n l y s l i g h t l y r e d u c e d i f 0 . 0 5 mM NDF w a s a p p l i e d t o g e t h e r w i t h 0 . 5 mM h e x a -
m e t h o n i u m . I f t h e c e l l w e r e t o b e e x p o s e d t o NDF a l o n e , t h e r es p o n s e t o 0 . 0 5mM c a r b a m y l c h o l i n e w o u l d b e a b s e n t ( s e e . ' P i g . 1 ) . H e x a m e t h o n i u m i s a b o u t
h a l f a s e f f e c t i v e i n t h e p r o t e c t i o n o f t h e b i n d i n g s i t e s a g a i n s t TD P t h a n a g a i n s tNDF ( F i g . 2 A ) ; t h i s i s m e a s u r e d b y t he r e s p o n s e t o c a r b a m y l c h o l i n e a f t e r e x -p o s u r e t o 0 . 5 mM h e x a m e t h o n i u m i n t h e p r e s e n c e o f t h e t w o a n a l o g s .
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F I G . 2 . - o m p e t i t i o n b e t w e e n h e x a m e t h o n i u m a n d d i a z o n i u m c o m p o u n d s .( A ) A f t e r t he t e s t r e s p o n s e t o 0 . 0 5 mM c a r l 1 g m y l c h o l i n e , t h e c e l l w a s i n c u b a t e d
i n 0 . 5 mM h e x a m e t h o n ~ i u m f o r 1 5 m m n f o l l o W e d b y 0 . 5 mM h e x a m e t h o n i u m p l u s0 . 0 5 mM TD F f o r 2 0 m m n ; t h e n t h e c e l l w ~ s r i n s e d i n n o r i i a l R i n g e r ' s ( N . R . )
f o r a n o t h e r 2 0 m i n t o r e m o v e t h e h e x a m e t h e i i i u m . F u r t h e r a p p l i c a t i o n s o f c a r -b a m y l c h o l i n e d e p o l a r i ze d t h e c e l l p a r t i a l l y . ( B ) Same e x p e r i m e n t a s i n A , e x c e p t
t h a t NDF w a s u s e d i n s t e a d o f T D F . . T h e , - s e c o n d a n d t h i r d e x p o s u r e s t o c a r -b a m y l c h o l i n e g i v e a d e c r e a s e d r e s p o n s e , b u t r i o t a s much a s a f t e r i n c u b a t i o n w i t hTDF.
A s r e p o r t e d b y P o d l e s k i e t a l . , 1 6 TD F b e c o m e s a v e r y p o t e n t , r e v e r s i b l e r e c e p t o ra c t i v a t o r a f t e r e x p o s u r e o f t h e c e l l t o d i t h i o t h r e i t o l , w h i c h r e d u c e s S - S b o n d s i nt h e v i c i n i t y o f t h e r e c e p t o r s i t e . 1 7 NDF, e v r e n a t a c o n c e n t r a t i o n o f 1 m M I , d o e s
n o t d e p o l a r i z e t h e e l e c t r o p l a x m e m b r a n e a f t e r d i t h i o t h r e i t o l , b u t i n h i b i t s i r -
r e v e r s i b l y t h e d e p o l a r i z a t i o n c a u s e d b y TDF, r e g a r d l e s s o f w h e t h e r i t i s a p p l i e d
b e f o r e o r a f t e r d i t h i o t h r e i t o l ( F i g . 3 ) . M f t e r d i t h i o t h r e i t o l t he r e s p o n s e t o 0 . 0 4m M d c a r b a m y l c h o l i n e i s r e d u c e d , w h i l e 0 . 0 0 5 mM TD F c a u s e s a f a s t d e p o l a t i z a -t i o n w h i c h i s r e v e r s e d i m m e d i a t e l y a n d c o m p l e t e l y o n a d d i t i o n o f 0 . 1 mM NDF
i n t h e p r es e n c e o f 0 . 0 0 5 mM TDF. M f t e r r e m o v a l o f NDF f r o m t h e t e s t s o l u t i o n ,TD F n o l o n g e r d e p o l a r i z e s . T h e s a m e e f f e c t s a r e o b s e r v e d w h e n t h e r e d u c e d c e l l
i s i n c u b a t e d i n 0 . 0 5 mM N D F ? f o r 1 0 m m n a n d w a s h e d i n R i n g e r ' s : t h e d e p o l a r i z -i n g a c t i o n o f TD F i s a b o l i s h e d . TD F i s s e l f - i n h i b i t o r y . M f t e r i n c u b a t i o n o f t h e
c e l l i n 0 . 1 mM TD F f o r 2 0 m m n b e f o r e r e d u c t i o n , t h e d e p o l a r i z i n g a c t i o n o f TDFi s a l s o a b o l i s h e d .
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V o L . 6 7 , 1 9 7 0 ACETYLCHOLrNE RECEPTOR AND ESTERASE 7 7
- m V
90
CC 4xO5 DTT IxO3 TD F 5 x I O
80- 6 CC 4x0-5 | TD F 5 x 1 O 6
F I G . 3 . - A n t a g o n i s m b e t w e e n 7 0
TD F a n d NDF a f t e r d i s u l f i d er e d u c t i o n b y d i t h i o t h r e i t o l . F o r 6 0 - R .e x p l a n a t i o n s e e t e x t . C C , c a r - lb a m y l c h o l i n e ; DTT, d i t h i o - 5 0 N . R .t h r e i t o l .
D F 5 x 1 O 6 + N D F I x I O 4
I N . R .30L8 1 2 1 6 2 8 32 36 4 4 48 52 5 6
T I M E I N MINUTES
D i s c u s s i o n . T h e s t a b i l i t i e s o f t h e v a r i o u s a n a l o g s i n e e l R i n g e r ' s s o l u t i o n ,a t pH 6 , d e c r e a s e f r o m DDF t o TD F t o NDF t o ADF. The h a l f - l i f e o f AD F i s
4 0 m i n c o m p a r e d t o 6 9 m i n f o r N I D F ( T a b l e 1 ) . S i n c e t h e s o l u t i o n s w e r e p r e -
T A B L E 1 . D e c o m p o s i t i o n o f p - t r i m e t h y l a m m o n i u m b e n z e n e f l u o r o b o r a t e ( T D F ) a n d i t s p -
d i m e t h y l ( D D F ) , p - n i t r o ( N D F ) , a n d p - a c e t o x y ( A D F ) a n a l o g s i n e e l R i n g e r ' ss o l u t i o n ( p H 6 , 2 5 0 C ) .
Compound k O b s / m i l n H a l f - l i f e ( m i n )
TD F 4 . 5 X 1 0 - ' 1 5 0
DDF ) 9 . 0 X 1 0 - 5 7 7 0 0NDF 1 0 X 1 0 - 3 6 9
AD F 1 7 X 1 0 - 3 4 1
p a r e d i m m e d i a t e l y b e f o r e a p p l i c a t i o n t o t h e e l e c t r o p l a x , t h e d i f f e r e n c e i n p o t e n c y
c a n n o t b e a c c o u n t e d f o r b y t h e d i f f e r e n t h a l f - l i v e s . T h e s t r i k i n g l a c k o f r e a c t i v -i t y o f DDF m u s t b e d u e t o t h i s c o m p o u n d b e i n g p r e d o m i n a n t l y i n t he p a r a -
q u i n o i d r e s o n a n c e f o r m . I t w a s n o t e d p r e v i o u s l y t h a t t h e s p e c t r u m o f DDF
r e s e m b l e s t h a t o f TD F o n l y i n v e r y - s t r o n g a c i d . 1 8
T h e o b s e r v a t i o n t h a t NDF i s a s p o t e n t a s TD F i n i n h i b i t i n g t h e c a r b a m y l -
c h o l i n e r e s p o n s e i n d i c a t e s t h a t t h e t r i m e t h y l a m m o n i u m g r o u p o f TD F i s n o t
c o n t r i b u t i n g t Q t h e b l o c k i n g a c t i o n b y t h i s c o m p o u n d , a s s e e m e d r e a s o n a b l e t oa s s u m e i n v i e w o f i t s s i m i l a r s t r u c t u r e t o p h e n y l t r i m e t h y l a m m o n i u m , a p o t e n t
d e p o l a r i z i n g c o m p o u n d . The d i a z o n i u m n i t r o g e n h a s a s t r o n g p o s i t i v e c h a r g e
w h o s e m a g n i t u d e d e p e n d s o n t h e p a r a s u b s t i t u t i o n o f t h e p h e n y l r i n g . T h e
p - t r i m e t h y l a m m o n i u m a n d t h e - n i t r o g r o u p s h a v e s i m i l a r l y h i g h e l e c t r o n -w i t h d r a w i n g p r o p e r t i e s , w h i l e t h e p - a c e t o x y g r o u p i s much w e a k e r . T h u s ,
t h e i n h i b i t o r y p o t e n c y o f t h e s e c o m p o u n d s may b e e x p l a i n e d b y t h e e a s e o fc o u p l i n g w i t h t h e d i a z o n i u m e n d .
T h e o b s e r v a t i o n s 8 t h a t d - t u b o c u r a r i i n e a n d p h e n y l t r i m e t h y l a m m o n i u m p r o t e c tt h e b i n d i n g s i t e s o f a c e t y l c h o l i n e a g a i n s t t h e a c t i o n o f TDF w e r e c o n f i r m e d ;
t h e s e c o m p o u n d s a l s o p r o t e c t t h e e b i n d i n g s i t e s a g a i n s t t h e a c t i o n o f NDF.T h u s , o n e i s l e d t o t h e c o n c l u s i o n t h a t TD F a n a l o g s d o i n d e e d i n t e r a c t w i t h t h e
b i n d i n g s i t e s o f t h e r e c e p t o r , b u t t ha t t he d i a z o n i u m g r o u p i t s e l f , r a t h e r t h a n t h e
t r i m e t h y l a m m o n i u m g r o u p , i s i m p l i c a t e d i n a t t a c h m e n t t o t h e r e c e p t o r b i o -p o l y m e r .
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7 8 BIOCHEMISTRY: MAUTNER AND BARTELS P R o c . N . A . S .
A f t e r e x p o s u r e o f t h e e l e c t r o p l a x c e l l t o d i t h i o t h r e i t o l , TD F b e c o m e s a p o t e n t
r e v e r s i b l e r e c e p t o r - a c t i v a t o r a t c o n c e n t r a t i o n s 1 0 0 t i me s l o w e r t h a n w e r e n e c e s -s a r y f o r t h e i r r e v e r s i b l e i n h i b i t i o n o f t h e c a r b a m y l c h o l i n e r e s p o n s e . NDF
d o e s n o t d e p o l a r i z e t h e m e m b r a n e a f t e r d i t h i o t h r e i t o l , b u t i n h i b i t s i r r e v e r s i b l yt h e d e p o l a r i z a t i o n i n d u c e d b y TDF. T h e s e o b s e r v a t i o n s s u g g e s t t h a t i t i s i n -
d e e d t h e t r i m e t h y l a m m o n i u m g r o u p o f TD F w h i c h s p e c i f i c a l l y i n d u c e s t h e d e -
p o l a r i z a t i o n o f t h e r e d u c e d r e c e p t o r . TD F i s s e l f - i n h i b i t o r y ; i f a p p l i e d b e f o r e
r e d u c t i o n i t n o l o n g e r c a u s e s d e p o l a r i z a t i o n . T h i s s u g g e s t s t h a t e i t h e r e n d o f
t h e m o l e c u l e c a n i n t e r a c t w i t h t h e s a m e a c t i v e s i t e o n t h e r e c e p t o r , b u t w i t h
d i f f e r e n t a f f i n i t i e s .
T h e o b s e r v a t i o n t h a t NDF i n h i b i t s a c e t y l c h o l i n e s t e r a s e a b o u t a s e f f i c i e n t l y a sd o e s TD F ( T a b l e 2 ) s u g g e s t s t h a t , h e r e t o o , t h e t r i m e t h y l a m m o n i u m g r o u p o f
TDF i s n o t n e c e s s a r y f o r t h e r e a c t i o n w i t h t h e a c t i v e s i t e o f t h e e n z y m e .
T A B L E 2 . P e r c e n t a g e i n h i b i t i o n o f a c e t y h c h o l i n e s t e r a s e b y T D F , DDF, a n d NDF.
C o n c e n t r a t i o n ( p M ) TDF DDF NDF
0 . 2 ... ... 2 1
0 . 5 2 0 . . . 7 4
1 5 9 . . . 1 0 0
2 8 4 0 . . .
5 9 2 0 ...
1 0 1 0 0 0 . . .
We w i s h t o t h a n k P r o f . D a v i d N a c h m a n s o h n a n d D r . H . W. C h a n g f o r v a l u a b l e a n d e n j o y -a b l e d i s c u s s i o n s a n d D r . C h a n g f o r s y n t h e s i z i n g ADF.
A b b r e v i a t i o n s : T D F , p - t r i m e t h y l a m m o n i u m b e n z e n e d i a z o n i u m f l u o r o b o r a t e ; DDF, i t sd i m e t h y l a n a l o g ; NDF, i t s p - n i t r o a n a l o g ; ADF, i t s p - a c e t o x y a n a l o g .
* T h i s w o r k w a s s u p p o r t e d , i n p a r t , b y a g r a n t f r o m t h e N a t i o n a l I n s t i t u t e f o r N e u r o l o g i c a lD i s e a s e s N S - 0 7 8 5 3 , f r o m t h e N a t i o n a l S c i e n c e F o u n d a t i o n g r a n t s N S F - G B - 6 8 3 5 a n d NSF-GB-
7 1 4 9 , b y t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , g r a n t N S - 0 3 3 0 4 , a n d b y t h e Ne w Y o r k H e a r t
A s s o c i a t i o n I n c .1 N a c h m a n s o h n , D . , i n C h e m i c a l a n d M o l e c u l a r B a s i s o f N e r v e A c t i v i t y ( N e w Y o r k : A c a -
d e i u c P r e s s , 1 9 5 9 ) , p . 2 3 5 .2 N a c h m a n s o h n , D . , " P r o t e i n s o f e x c i t a b l e m e m b r a n e s , " P r o c e e d i n g s o f t h e T e n t h A n n u a l
B a s i c S c i e n c e S y m p o s i u m o n M e m b r a n e P r o t e i n s , N o v . 2 9 - 3 0 , 1 9 6 8 , Ne w Y o r k H e a r t A s s o -
c i a t i o n . J . G e n . P h y s i o l . , 5 4 , S 1 8 7 - 2 2 4 ( 1 9 6 5 ) .3 O ' B r i e n , R . D . , a n d L . P . G i l m o u r , t h e s e P R O C E E D I N G S , 6 3 , 4 9 6 ( 1 9 6 9 ) .4 O ' B r i e n , R . D . , L . P . G i l m o u r , a n d M. E . E l d e f r a w i , t h e s e P R O C E E D I N G S , 6 5 , 4 3 8 ( 1 9 7 0 ) .6 T a k a g i , K . , M. I k a o , a n d A . T a k a h a s h i , L i f e S c i . , 4 , 2 1 6 5 ( 1 9 6 5 ) .6 G i l l , E . , a n d H . P . R a n g , M o l . P h a r m a c o l . , 2 , 2 8 4 ( 1 9 6 6 ) .7 K a r l i n , A . , a n d M. W i n n i k , t h e s e P R O C E E D I N G S , 6 0 , 6 6 8 ( 1 9 6 8 ) .8 C h a n g e u x , J . - P . , T . R . P o d l e s k i , a n d L . W o f s y , t h e s e P R O C E E D I N G S , 5 8 , 2 0 6 3 ( 1 9 6 7 ) .9 W o f s y , L . , a n d D . M i c h a e l i , t h e s e P R O C E E D I N G S , 5 8 , 2 2 9 6 ( 1 9 6 7 ) .
1 0 F e n t o n , J . W . , a n d S . J . S i n g e r , B i o c h e m . B i o p h y s . R e s . C o m m u n . , 2 0 , 3 1 5 ( 1 9 6 5 ) ." 1 T r a y l o r , P . S . , a n d S . J . S i n g e r , B i o c h e m i s t r y , 6 , 8 8 1 ( 1 9 6 7 ) .1 2 S c h i e m a n n , G . , a n d M. W i n k e l m u l l e r , C h e m . B e r . , 6 6 B , 7 2 7 ( 1 9 3 3 ) .1 3 S t a r k e y , E . B . , i n O r g a n i c S y n t h e s e s , C o i l . V o l . I I , e d . A . H . B l a t t . ( N e w Y o r k : J o h n
W i l e y & S o n s , 1 9 4 3 ) , p . 2 2 5 .1 4 S c h o f f e n i e l s , E . , B i o c h i m . B i o p h y s . A c t a , 2 6 , 5 8 5 ( 1 9 5 7 ) .1 6 H i g m a n , H . B . , T . R . P o d l e s k i , a n d E . B a r t e l s , B i o c h i m . B i o p h y s . A c t a , 7 5 , 1 8 7 ( 1 9 6 3 ) .1 6
P o d l e s k i ,T .
R . ,J . - C . M e u n i e r , a n d J . - P . C h a n g e u x , t h e s e P R O C E E D I N G S , 6 3 , 1 2 3 9 ( 1 9 6 9 ) .
1 7 K a r l i n , A . , a n d E . B a r t e l s , B i o c h i m . B i o p h y s . A d a , 1 2 6 , 5 2 5 ( 1 9 6 6 ) .1 8 K a z i t s y n a , L . A . , A . V . K n z n e t s o v a , 0 . A . K o r y t i n a , a n d O . A . R e n t o v , D o k l . A k a d .
V a u k . , S . S . S . R . , 1 5 4 , 3 7 9 ( 1 9 6 4 ) ; C h e m . A b s t r . , 6 0 , 9 1 7 7 b ( 1 9 6 4 ) .1 9 M a r c h , J . , A d v a n c e d O r g a n i c C h e m i s t r y ( N e w Y o r k : M c G r a w - H i l l Book C o . 1 9 6 8 ) .
