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On the specificity of the delta-endotoxin of bacillus thuringiensis
578
8th World C o q r ~
on Animal, Plant and Mi=obid T o ~
ACE£~LCHOLINE RECEPTORS 0Y HUMAN SKELETAL MUSCLE: A SPECIES DI~wK-RwCE DETECTED BY SNAKE NEUROTOXINS By Y. Ishikawa I, M. Kano 2 , N. Taaiya 3 and Y. Shimada I. IDept of Anatoay, School of M e d i c i n e , Chiba U n i v e r s i t y , Chiba 280, J a p a n . 2Dept o f P h y s i o l o g y , School o f M e d i c i n e , F~taanto U n i v e r s i t y , Kanagawa 228, J a p a n . 3Dept o f C h e ~ e t r y , F a c u l t y o f S c i e n c e , Tohoku U n i v e r s i t y , Senda£ 980, J a p a n . The n i c o t i n i c e c e t y l c h o l t n e r e c e p t o r s (AChRs) o f h m u n s k e l e t a l R u e c l e h a v e a t t r a c t e d a s p e c i a l a t t e n t i o n , s i n c e a r e d u c t i o n i n J u n c t i o n a l AChRs was found i n m y a s t h e n i a g r a v i s . The b i o c h e m i c a l and p h a r m a c o l o g i c a l p r o p e r t i e s o f t h e AChRs o f human m u s c l e h a v e bean r e p o r t e d t o be h i g h l y s i m i l a r t o t h o s e o f a l l v e r t e b r a t e s p r e v i o u s l y s t u d i e d . We i n v e s tigated the binding abilities o f t h e AChRs o f t h e s k e l e t a l m u s c l e s o f man and o t h e r v e r t e b r a t e s to two t y p i c a l c u r s r e m / a e t i c t o x i n s , e r a b u t o x i n b (Eb) and a - b u n g a r o t o x i u (a-BT). F l u o r e s c e n t m i c r o s c o p y u s i n g r h o d a m i n e - l a b e l e d e r a b u t o x i n b (TMR-Eb) and F 1 T C - l a b e l e d a b u n g a r o t o x i n (FITC-a-BT) r e v e a l e d t h a t AChRs o f human and c h i m p a n z e e m u s c l e s w~re s t a i n e d w i t h FITC-~-BT, b u t n o t w i t h TMR-Eb. I n c o n t r a s t , t h e AChRs o f f i s h , f r o g , c h i c k e n , mouse, r a t , e a t and monkey were s t a i n e d w i t h b o t h f l u o r e s c e n t t o x i n s . The s t a i n i n g s o f h t ~ a n and c h i m p a n z e e AChRs w i t h FITC-a-BT were i n h i b i t e d by p r e i n c u b a t i o n w i t h , - - , n d i f i e d • -BT, b u t n o t w i t h e i t h e r ,~,=-~dified Eb o r o t h e r s h o r t - c h a i n n e u r o t o x i n e . Binding experiwteute u s i n g 1 2 5 I - l a b e l e d Eb ( 1 2 5 I - E b ) and 1 2 5 I - l a b e l e d a-BT ( 1 2 5 I - a - B T ) showed t h a t t h e a f f i n i t y o f b , - - - - AChRs f o r 125I-Eb was u n u s u a l l y low. E l e c t r o p h y e l o l o g i c a l e x p e r i m e n t s showed that both acetylcholine potential and end-plate potential of b,mmn muscle were blocked by addition of a-BT, but not by Eb. On the contrary, acetylchollne potential of rat muscle was blocked by addition of Eb. All these results indicate that AChRs of human end chimpanzee muscles are different from those of other enlmals in having an exceptionally low affinity for Eb and other short-chain nsurotoxlns. The results suggest a hate r o g A n e i t y among v e r t e b r a t e AChRs c o n c e r n i n g t h e i r r e a c t i v t t i e s t o curarez~L~etlc t o x i n s . REFERENCES I e h i k a w a , Y. and Shimada, Y. (1983) B r a i n Res. 266, 159. KEY WORDS snake neurotoxin; acetylcholine receptor; n s u r o s m s c u l a r J u n c t i o n ; human s k e l e t a l m u s c l e
O N THE S P E C I F I C I T Y OF THE D E L T A - E N D O T O X I N
OF B A C I L L U S T H U R I N G I E N S I S
By F. JAQUET, P. LUETHY, and R. HUETTER. CH-8092 Zurich, Switzerland.
Institute o f M i c r o b i o l o g y ETH,
The c r y s t a l l i n e p r o t e i n p r o d u c e d d u r i n g s p o r u l a t i o n by B a c i l l u s thur l n ~ l e n s i s is r e s p o n s i b l e for i n s e c t i c i d a l a c t i v i t y against larvae of m a n y species of the order L e p i d o p t e r a to w h i c h b e l o n g a c o n s i d e r a b l e number of important pest insects. The crystals c o n s i s t o f subunlts with an average m o l e c u l a r w e i g h t of 130'000. The crystals and the subunits have to be cons i d e r e d as p r o t o x l n s r e q u i r i n g p r o t e o l y t l c a c t i v a t i o n in o r d e r to d e s t r o y the m i d g u t e p i t h e l i a of target insects. The d e l t a - e n d o t o x i n is k n o w n to be h i g h l y specific. The degree of s p e c i f i c i t y is not only d e t e r m i n e d by the insect host but also by the subspecies of B. t h u r l n ~ i e n s l s or even by a given strain, T h i r t e e n strains b e l o n g i n g to Ii subspecies were tested against Pierls brasslcae, H e l i o t h l s v i r e s c e n s and S p o d o p t e r a llttoralls. The P. b r a s s i c a e larvae were m o s t s u s c e p t i b l e to the toxins of strains b e l o n g i n g to the subspecies thurin~iensis and m o r r l s o n l w h e r e a s H. virescens r e s p o n d e d best to the subspecies ken~ae and kurstakl. On the other hand, e n t o m o c i d u s gave the best results in the b l o a s s a y against S. littoralls. C o n s i d e r a b l e d i f f e r e n c e s were o b t a i n e d when crystals, p r o t o x l n and a c t i v a t e d toxin w e r e compared. The h i g h e s t a c t i v i t y was r e c o r d e d in all cases with the a c t i v a t e d form of the d e l t a - e n d o t o x i n when b l o a s s a y e d a g a i n s t H. v l r e s c e n s and S. llttoralis. The d i f f e r e n c e s of the three toxin forms were m i n o r in the case of P. b r a s s i c a e as target species. KEY W O R D S Bacillus
thurlnglensis,
delta-endotoxin,
specificity
8th World C o q r ~
on Animal, Plant and Mi=obid T o ~
ACE£~LCHOLINE RECEPTORS 0Y HUMAN SKELETAL MUSCLE: A SPECIES DI~wK-RwCE DETECTED BY SNAKE NEUROTOXINS By Y. Ishikawa I, M. Kano 2 , N. Taaiya 3 and Y. Shimada I. IDept of Anatoay, School of M e d i c i n e , Chiba U n i v e r s i t y , Chiba 280, J a p a n . 2Dept o f P h y s i o l o g y , School o f M e d i c i n e , F~taanto U n i v e r s i t y , Kanagawa 228, J a p a n . 3Dept o f C h e ~ e t r y , F a c u l t y o f S c i e n c e , Tohoku U n i v e r s i t y , Senda£ 980, J a p a n . The n i c o t i n i c e c e t y l c h o l t n e r e c e p t o r s (AChRs) o f h m u n s k e l e t a l R u e c l e h a v e a t t r a c t e d a s p e c i a l a t t e n t i o n , s i n c e a r e d u c t i o n i n J u n c t i o n a l AChRs was found i n m y a s t h e n i a g r a v i s . The b i o c h e m i c a l and p h a r m a c o l o g i c a l p r o p e r t i e s o f t h e AChRs o f human m u s c l e h a v e bean r e p o r t e d t o be h i g h l y s i m i l a r t o t h o s e o f a l l v e r t e b r a t e s p r e v i o u s l y s t u d i e d . We i n v e s tigated the binding abilities o f t h e AChRs o f t h e s k e l e t a l m u s c l e s o f man and o t h e r v e r t e b r a t e s to two t y p i c a l c u r s r e m / a e t i c t o x i n s , e r a b u t o x i n b (Eb) and a - b u n g a r o t o x i u (a-BT). F l u o r e s c e n t m i c r o s c o p y u s i n g r h o d a m i n e - l a b e l e d e r a b u t o x i n b (TMR-Eb) and F 1 T C - l a b e l e d a b u n g a r o t o x i n (FITC-a-BT) r e v e a l e d t h a t AChRs o f human and c h i m p a n z e e m u s c l e s w~re s t a i n e d w i t h FITC-~-BT, b u t n o t w i t h TMR-Eb. I n c o n t r a s t , t h e AChRs o f f i s h , f r o g , c h i c k e n , mouse, r a t , e a t and monkey were s t a i n e d w i t h b o t h f l u o r e s c e n t t o x i n s . The s t a i n i n g s o f h t ~ a n and c h i m p a n z e e AChRs w i t h FITC-a-BT were i n h i b i t e d by p r e i n c u b a t i o n w i t h , - - , n d i f i e d • -BT, b u t n o t w i t h e i t h e r ,~,=-~dified Eb o r o t h e r s h o r t - c h a i n n e u r o t o x i n e . Binding experiwteute u s i n g 1 2 5 I - l a b e l e d Eb ( 1 2 5 I - E b ) and 1 2 5 I - l a b e l e d a-BT ( 1 2 5 I - a - B T ) showed t h a t t h e a f f i n i t y o f b , - - - - AChRs f o r 125I-Eb was u n u s u a l l y low. E l e c t r o p h y e l o l o g i c a l e x p e r i m e n t s showed that both acetylcholine potential and end-plate potential of b,mmn muscle were blocked by addition of a-BT, but not by Eb. On the contrary, acetylchollne potential of rat muscle was blocked by addition of Eb. All these results indicate that AChRs of human end chimpanzee muscles are different from those of other enlmals in having an exceptionally low affinity for Eb and other short-chain nsurotoxlns. The results suggest a hate r o g A n e i t y among v e r t e b r a t e AChRs c o n c e r n i n g t h e i r r e a c t i v t t i e s t o curarez~L~etlc t o x i n s . REFERENCES I e h i k a w a , Y. and Shimada, Y. (1983) B r a i n Res. 266, 159. KEY WORDS snake neurotoxin; acetylcholine receptor; n s u r o s m s c u l a r J u n c t i o n ; human s k e l e t a l m u s c l e
O N THE S P E C I F I C I T Y OF THE D E L T A - E N D O T O X I N
OF B A C I L L U S T H U R I N G I E N S I S
By F. JAQUET, P. LUETHY, and R. HUETTER. CH-8092 Zurich, Switzerland.
Institute o f M i c r o b i o l o g y ETH,
The c r y s t a l l i n e p r o t e i n p r o d u c e d d u r i n g s p o r u l a t i o n by B a c i l l u s thur l n ~ l e n s i s is r e s p o n s i b l e for i n s e c t i c i d a l a c t i v i t y against larvae of m a n y species of the order L e p i d o p t e r a to w h i c h b e l o n g a c o n s i d e r a b l e number of important pest insects. The crystals c o n s i s t o f subunlts with an average m o l e c u l a r w e i g h t of 130'000. The crystals and the subunits have to be cons i d e r e d as p r o t o x l n s r e q u i r i n g p r o t e o l y t l c a c t i v a t i o n in o r d e r to d e s t r o y the m i d g u t e p i t h e l i a of target insects. The d e l t a - e n d o t o x i n is k n o w n to be h i g h l y specific. The degree of s p e c i f i c i t y is not only d e t e r m i n e d by the insect host but also by the subspecies of B. t h u r l n ~ i e n s l s or even by a given strain, T h i r t e e n strains b e l o n g i n g to Ii subspecies were tested against Pierls brasslcae, H e l i o t h l s v i r e s c e n s and S p o d o p t e r a llttoralls. The P. b r a s s i c a e larvae were m o s t s u s c e p t i b l e to the toxins of strains b e l o n g i n g to the subspecies thurin~iensis and m o r r l s o n l w h e r e a s H. virescens r e s p o n d e d best to the subspecies ken~ae and kurstakl. On the other hand, e n t o m o c i d u s gave the best results in the b l o a s s a y against S. littoralls. C o n s i d e r a b l e d i f f e r e n c e s were o b t a i n e d when crystals, p r o t o x l n and a c t i v a t e d toxin w e r e compared. The h i g h e s t a c t i v i t y was r e c o r d e d in all cases with the a c t i v a t e d form of the d e l t a - e n d o t o x i n when b l o a s s a y e d a g a i n s t H. v l r e s c e n s and S. llttoralis. The d i f f e r e n c e s of the three toxin forms were m i n o r in the case of P. b r a s s i c a e as target species. KEY W O R D S Bacillus
thurlnglensis,
delta-endotoxin,
specificity