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Lipid protein interactions in bovine erythrocyte acetylcholinestrase Sekar, Chandra, M. 1979

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L I P I D PROTEIN INTERACTIONS IN BOVINE ERYTHROCYTE ACETYLCHOLINES TRASE by M.CHANDRA^SEKAR THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i r i THE FACULTY"OF GRADUATE STUDIES he D i v i s i o n o f P h a r m a c e u t i c a l C h e m i s t r y f t h e F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA November 1979 (c) M. C h a n d r a S e k a r , 1979 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 ABSTRACT I n v o l v e m e n t o f l i p i d i n t h e a c t i v i t y o f mammalian e r y t h r o c y t e a c e t y l c h o l i n e s t e r a s e (AChE) has been»*proposed by v a r i o u s w o r k e r s . I n b o v i n e e r y t h r o c y t e AChE a t i g h t l y bound f r a c t i o n o f c a r d i o l i p i n (CL) was proposed" t o be i n v o l v e d i n t h e m o d u l a t i o n o f AChE c a t a l y t i c a c t i v i t y . M e t h ods p r e v i o u s l y u s e d f o r t h e i s o l a t i o n o f CL r e s u l t e d i n enzyme d e n a t u r a t i o n . I n t h e p r e s e n t s t u d y v a r i o u s m ethods f o r t h e s e p a r a t i o n o f CL u n d e r n o n - d e n a t u r i n g c o n d i t i o n s h a v e b e e n i n v e s t i g a t e d . I t was r e p o r t e d e a r l i e r t h a t a l l t h e l i p o p r o t e i n f o r m s o f t h e enzyme c o n t a i n i n g CL showed a b i p h a s i c A r r h e n i u s p l o t w i t h a b r e a k a r o u n d 20°C. I t was s u g g e s t e d t h a t t r e a t m e n t of t h e enzyme w i t h 1.8M s o d i u m c h l o r i d e , 2mM s o d i u m p h o s p h a t e , pH 7.4 or IM s o d i u m b i c a r b o n a t e pH 8-10, c a u s e d d i s s o c i a t i o n o f c a r d i o l i p i n and i t was a c c o m p a n i e d by a b o l i t i o n o f t h e A r r h e n i u s p l o t b r e a k . Methods u s e d f o r t h e s e p a r a t i o n of CL f r o m AChE were b a s e d on t h e d i f f e r e n c e i n s i z e and d e n s i t y b e t w e e n t h e two c o m p o n e n t s . Enzyme was t r e a t e d w i t h " h i g h s a l t " c o n d i t i o n s w h i c h were p o s t u l a t e d t o c a u s e d i s s o c i a t i o n o f CL. The r e s u l t i n g m i x t u r e was p a s s e d t h r o u g h a S ephadex g e l c o l u m n so t h a t CL c a n be s e p a r a t e d f r o m t h e enzyme b e c a u s e o f i t s s i z e d i f f e r e n c e . The enzyme o b t a i n e d f r o m t h e S e p h a d e x g e l c o l u m n gave a p a r t i a l s p e c i f i c v o l u m e o f 0.81 m l / g , w h i c h i s h i g h e r t h a n t h a t e x p e c t e d f r o m t h e amino a c i d c o m p o s i t i o n i i i o f t h e p r o t e i n , i n d i c a t i n g t h a t CL i s s t i l l bound t o t h e enzyme. I n a n o t h e r e x p e r i m e n t ( " f l o t a t i o n e x p e r i m e n t " ) a n a t t e m p t was made t o s e p a r a t e t h e d i s s o c i a t e d CL f r o m t h e enzyme on a s u c r o s e g r a d i e n t , b a s e d on t h e d e n s i t y d i f f e r e n c e b e t w e e n t h e p h o p h o l i p i d and p r o t e i n . A r r h e n i u s p l o t s were o b t a i n e d a t d i f f e r e n t t i m e i n t e r v a l s on t h e enzyme r e c o v e r e d f r o m t h e s u c r o s e g r a d i e n t . A l i n e a r A r r h e n i u s p l o t was o b s e r v e d a f t e r 24 h. S t o r a g e o f t h e enzyme f o r 5 t o 8 d a y s gave r i s e t o a g i s t i n c t b r e a k i n t h e A r r h e n i u s p l o t . The r e a p p e r a r a n c e o f t h e b r e a k was o b s e r v e d even when t h e c e n t r i f u g a t i o n was done i n t h e p r e s e n c e o f 0.09% T r i t o n X-100. T h i s was i n t e r p r e t e d t o i n d i c a t e t h a t e ndogeneous CL was bound t o t h e enzyme t h r o u g h i o n i c and h y d r o p h o b i c i n t e r a c t i o n . " H i g h s a l t " t r e a t m e n t may a b o l i s h t h e i o n i c i n t e r a c t i o n , c a u s i n g " f u n c t i o n a l d i s s o c i a t i o n " of CL ( a s shown"by d i s a p p e a r a n c e o f t h e A r r h e n i u s p l o t b r e a k ) b u t s i m u l t a n e o u s s t r e n g t h e n i n g o f t h e h y d r o p h o b i c i n t e r a c t i o n s may a c c o u n t f o r t h e r e a p p e a r a n c e o f t h e b r e a k . The n e x t method a t t e m p t e d f o r t h e s e p a r a t i o n o f CL was b a s e d on t h e p r i n c i p l e t h a t , i f enzyme c o u l d be bound t o a s o l i d s u p p o r t , t h e n w a s h i n g o f t h e enzyme w i t h c h a o t r o p i c ; a g e n t s , d e t e r g e n t s and " h i g h s a l t " may r e s u l t i n t h e r e l e a s e o f CL. The s u i t a b i l i t y o f t h e N - m e t h y l a c r i d i n i u m (MAC) a f f i n i t y c o l u m n as a s o l i d s u p p o r t f o r t h i s enzyme w a s ' i n v e s t i g a t e d . The c h o i c e of MAC as an a f f i n i t y l i g a n d was b a s e d on t h e r e c e n t r e p o r t s r e g a r d i n g i t s s u i t a b i l i t y as an a f f i n i t y l i g a n d f o r p u r i f i c a t i o n o f e e l and p i g b r a i n AChE. The e l u t i o n p r o f i l e o f t h e enzyme i n 0.IM N a C l , 20mM s o d i u m p h o s p h a t e , pH 7.4 i v a t d i f f e r e n t l i g a n d (MAC) c o n c e n t r a t i o n s i n d i c a t e d t h a t a minimum of 2.8 u m o l e / m l g e l r e q u i r e d f o r s u f f i c i e n t r e t e n t i o n of t h e enzyme. As t h e a f f i n i t y o f t h e l i g a n d f o r t h e enzyme w i l l f u r t h e r d e c r e a s e w i t h i n c r e a s i n g i o n i c s t r e n g t h , t h e MAC a f f i n i t y column, i s u n s u i t a b l e as a s o l i d s u p p o r t f o r b o v i n e e r y t h r o c y t e AChE. I t was f o u n d t h a t t h e l o w e r r e t e n t i o n of t h e b o v i n e e r y t h r o c y t e AChE compared t o t h e e e l enzyme on t h e MAC a f f i n i t y c o l u m n s was due t o t h e l o w e r a f f i n i t y o f t h e f o r m e r f o r t h e l i g a n d , r a t h e r t h a n t o any s t r u c t u r a l d i f f e r e n c e o r a d i f f e r e n t mode o f b i n d i n g . P r e l i m i n a r y f i n d i n g s s u g g e s t t h a t CL d i s s o c i a t i o n d i d n o t a l t e r t h e a f f i n i t y o f t h e enzyme f o r t h e l i g a n d . F i n a l l y , as a p r i m a r y r e q u i r e m e n t f o r t h e p r e p a r a t i o n o f l a r g e quantities', o f p u r e AChE, so t h a t CL c a n be e x c h a n g e d by a d e t e r g e n t e x c h a n g e m e t h o d , v a r i o u s s t e p s f o r t h e p u r i f i c a t i o n o f t h e n a t i v e f o r m s of AChE by a d e t e r g e n t f r e e method have been c h a r a c t e r i z e d . The f o l l o w i n g f i n d i n g s were made. B u t a n o l t r e a t -ment e n h a n c e d t h e enzyme r e l e a s e .from t h e membrane, f r o m 40 t o 80 p e r c e n t , by e x t r a c t i n g t h e " m o b i l e " p h o s p h o l i p i d s . The e x t r a c t i o n o f AChE c a n be i n c r ea s ed ..-b y i n c r e a s i n g t h e i o n i c s t r e n g t h o f t h e medium and by c a l c i u m c h e l a t i o n . P u r i f i c a t i o n o f t h e above enzyme c a n be a c h e i v e d by a f f i n i t y p u r i f i c a t i o n b u t optimum c o n d i t i o n s r e q u i r e d f o r t h e above p u r i f i c a t i o n a r e s t i l l u n d e r i n v e s t i g a t i o n . C h a r a c t e r i z a t i o n o f t h e m o l e c u l a r f o r m s o f t h e enzyme on s u c r o s e d e n s i t y g r a d i e n t i n d i c a t e s e x t e n s i v e a g g r e g a t -i o n a t low' i o n i c s t r e n g t h , w h i l e a l o w e r d e g r e e o f a g g r e g a t i o n w i t h a p r o m i n a n t 11S peak was o b s e r v e d i n t h e p r e s e n c e o f 0.IM s o d i u m c h l o r i d e , 20mM s o d i u m p h o s p h a t e . V TABLE OF CONTENTS Page ABSTRACT i i L I S T OF TABLES v i i L I S T OF FIGURES v i i i ACKNOWLEDGEMENT x INTRODUCTION 1 LITERATURE REVIEW 4 I s o l a t i o n and p u r i f i c a t i o n o f a c e t y l c h o l i n e s t e r a s e 4 A c e t y l c h o l i n e s t e r a s e s t r u c t u r e 6 L i p i d - p r o t e i n i n t e r a c t i o n s i n a c e t y l c h o l i n e s t e r a s e 9 Membrane s t r u c t u r e 12 T h e r m a l p h a s e t r a n s i t i o n and p h a s e s e p a r a t i o n 15 i n membrane l i p i d s F a c t o r s e f f e c t i n g p h a s e t r a n s i t i o n and p h a s e s e p a r a t i o n 17 B o u n d a r y l i p i d and o t h e r l i p i d s 19 L i p i d m o d u l a t i o n o f enzyme a c t i v i t y 20 The A r r h e n i u s p l o t : s i g n i f i c a n c e and l i m i t a t i o n s 22 METHODS AND MATERIALS 25 M a t e r i a l s 25 " F l o t a t i o n " t e c h n i q u e f o r c a r d i o l i p i n s e p a r a t i o n 25 C a r d i o l i p i n s e p a r a t i o n by S e p h a d e x g e l f i l t r a t i o n 26 P a r t i a l s p e c i f i c v o l u m e 26 A f f i n i t y c h r o m a t o g r a p h y o f a c e t y l c h o l i n e s t e r a s e on 27 MAC a f f i n i t y c o l u m n s P r o t e i n e s t i m a t i o n 29 R a d i o m e t r i c a s s a y o f a c e t y l c h o l i n e s t e r a s e 29 K i n e t i c s t u d i e s 30 I s o l a t i o n and p u r i f i c a t i o n o f a c e t y l c h o l i n e s t e r a s e f r o m b o v i n e b l o o d S e d i m e n t a t i o n c o e f f i c i e n t s P h o s p h o l i p i d e x t r a c t i o n and e s t i m a t i o n R e s u l t s P a r t i a l s p e c i f i c v o l u m e o f " h i g h s a l t " t r e a t e d a c e t y l c h o l i n e s t e r a s e a f t e r S e p h a d e x G-200 g e l f i l t r a t i o n C h a r a c t e r i z a t i o n o f c a r d i o l i p i n - a c e t y l c h o l i n e s t e r a s e i n t e r a c t i o n s by A r r h e n i u s p l o t s K i n e t i c s o f MAC i n h i b i t i o n o f b o v i n e e r y t h r o c y t e " - . e a c e t y l c l i o l i n e s t e r as e R o l e o f t i g h t l y bound c a r d i o l i p i n ' i n ; .MAC a f f i n i t y P h o s p h o l i p i d a n a l y s i s R e l e a s e o f a c e t y l c h o l i n e s t e r a s e f r o m b u t a n o l t r e a t e d b o v i n e e r y t h r o c y t e membranes A f f i n i t y p r u i f i c a t l o n o f a c e t y l c h o l i n e s t e r a s e on PTA colum n S u c r o s e d e n s i t y s e d i m e n t a t i o n DISCUSSION C a r d i o l i p i n m o d u l a t i o n o f a c e t y l c h o l i n e s t e r a s e c a t a l y s i s A f f i n i t y c h r o m a t o g r a p h y o f a c e t y l c h o l i n e s t e r a s e on MAC a f f i n i t y c o l u m n s I s o l a t i o n and p u r i f i c a t i o n o f b o v i n e e r y t h r o c y t e a c e t y l c h o l i n e s t e r a s e BIBLIOGRAPHY v i i L I S T OF TABLES T a b l e Page I C r i t e r i a f o r d i s t i n g u i s h i n g p e r i p h e r a l and 14 i n t e g r a l membrane p r o t e i n s I I Q u a n t i t a t i v e e s t i m a t i o n o f p h o s p h o l i p i d 54 c o n t e n t I I I E x t r a c t i o n o f a c e t y l c h o l i n e s t e r a s e f r o m 56 b u t a n o l t r e a t e d e r y t h r o c y t e g h o s t s w i t h d i f f e r e n t i o n i c s t r e n g t h m e d i a V I 1 X F i g . 1. 2. 3. 9A. 9B, 10 L I S T OF FIGURES P r o p o s e d s t u r c t u r e f o r t h e 18S f o r m o f e e l e l e c t r i c o r g a n a c e t y l c h o l i n e s t e r a s e P l a t e model o f b i o l o g i c a l membrane P a r t i a l s p e c i f i c v o l u m e o f t h e a c e t y l c h o l i n e -s t e r a s e a f t e r S ephadex g e l c h r o m a t o g r a p h y A r r h e n i u s p l o t s a t d i f f e r e n t t i m e p e r i o d s on t h e .enzyme o b t a i n e d f r o m s u c r o s e g r a d i e n t i n t h e a b s e n c e o f T r i t o n X-100 A r r h e n i u s p l o t s a t d i f f e r e n t t i m e p e r i o d s i n t h e p r e s e n c e and a b s e n c e o f T r i t o n X-100 i n t h e c e n t r i f u g a t i o n m e d i a E l u t i o n p r o f i l e o f a c e t y l c h o l i n e s t e r a s e a t d i f f e r e n t l i g a n d c o n c e n t r a t i o n s on MAC a f f i n i t y c o l u m n s L i n e w e a v e r - B u r k p l o t o f a c e t y l c h o l i n e s t e r a s e a t d i f f e r e n t MAC c o n c e n t r a t i o n s S-lope and i n t e r c e p t r e p l o t o f t h e L i n e w e a v e r - B u r k p l o t E f f e c t o f C a C l 2 on MAC i n h i b i t i o n i n t h e a b s e n c e o f a dded s a l t s D i x o n p l o t o f t h e e f f e c t o f C a C l 2 on MAC i n h i b i t i o n D i x o n p l o t o f t h e e f f e c t o f C a C l 2 on MAC i n h i b i t i o n i n t h e p r e s e n c e o f 40mM N a C l Page 7 13 39 42 43 45 46 47 48 49 i x F i g . Page 11. E f f e c t o f e n d o g e n o u s l y bound c a r d i o l i p i n on 51 MAC a f f n i t y 12. F l o w s h e e t f o r t h e p u r i f i c a t i o n o f a c e t y l c h o l i n e s - 52 t e r a s e f r o m b o v i n e e r y t h r o c y t e g h o s t s 13. T h i n l a y e r c h r o m a t o g r a p h y o f f r e e z e d r i e d 53 e r y t h r o c y t e g h o s t s b e f o r e and a f t e r b u t a n o l t r e a t m e n t 14. A f f i n i t y p u r i f i c a t i o n o f a c e t y l c h o l i n e s t e r a s e 57 on PTA a f f i n i t y c o l u m n 15. S e d i m e n t a t i o n p r o f i l e o f a c e t y l c h o l i n e s t e r a s e 59 i n l o w i o n i c s t r e n g t h m e d i a 16. S e d i m e n t a t i o n p r o f i l e o f a c e t y l c h o l i n e s t e r a s e 60 i n t h e p r e s e n c e o f 0.IM s o d i u m c h l o r i d e , 20mM s o d i u m p h o s p h a t e ACKNOWLEDGEMENTS I wish to express my g r a t i t u d e to Dr. B.D. Ro u f o g a l i s f o r h i s e x c e l l e n t guidance and a s s i s t a n c e throughout! the course of t h i s work and t o Dr. G. Webb f o r h i s many h e l p f u l suggestions and d i s c u s s i o n s at va r i o u s stages of t h i s study. 1 INTRODUCTION A c e t y l c h o l i n e s t e r a s e (AChE) p l a y s a c r i t i c a l r o l e i n t e r m i n a t i n g t h e a c t i o n o f t h e n e u r o t r a n s m i t t e r , a c e t y l c h o l i n e ^ l i b e r a t e d a t c h o l i n e r g i c s y n a p s e s ( 1 , 2 ) . R e c e n t l y , t h i s enzyme has a l s o b e e n c o n s i d e r e d t o be a p a r t o f t h e b a s i c e x c i t a t i o n u n i t o r " g a t e w a y " r e g u l a t i n g i o n f l o w ( 3 ) . AChE has b;een i s o l a t e d f r o m a w i d e v a r i e t y o f s o u r c e s , i n c l u d i n g m u s c l e , n e r v e , e l e c t r i c o r g a n , b r a i n , e r y t h r o c y t e s and o t h e r s ( 4 , 1 0 3 ) . B e c a u s e o f t h e l a r g e s c a l e a v a i l a b i l i t y o f t h i s enzyme i n t h e e l e c t r i c o r g a n o f E l e c t r o p h o r o u s r . e l e c t r i c u s ( E e l ) and T o r p e d o ma rmor a t a, mo s t work h a s been done on':the enzyme i s o l a t e d f r o m t h e s e s o u r c e s ( 5 - 8 ) . The l a c k o f c o m p l e x i n t r a c e l l u l a r o r g a n i s a t i o n i n e r y t h r o c y t e s makesithem an i d e a l s y s t e m f o r p l a s m a membrane s t u d i e s . E v i d e n c e a v a i l a b l e so f a r s u g g e s t s t h a t AChE i s a membrane bound enzyme i n e r y t h r o c y t e s ( 9 - 1 2 ) ; ' t h i s makes t h e enzyme s u i t a b l e f o r s t u d y i n g l i p i d - p r o t e i r i i n t e r a c t i o n . S i h o t a n g (13) f i r s t s u g g e s t e d t h e r e q u i r e m e n t of p h o s p h o l i p i d f o r t h e a c t i v i t y o f human e r y t h r o c y t e AChE. B a s e d on t h e i r s t u d i e s w i t h b o v i n e e r y t h r o c y t e AChE, B e a u r e g a r d and R o u f o g a l i s ( 1 4 , 1 5 ) p o s t u l a t e d t h a t t i g h t l y bound c a r d i o l i p i n was i n v o l v e d i n m o d u l a t i n g t h e a c t i v i t y of b o v i n e e r y t h r o c y t e AChE. The s e p a r a t i o n o f c a r d i o l i p i n f r o m AChE (14,15) was a c h i e v e d by t r e a t m e n t o f t h e enzyme w i t h " h i g h s a l t " (1.8M s o d i u m c h l o r i d e 2 and 2mM p h o s p h a t e ) " o r IM ammonium h y d r o x i d e , f o l l o w e d by c h l o r o f o r m - m e t h a n o l e x t r a c t i o n . The a b o v e m e n t i o n e d c o n d i t i o n s of e x t r a c t i o n l e d to enzyme d e n a t u r a t i o n . In o r d e r t o obtain a b e t t e r u n d e r s t a n d i n g ; of t h e r o l e o f c a r d i o l i p i n , i t i s e s s e n t i a l to s e p a r a t e c a r d i o l i p i n f r o m AChE under non d e n a t u -r i n g c o n d i t i o n s . I n the p r e s e n t s t u d y t h e f o l l o w i n g methods f o r r e m o v a l o f c a r d i o l i p i n w i t h o u t d e n a t u r i n g the enzyme were examined. 1. " F l o t a t i o n " method: T h i s method i s b a s e d on the p r i n c i p l e t h a t d u r i n g c e n t r i f u g a t i o n i n a g r a d i e n t w i t h d i s i t i n c t d e n s i t y r e g i o n s , t h e l i g h t e r component c a r d i o l i p i n , w h ich has been p o s t u l a t e d to be d i s s o c i a t e d f r o m the enzyme i n IM sodium b i c a r a b o n a t e or 1.8M sodium c h l o r i d e and 2mM sodium p h o s p h a t e ( h i g h s a l t ) w i l l f l o a t to the t o p , w h i l e t h e h e a v i e r component, AChE, w i l l s t a y at t h e b o t t o m . A somewhat s i m i l a r t e c h n i q u e has been used by W a t k i n s e_t a l . (16) f o r l i p i d b i n d i n g s t u d i e s w i t h AChE. 2. G e l f i l t r a t i o n : Sigma AChE (0.5 mg/ml) was t r e a t e d w i t h " h i g h s a l t " , c o n d i t i o n s w h i c h were p o s t u l a t e d to d i s s o c i a t e the c a r d i o l i p i n from the enzyme, and p a s s e d t h r o u g h a Sephadex G-200 column e q u i l i b r a t e d w i t h " h i g h s a l t " . D u r i n g t h i s p r o c e s s , b e c a u s e of t h e l a r g e m o l e c u l a r s i z e of the p r o t e i n , i t w i l l a p p e a r i n t h e v o i d volume, w h i l e c a r d i o l i p i n b e i n g much s m a l l e r w i l l be r e t a r d e d by t h e column. The a v a i l a b i l i t y of the d e l i p i d a t e d p r e p a r a t i o n of the enzyme would a l l o w e x a m i n a t i o n of t h e f o l l o w i n g a s p e c t s of t h e l i p i d - p r o t e i n i n t e r a c t i o n i n AChE: 3 (a) S p e c i f i c i t y o f t h e c a r d i o l i p i n f o r t h e e n z y m a t i c a c t i v i t y . (b) Minimum number o f p h o s p h o l i p i d m o l e c u l e s e s s e n t i a l f o r t h e e n z y m a t i c a c t i v i t y . ( c ) M e c h a n i s m r e s p o n s i b l e f o r t h e b r e a k i n t h e A r r h e n i u s p l o t ; w h e t h e r i t i s due t o t h e a l t e r a t i o n i n t h e f l u i d i t y o f t h e f a t t y a c y l c h a i n o r due t o a l t e r a t i o n i n t h e c o n f i g u r a t i o n o f c a r d i o l i p i n f r o m h e x a g o n a l (Hj^) t o b i l a y e r p h a s e ( 1 7 ) . 4 LITERATURE REVIEW I s o l a t i o n and P u r i f i c a t i o n Of AChE T h e r e a r e c o n t r a d i c t o r y r e p o r t s r e g a r d i n g t h e b i n d i n g o f AChE t o membrane. Many w o r k e r s c o n s i d e r ' AChE t o be an i n t e g r a l membrane p r o t e i n o f t h e e r y t h r o c y t e ( 9 - 1 2 ) . The enzyme can be r e l e a s e d f r o m human e r y t h r o c y t e g h o s t s > b y ; d e t e r g e n t s ( 1 1 , 1 2 ) o r h i g h s a l t t r e a t m e n t ( 9 , 1 2 ) , b u t h i g h s a l t t r e a t m -e n t d o e s n ' t r e l e a s e t h e enzyme f r o m i n t a c t human e r y t h r o c y t e ( 1 0 ) . The above d i f f e r e n c e c a n be a t t r i b u t e d t o an a l t e r a t i o n i n membrane s t r u c t u r e r e s u l t i n g f r o m h e m o l y s i s . B o v i n e e r y t h r o c y t e AChE a p p e a r s t o be l e s s t i g h t l y bound t o t h e membrane as i t can a l s o be r e l e a s e d f r o m t h e membrane i n a l i p o p r o t e i n f o r m i n h y p o - o s m o t i c m e d i a ( 1 8 ) . O t h e r methods f o r t h e e x t r a c t i o n o f e r y t h r o c y t e AChE h a v e a l s o b e e n u s e d r e c e n t l y . I n c u b a t i o n o f p h o s p h a t i d y l c h o l i n e v e s i c l e s w i t h i n t a c t human e r y t h r o c y t e s r e s u l t s i n t h e e x t a c t i o n o f more t h a n 80% o f t h e AChE w i t h few o t h e r o u t e r membrane p r o t e i n s ( 2 0 ) . T r e a t m e n t o f t h e i n t a c t ox e r y t h r o c y t e w i t h 10 u n i t s / m l o f p h o s p h a t i d y l i n o s i t o l - s p e c i f i c p h o s p h o l i p a s e C c a u s e s a l m o s t t h e c o m p l e t e r e l e a s e o f AChE ( 2 1 ) , i n d i c a t i n g that p h o s p h a t i d y l i n o s i t o l may be i n v o l v e d i n t h e b i n d i n g o f AChE t o t h e membrane. The i n t r o d u c t i o n o f a f f i n i t y c h r o m a t o g r a p h y f o r t h e p u r i f i c a t i o n o f AChE ( 7 , 1 9 , 2 5 ) h a s c o m p l e t e l y e c l i p s e d more 5 c o n v e n t i o n a l m e t h o d s , s u c h as ammonium s u l f a t e p r e c i p i t a t i o n ( 2 4 ) , g e l c h r o m a t o g r a p h y (23) and i o n e x c h a n g e c h r o m a t o g r a p h y (22) as a method o f c h o i c e f o r t h e p u r i f i c a t i o n o f t h i s enzyme. Berman and Young (25) p u r i f i e d t h e e e l and b o v i n e e r y t h r o c y t e AChE t o a s p e c i f i c a c t i v i t y o f 10,000 and 3,500 u n i t s , r e s p e c t i -v e l y , u s i n g N - t r i m e t h y l ( a m i n o p h e n y l ) a m m o n i u m (PTA) d e r i v a t i v e as an a f f i n i t y l i g a n d . F o r optimum r e t e n t i o n t h e two enzymes r e q u i r e d i f f e r e n t i s o m e r s ; w h i l e t h e e e l enzyme i s o p t i m a l l y r e t a i n e d w i t h t h e p a r a - a m i n o i s o m e r , b o v i n e AChE r e q u i r e s t h e meta- i s o m e r f o r r e t e n t i o n . N i d a y e_t £il_. (19) p u r i f i e d t h e human e r y t h r o c y t e AChE t o a s p e c i f i c a c t i v i t y o f 418 u n i t s / m g by d o u b l e c h r o m a t o g r a p h y on a PTA c o l u m n . A t e n - f o l d h i g h e r s p e c i f i c a c t i v i t y o f t h e AChE f r o m human e r y t h r o c y t e was r e p o r t e d by O t t and B r o d b e c k (26) ( 3 500-4000 u n i t s / m g ) u s i n g d e t e r g e n t s o l u b i l i s a t i o n and ammonium s u l f a t e p r e c i p i t a t i o n . T h i s h i g h s p e c i f i c a c t i v i t y was a t t r i b u t e d by N i d a y e_t a l . (19) t o a t e n - f o l d u n d e r e s t i m a t i o n of p r o t e i n due t o t h e p r e s e n c e o f T r i t o n X-100. The m a i n d r a w b a c k s of t h e PTA l i g a n d a r e i t s l o w a f f i n i t y f o r AChE a t h i g h i o n i c s t r e n g t h (28) and a h i g h i o n i c s t r e n g t h d e p e n d e n c e o f t h e a f f i n i t y of t h e l i g a n d - f o r AChE ( 2 7 ) . The use of N - m e t h y l a c r i d i n i u m (MAC) as an a f f i n i t y l i g a n d ( 2 8 ) o v e r c a m e t h e above p r o b l e m s w i t h e e l AChE. R e c e n t l y , R e a v i l l and Plummer (29) h a v e a l s o r e p o r t e d t h e s u i . t a . b i . l i t y o f t h i s l i g a n d f o r t h e p u r i f i c a t i o n o f p i g b r a i n AChE. I n t h e p r e s e n t 6 s t u d y t h e use o f MAC f o r t h e p u r i f i c a t i o n o f b o v i n e e r y t h r o c y t e AChE has been i n v e s t i g a t e d . AChE S t r u c t u r e Most o f t h e p r e s e n t k n o w l e d g e o f AChE s t r u c t u r e i s b a s e d on t h e enzyme o b t a i n e d f r o m e e l e l e c t r i c o r g a n . A f r e s h s o l u b i l i -s e d p r e p a r a t i o n o f e e l AChE c o n s i s t s o f a m i x t u r e o f t h r e e f o r m s o f t h e enzyme ( 6 ) , t e r m e d A, C and D, w i t h s e d i m e n t a t i o n c o e f f i c i e n t s o f 9.2S, 14.2S and 18.4S. The a s y m m e t r i c n a t u r e o f t h e t h r e e f o r m s were shown by e l e c t r o n m i c r o s c o p y (6) and c o l u m n c h r o m a t o g r a p h y ( 3 0 ) . I n R o s e n b e r r y 1 s m o d e l ( f i g . 1, i n w h i c h i s shown t h e D f o r m ) t h e A, C and D f o r m s c o n s i s t o f one, two o r t h r e e t e t r a m e r s , r e s p e c t i v e l y , a t t a c h e d t o g e t h e r by a c o l l a g e n l i k e t a i l ( 8 ) . The t a i l h a s b een i m p l i c a t e d i n b i n d i n g t o t h e s y n a p t i c c l e f t (31) and i n a g g r e g a t i o n ( 3 2 ) . The a p p e a r a n c e o f t h e m u l t i p l e m o l e c u l a r f o r m s o f AChE has been shown i n enzymes o b t a i n e d f r o m i ' O t h e r s o u r c e s s u c h as r a t d i a p h r a g m ( 1 3 3 ) , mammalian b r a i n ( 3 3,120) and e r y t h r o c y t e s ( 3 4 ) . I n r a t b r a i n (33) t h e o r i g i n o f m u l t i p l e m o l e c u l a r f o r m o f AChE was f o u n d t o depend on t h e i o n i c s t r e n g t h o f t h e m i c r o e n v i o r n m e n t . O t t and B r o d b e c k (34) showed t h a t t h e s e d i m e n t a t i o n p r o f i l e o f t h e human e r y t h r o c y t e AChE depended on t h e c o n d i t i o n o f s t u d y . I n t h e p r e s e n c e o f T r i t o n X-100 a s i n g l e m o l e c u l a r f o r m w i t h a s e d i m e n t a t i o n c o e f f i c i e n t o f 6.3S i s o b t a i n e d , w h i l e i n t h e a b s e n c e o f d e t e r g e n t f i v e f o r m s of t h e enzyme v a r y i n g f r o m 6.3S t o 16S were o b s e r v e d . T r i t o n X-100 i s v e r y o f t e n u s e d i n t h e e x t r a c t i o n o f F i g . 1. Model of the 18S a s y m m e t r i c form o f e e l e l e c t r i c o r g a n AChE as p r o p o s e d by R o s e n b e r r y and R i c h a r d s o n ( 8 ) . 8 e r y t h r o c y t e AChE and t h e f a c t t h a t T r i t o n X-100 c a n c a u s e d i s s o c i a t i o n o f m u l t i p l e m o l e c u l a r f o r m s i n t o d i m e r s i n v i t r o , l e a v e s t h e q u e s t i o n o f t h e n a t u r e of t h e n a t i v e f o r m o f AChE i n e r y t h r o c y t e membrane u n a n s w e r e d . The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e a p p e a r a n c e o f v a r i o u s m u l t i m e r i c f o r m s o f AChE i s unknown. A p p e a r a n c e o f a new m o l e c u l a r f o r m o f t h e enzyme i n r e c t a l t i s s u e was s e e n u n d e r t h e c o n d i t i o n o f H i r s p r u g ' s d i s e a s e ( 3 5 ) . I n b i r d s and mammals, t h e a s y m m e t r i c 16S f o r m l o c a l i s e d i n m u s c l e was f o u n d t o d i s a p p e a r on d e n e r v a t i o n ( 3 6 ) . D i f f e r e n t m o l e c u l a r f o r m s o f E l e c t r o p h o r u s , T o r p e d o , r a t and c h i c k e n were f o u n d t o be c a t a l y t i c a l l y e q u i v a l e n t ( 3 7 ) . S i m i l a r t u r n o v e r r a t e s f o r g l o b u l a r and a s y m m e t r i c f o r m s o f the- e e l enzyme (37) s u g g e s t s t h a t q u a t e r n a r y i n t e r a c t i o n s among t h e c a t a l y t i c s u b u n i t s or w i t h t h e t a i l do n o t i n f l u e n c e t h e c a t a l y t i c e f f i c i e n c y . The c a t a l y t i c domain was f o u n d t o be r e l a t i v e l y i n s e n s i t i v e t o t h e t e r t i a r y s t r u c t u r e of t h e enzyme. S u b u n i t s t r u c t u r e o f e r y t h r o c y t e AChE: Berman and Young (25) r e p o r t e d t h e d i s s o c i a t i o n o f a p a r t i a l l y p u r i f i e d b o v i n e AChE o f a p p a r e n t m o l e c u l a r w e i g h t 200,000 t o two u n e q u a l s u b u n i t s o f m o l e c u l a r w e i g h t 126,000 and 75,000 on SDS g e l e l e c t r o p h o r e s i s . B o t h of t h e s e s u b u n i t s c o n t a i n e d one c a t a l y t i c a c t i v e s i t e . S h a f a i and C o r t n e r (39) h a v e a l s o r e p o r t e d t h e p r e s e n c e o f two d i s s i m i l a r <j( and *|i s u b u n i t s f r o m e e l AGhE. E x c e p t f o r t h e above two r e p o r t s , most of t h e o t h e r f i n d i n g s ( 1 9 , 4 0 , 1 3 2 ) s u p p o r t t h e p r e s e n c e o f e q u i v a l e n t s u b u n i t s i n v a r i o u s m o l e c u l a r f o r m s o f t h e enzyme. B e l l h o r n e t a l . (40) and N i d a y je_t _ a l . (19) f o u n d t h e m o l e c u l a r w e i g h t o f human e r y t h r o c y t e AChE on SDS g e l e l e c t r o p h o r e s i s t o be 180,000 and 1 6 0 , 0 0 0 , r e s p e c t i v e l y , w h i c h d i s s o c i a t e d i n t o two e q u a l s u b u n i t s o f 90,000 and 80,000 i n SDS and m e r c a p t o e t h a n o l . N i d a y e_t a_l. (19) and 0 f t ' e.t a l . (34). reported t h e human e r y t h r o c y t e enzyme t o be g l y c o p r o t e i n i n n a t u r e . The l i p o p r o t e i n f o r m o f t h e b o v i n e e r y t h r o c y t e enzyme o b t a i n e d by L u b r o l WX s o l u b i l i s -a t i o n (15) was f o u n d t o h a v e a m o l e c u l a r w e i g h t o f 156,000 w i t h a s e d i m e n t a t i o n c o e f f i c i e n t o f 7.3S and S t o k e s r a d i u s o f 6 nm. I n summary, e r y t h r o c y t e AChE a p p e a r s t o be a t a i l l e s s d i m e r o f m o l e c u l a r w e i g h t b e t w e e n 142,000 and 200,000 w i t h a s e d i m e n t a t i o n c o e f f i c i e n t o f 6.3-7.3S, w h i c h can f u r t h e r a g g r e -g a t e a t low i o n i c s t r e n g t h t o l e s s s y m m e t r i c f o r m s o f h i g h e r m o l e c u l a r w e i g h t . L i p i d - P r o t e i n I n t e r a c t i o n s I n AChE AChE i s embedded i n t h e membrane and i t c a t a l y s e s a c e t y l c h o l i n e h y d r o l y s i s on t h e e x t e r n a l s u r f a c e o f t h e p l a s m a membrane ( 9 , 1 0 ) . The e x t e n t o f i t s membrane a s s o c i a t i o n i s n o t c l e a r . R e l e a s e o f b o v i n e e r y t h r o c y t e AChE by h y p o t o n i c s h o c k (15) o r by e x t r a c t i o n w i t h h i g h s a l t f r o m human e r y t h r o c y t e g h o s t s ( 9 , 1 2 ) s u g g e s t s t h a t i t i s an e x t r i n s i c membrane p r o t e i n . H owever, o t h e r f i n d i n g s , s u c h as t h e r e q u i r e m e n t o f d e t e r g e n t f o r e x t r a c t i o n of. AChE f r o m i n t a c t human e r y t h r o c y t e s ( 1 0 ) , 10 t h e r e l e a s e o f AChE i n l i p o p r o t e i n f o r m ( 1 2 , 1 3 , 1 5 , 1 9 ) , e x t r a c t i o n o f AChE by p h o s p h a t i d y l c h o l i n e v e s i c l e s (20) and t h e r e l e a s e o f t h e enzyme by t r e a t m e n t w i t h p h o s p h a t i d y 1 i n o s i t o l s p e c i f i c p h o s p h o l i p a s e C (21) s u g g e s t t h a t AChE i s an i n t e g r a l membrane p r o t e i n . The p r e s e n c e o f " t i g h t l y a s s o c i a t e d " l i p i d w i t h mammalian e r y t h r o c y t e AChE has b e e n p r o p o s e d by v a r i o u s w o r k e r s ( 1 3 - 1 5 ) . S i h o t a n g (13) f o u n d t h a t t h e maximum s p e c i f i c a c t i v i t y o f t h e r e l e a s e d AChE f r o m human e r y t h r o c y t e was o b t a i n e d a t a d e o x y c h o l a t e c o n c e n t r a t i o n o f 60mM; f u r t h e r i n c r e a s e i n d e o x y c h o l a t e c o n c e n t r a t i o n r e s u l t e d i n d e c r e a s e d e n z y m a t i c a c t i v i t y a c c o m p a n i e d by a s i m u l t a n e o u s d e c r e a s e i n t h e p h o s p h o -l i p i d c o n c e n t r a t i o n . A t maximum s p e c i f i c a c t i v i t y t h e p h o s p h o l i p i d c o n t e n t o f t h e enzyme was f o u n d t o be 0.3+0.1 ug/ug o f p r o t e i n . The d e l i p i d a t e d enzyme p r e p a r a t i o n o b t a i n e d a t a h i g h e r d e o x y c h o l a t e c o n c e n t r a t i o n can be r e a c t i v a t e d by a d d i n g b a c k membrane l i p i d s o r p h o s p h a t i d y l s e r i n e ( 1 3 ) . I n b o v i n e e r y t h r o c y t e AChE c a r d i o l i p i n was f o u n d t o be t i g h t l y bound t o t h e enzyme (14) and b a s e d on t h e f o l l o w i n g o b s e r v a t i o n s i t was s u g g e s t e d t o be i n v o l v e d i n t h e m o d u l a t i o n o f AChE a c t i v i t y , I f t h e A r r h e n i u s p l o t was done on " S i g m a " o r L u b r o l s o l u b i l i s e d b o v i n e e r y t h r o c y t e AChE i n d i s t i l l e d w a t e r , a b r e a k i n t h e A r r h e n i u s p l o t was o b s e r v e d ; u n d e r t h e s e c o n d i t i o n s c a r d i o l i p i n c a n n o t be e x t r a c t e d by c h l o r o f o r m - m e t h a n o 1 t r e a t m e n t ( 1 4 ) . T r e a t m e n t o f t h e enzyme w i t h 1.8M s o d i u m c h l o r i d e . ; , 2mM p h o s p h a t e pH 7.4 , IM s o d i u m b i c a r b o n a t e pH 8.0 or 0.7M s o d i u m p h o s p h a t e pH 7.4 a b o l i s h e d t h e A r r h e n i u s p l o t 11 b r e a k ; t h e s e c o n d i t i o n s a l s o made p o s s i b l e t h e e x t r a c t i o n o f c a r d i o l i p i n by c h l o r o f o r m - m e t h a n o l . From a s t u d y o f t h e e f f e c t o f e ndogenous c a r d i o l i p i n (43) on v a r i o u s s t e p s i n v o l v e d i n a c e t y l c h o l i n e h y d r o l y s i s , i t was p o s t u l a t e d t h a t c a r d i o l i p i n m o d u l a t e d t h e c o n f o r m a t i o n a l r e a r r a n g m e n t o f t h e e n z y m e - s u b s t r a t e c o m p l e x , w h i c h i s t h e r a t e d e t e r m i n i n g s t e p a t s u b s a t u r a t 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 ( 4 3 ) . The t i g h t l y bound c a r d i o l i p i n was p r o p o s e d t o be b u r i e d i n s i d e t h e t h r e e d i m e n s i o n a l s t r u c t u r e o f t h e enzyme, r a t h e r t h a n f o r m i n g an a n n u l a r r i n g as i n c y t o c h r o m e o x i d a s e ( 6 7 , 9 0 ) . T h i s was c o n c l u d e d f r o m t h e i n a c c e s s i b i l i t y o f t h e c a r d i o l i p i n t o pho spho 1 i p as e as w e l l as t h e f a i l u r e o f h i g h s a l t t r e a t m e n t t o a l t e r t h e S t o k e s r a d i u s ( 1 3 , 4 4 ) . Wiedmer et^ J L L . (45) h a v e r e c e n t l y s t u d i e d t h e i n c o r p o r a -t i o n o f b o v i n e and e e l AChE i n l i p i d m o n o l a y e r s . An i n c r e a s e i n s u r f a c e p r e s s u r e due t o i n c o r p o r a t i o n of- t h e enzyme was o b s e r v e d b o t h w i t h b o v i n e and e e l enzyme. T h i s i s c o n t r a r y t o e x p e c t a t i o n s , as e e l enzyme i s c o n s i d e r e d t o be a p e r i p h e r a l membrane p r o t e i n . On t h e o t h e r h a n d , i n c o r p o r a t i o n o f b o v i n e AChE was much l e s s t h a n e x p e c t e d f r o m i t s p r o p o s e d i n t e g r a l n a t u r e . The l o w e r i n c o r p o r a t i o n o f b o v i n e AChE was a t t r i b u t e d t o t h e a g g r e g a t i o n of t h e d e t e r g e n t - d e p l e t e d f o r m of t h e b o v i n e enzyme, t h u s m a s k i n g t h e h y d r o p h o b i c s i t e i n t h e l i p i d i n t e r a c t i o n . A n o t h e r u n a c c o u n t a b l e o b s e r v a t i o n was t h e p e n e t r a t i o n t o e q u a l e x t e n t by b o t h g l o b u l a r and a s y m m e t r i c f o r m s o f t h e e e l enzyme, t h u s m a k i n g t h e t a i l ; n o n e s s e n t i a l f o r l i p i d b i n d i n g . T h i s i s c o n t r a r y t o t h e f i n d i n g s o f W a t k i n s e_t a_l. (16) who showed t h e s p e c i f i c b i n d i n g o f 12 s p h i n g o m y e l i n t o t h e t a i l r e g i o n o f t h e a s y m m e t r i c f o r m . Membrane S t r u c t u r e The " f l u i d m o s a i c " model p r o p o s e d by S i n g e r and N i c o l s o n (46) r e g a r d s t h e b i o l o g i c a l membrane as a homogeneous two d i m e n s i o n a l f l u i d , w i t h g l o b u l a r p r o t e i n s f l o a t i n g on o r i n a " s e a of l i p i d " . The b a s i c a s s u m p t i o n i n h e r e n t i n t h e f l u i d m o s a i c m o d e l i s t h a t t h e r e i s l i t t l e o r no s p e c i f i c i n t e r m o l e c u l a r i n t e r a c t i o n s among membrane c o m p o n e n t s , w h i c h i s c o n t r a r y t o t h e p r e s e n t day c o n c e p t o f p r o t e i n - l i p i d o r p r o t e i n - p r o t e i n i n t e r a c t i o n s i n t h e membrane. The a b o v e l i m i t a t i o n was overcome i n t h e " p l a t e m o d e l " p r o p o s e d by J a i n and W h i t e ( 4 7 ) . A c c o r d i n g t o t h i s m o d e l t h e membrane i s a s p a t i a l and t e m p o r a l m o s a i c o f s m a l l s c a l e a g g r e g a t e d s y s t e m s , w here t h e i n d i v i d u a l p l a t e s o r i s l a n d p a t c h e s ( F i g . 2) a r e i n r e l a t i v e m o t i o n w i t h r e s p e c t t o e a c h o t h e r . T h i s m o d e l a c c o u n t s f o r t h e p r e s e n c e of " a n n u l a r l i p i d " as w e l l as t h e c o e x i s t a n c e o f s e v e r a l p h a s e s i n t h e l i p i d b i l a y e r . Membrane p r o t e i n s a r e c l a s s i f i e d i n t o two o p e r a t i o n a l c a t e g o r i e s i ) p e r i p h e r a l p r o t e i n s and i i ) i n t e g r a l p r o t e i n s ( 4 8 ) . P e r i p h e r a l p r o t e i n s a r e t h o s e t h a t a p p e a r t o be o n l y s u p e r f i c i -a l l y bound t o t h e membrane, and t h e i r a t t a c h m e n t t o membrane i s b r o u g h t a b o u t by i n t e r a c t i o n w i t h i n t e g r a l membrane p r o t e i n s r a t h e r t h a n t o membrane l i p i d s ( 4 8 ) . The r e s u l t i s a weak i n t e r a c t i o n w h i c h can be d i s r u p t e d by r e l a t i v e l y g e n t l e t e c h n i q u e s , s u c h as s o n i c a t i o n o r a l t e r a t i o n i n i o n i c s t r e n g t h . 13 F i g . 2 . P l a t e m o d el o f b i o l o g i c a l membrane as p r o p o s e d by J a i n and W h i t e ( 4 7 ) a) O r g a n i z e d l i p i d m o l e c u l e s may f o r m d i s c r e e t p l a t e s ( w h i t e c i r c l e s ) t h a t a r e s e p a r a t e d f r o m e a c h o t h e r by r e g i o n s o f r e l a t i v e l y d i s o r g a n i z e d l i p i d s ( h a t c h e d c i r c l e s ) b) The o r g a n i z e d and d i s o r g a n i z e d r e g i o n s a r e v i e w e d as p l a t e s , e a c h h a v i n g c h a r a c t e r i s t i c - s y s t e m p r o p e r t i e s s p e c i f i e d by i t s c o m p o n e n t s . c) V a r i o u s m o l e c u l e s ( w i t h i n t h e b i l a y e r of biomembrane) i n t e r a c t h y d r o p h o b i c a l l y and a r e d i s t r i b u t e d asymm-e t r i c a l l y . 14 I n t e g r a l membrane p r o t e i n s on t h e o t h e r hand a r e more s t r o n g l y bound to the membrane by d i r e c t h y d r o p h o b i c and i o n i c i n t e r a c t i o n s w i t h membrane l i p i d s . These p r o t e i n s r e q u i r e more d r a s t i c c o n d i t i o n s , such as d e t e r g e n t s or o r g a n i c s o l v e n t s , f o r t h e i r s o l u b i l i s a t i o n . T a b l e 1 shows some of the b a s i c d i f f e r e n c e s i n p r o p e r t i e s between p e r i p h e r a l ' arid'i i n t e g r a l membrane p r o t e i n s . T a b l e 1 C r i t e r i a f o r d i s t i n g u i s h i n g p e r i p h e r a l and i n t e g r a l membrane p r o t e i n s P r o p e r t y P e r i p h e r a l p r o t e i n I n t e g r a l p r o t e i n R e q u i r e m e n t s f o r ' d i s s o c i a t i o n from membrane M i l d t r e a t m e n t s s u f f i c i e n t : h i g h i o n i c s t r e n g t h , m e t a l i o n c h e l a t i n g a g e n t s H y d r o p h o b i c bond -b r e a k i n g a g e n t s r e q u i r e d : d e t e r g e n t s , o r g a n i c s o l v e n t s , c h a o t r o p i c a g e n t s . U s u a l l y a s s o c i a t e d w i t h l i p i d s when s o l u b i l i z e d U s u a l l y i n s o l u b l e or a g g r e g a t e d i n n e u t r a l aqueous buf f e r s A s s o c i a t i o n w i t h l i p i d s when s o l u b i l i z e d S o l u b i l i t y a f t e r d i s s o c i a t i o n from memb rane U s u a l l y s o l u b l e , f r e e of l i p i d s S o l u b l e and m o l e c u -l a r l y d i s p e r s e d i n n e u t r a l aqueous b u f f e r s 15 T h e r m a l P h a s e T r a n s i t i o n And P h a s e S e p a r a t i o n I n Membrane L i p i d s L i p i d s a r e c a p a b l e o f e x i s t i n g i n two p h a s e s a) s o l i d g e l p h a s e and b) l i q u i d c r y s t a l l i n e p h a s e . I n t h e g e l p h a s e , f a t t y a c y l c h a i n s a r e a r r a n g e d i n a l l - t r a n s c o n f o r m a t i o n s ( 4 9 , 1 1 4 ) , w h i l e i n t h e l i q u i d c r y s t a l l i n e p h a s e t h e y show a g r e a t e r d e g r e e o f m o b i l i t y ( 4 9 , 5 2 , 1 1 4 ) . C o n v e r s i o n f r o m g e l t o l i q u i d c r y s t a l l i n e s t a t e i s a c c o m p a n i e d by l a t e r a l e x p a n s i o n and a l t e r a t i o n i n p e r m e a b i l i t y , c o m p r e s s i b i l i t y , p a c k i n g d e n s i t y and t h i c k n e s s o f t h e b i l a y e r , ( 1 0 5 ) . P h a s e t r a n s i t i o n s h a v e b e e n s t u d i e d i n many p u r e p h o s p h o l i p i d s y s t e m s ( 5 2 ) . I t h a s b e e n f o u n d t h a t u n l i k e " c o n v e n t i o n a l m e l t i n g p o i n t s " w h i c h a r e d i s c o n t i n u o u s , t h e g e l t o l i q u i d c r y s t a l l i n e p h a s e t r a n s i t i o n i s c o n t i n u o u s . I n " m e l t i n g " t h e s o l i d and l i q u i d p h a s e c o e x i s t i n e q u i l i b r i u m o n l y / a t a s i n g l e t e m p e r a t u r e c a l l e d t h e m e l t i n g p o i n t , b u t i n a c o n t i n u o u s t r a n s i t i o n , t h e e q u i l i b r i u m o c c u r s o v e r a w i d e r a n g e of t e m p e r a t u r e e.g. 1°C i n p u r e b i l a y e r ' - o f d i m y r i s -t oy 1-.-ph o sph a t i dy I c h o 1 i n e ( 109 ) .In a ..system c o n s i s t i n g o f a m i x t u r e o f l i p i d , s u c h as n a t u r a l l y o c c u r i n g membrane, t h e t r a n s i t i o n i s much b r o a d e r ( 1 1 4 ) . The t r a n s i t i o n t e m p e r a t u r e of a p u r e p h o s p h o l i p i d i s d e t e r m i n e d b o t h by t h e s i z e and n a t u r e o f t h e head g r o u p and t h e f a t t y a c y l c h a i n ( 5 2 , 1 1 4 ) . S i m i l a r t o m e l t i n g p o i n t o f an o r g a n i c compound, t r a n s i t i o n t e m p e r a t u r e i n c r e a s e s w i t h c h a i n l e n g t h and d e c r e a s e s w i t h t h e i n c o r p o r a t i o n o f a d o u b l e bond ( 1 1 0 , 1 1 4 ) . S i m i l a r t o o t h e r m e l t i n g p r o c e s s e s , p r e and p o s t t r a n s i t i o n 16 phenomenon a r e a s s o c i a t e d w i t h t h e p h a s e t r a n s i t i o n ( 5 3 ) . A t a l l t e m p e r a t u r e s above z e r o a s o l i d has d e f e c t s ; t h e number o f d e f e c t s i n c r e a s e s w i t h i n c r e a s i n g e n t r o p y , w h i c h i s a f u n c t i o n o f t e m p e r a t u r e . The d e f e c t s c a n be s i m p l e v a c a n t s i t e s o r c o m p l e x a r r a n g m e n t s , s u c h as g r a i n b o u n d a r i e s . A g r a i n b o u n d a r y i s t h e b o u n d a r y b e t w e e n two c r y s t a l s w i t h s l i g h t l y d i f f e r e n t o r i e n t a t i o n . The " d e f e c t s t r u c t u r e " f o r m e d d u r i n g p r e t r a n s i t i o n p r o v i d e s a s i m p l e e x p l a n a t i o n f o r g e l t o l i q u i d c r y s t a l l i n e p h a s e t r a n s i t i o n ( 5 3 ) . As t h e t e m p e r a t u r e a p p r o a c h e s t h a t o f t h e t r a n s i t i o n , s m a l l random c l o s e p a c k e d r e g i o n s of l i p i d w i l l f o r m t h r o u g h o u t t h e r e l a t i v e l y p e r f e c t g e l l a t t i c e . The l i q u i d c r y s t a l l i n e p h a s e w i l l be c o n t a i n e d i n s m a l l i s l a n d s o r domains so t h a t l o n g r a n g e o r d e r w i l l be r e t a i n e d i n t h e b i l a y e r . As t h e t e m p e r a t u r e i n c r e a s e s , t h e a r e a o c c u p i e d by t h e s e r e g i o n s w i l l grow, u n t i l a t some p o i n t t h e y w i l l c o a l e s c e . A t t h i s p o i n t , t h e c o n t i n u i t y of t h e g e l r e g i o n i s r e p l a c e d by c o n t i n u i t y of t h e l i q u i d c r y s t a l l i n e r e g i o n , w i t h t h e c o n s e q u e n t l o s s of r i g i d i t y a s s o c i a t e d w i t h t h e g e l p h a s e . The t e m p e r a t u r e a t w h i c h t h e c o n t i n u i t y of g e l p h a s e i s r e p l a c e d by l i q u i d c r y s t a l l i n e p h a s e i s c a l l e d t h e t r a n s i t i o n t e m p e r a t u r e (% c) (52) The g e l t o l i q u i d c r y s t a l l i n e p h a s e t r a n s i t i o n e x h i b i t s t h e phenomenon o f h y s t e r e s i s , s i n c e t h e t r a n s i t i o n f r o m g e l t o l i q u i d c r y s t a l l i n e s t a t e i s n o t i d e n t i c a l t o t h a t f r o m t h e c r y s t a l l i n e t o g e l s t a t e ( 5 4 ) . P h a s e S e p a r a t i o n : B i n a r y l e c i t h i n m i x t u r e s h a v i n g c o m ponents w h i c h v a r y by o n l y two c a r b o n a t o m s , s u c h as d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e and d i p a l m i t o y l p h o s p h a t i d y l -c h o l i n e , u s u a l l y c o c r y s t a l l i s e b e l o w t h e t r a n s i t i o n t e m p e r a t u r e ( 1 1 5 ) . H owever, when t h e p h o s p h o l i p i d s a r e s t r u c t u r a l l y so d i f f e r e n t t h a t p a c k i n g p r o b l e m s a r i s e , l a t e r a l p h a s e s e p a r a t i o n and c r y s t a l l i s a t i o n of t h e i n d i v i d u a l p h o s p h o l i p i d s w i l l o c c u r ( s o l i d p h a s e i m m i s c i b i l i t y ) . Such a phenomenon has b e e n d e m o n s t r a t e d i n model membranes c o n t a i n i n g a s p i n l a b e l l e d s t e r o i d d e r i v a t i v e ( 5 0 ) . A " v e r t i c a l " p h a s e s e p a r a t i o n may a l s o o c c u r b e l o w T >• by p r e f e r e n t i a l l y p a r t i t i o n i n g b a c k i n t o t h e w a t e r phase ( 5 1 ) . The s e p a r a t i o n o c c u r r i n g above T c ( f l u i d / f l u i d p h ase s e p a r a t i o n ) i s of g r e a t e r b i o l o g i c a l s i g n i f i c a n c e . L i m i t e d m i s c i b i l f t y a b o v e T c i s t y p i c a l l y o b s e r v e d f o r m i x t u r e s of p h o s p h o l i p i d s and n o n h y d r a t i n g l i p i d s , s u c h ; a s t r i g l y c e r i d e s o r c h o l e s t e r o l e s t e r s . A l a t e r a l p h a s e s e p a r a t i o n above has a l s o b e e n d e s c r i b e d f o r t h e b i n a r y m i x t u r e s of d i p a l m i t o y l p h o s -p h a t i d y l e t h a n o l a m i n e and d i e l a i d o y l p h o s p h a t i d y l c h o l i n e ( 5 5 ) . F a c t o r s e f f e c t i n g p h a s e t r a n s i t i o n and p h a s e s e p a r a t i o n : I f t h e p h a s e t r a n s i t i o n and p h a s e s e p a r a t i o n were p r o d u c e d o n l y t h r o u g h c h a n g e s i n t e m p e r a t u r e , i t may n o t be a b i o l o g i c a l l y i m p o r t a n t phenomenon. However, b o t h o f t h e above phenomenon c a n be p r o d u c e d by a l t e r i n g any of t h e f o l l o w i n g p a r a m e t e r s : a) C h o l e s t e r o l : E x c l u s i o n o f c h o l e s t e r o l f r o m t h e + 2 +2 " b o u n d a r y l i p i d " l a y e r o f (Mg +Ca )-ATPase h a s b e e n shown by W a r r e n et_ a _ l . ( 6 8 ) . R e c e n t s t u d i e s w i t h d i m y r i s t o y l l e c i t h i n and d i p a m i t o y l l e c i t h i n d i s p e r s i o n s i n d i c a t e t h a t t h e t r a n s i t -i o n e n t h a l p y d e c r e a s e s l i n e a r l y w i t h t h e i n c r e a s e i n 18 t h e p e r c e n t a g e of c h o l e s t e r o l and . v a n i s h e s a t 33 mole p e r c e n t c h o l e s t e r o l ( 5 6 ) . The a d d i t i o n of c h o l e s t e r o l a c t s as a p l a s t i c i z e r , a l l o w i n g b i l a y e r s t o e x i s t i n a s e m i f l u i d s t a t e o v e r a w i d e r a n g e o f t e m p e r a t u r e . C h o l e s t e r o l shows t h e f o l l o w i n g o r d e r of p r e f e r e n c e f o r p h o s p h o l i p i d h e a d g r o u p : s p h i n g o m y e l i n ^ p h o s p h a t i d y l c h o l i n e "y p h o s p h a t i d y 1 e t h a n o 1 a m i n e ( 1 1 9 ) . I n t h e c a s e of p h o s p h o l i p i d s w i t h , s i m i l a r h e a d g r o u p , c h o l e s t e r o l p r e f e r e n t i a l l y p a r t i t i o n s i n t o t h e p h o s p h o l i p i d h a v i n g more u n s a t u r a t e d - f a t t y a c y l c h a i n s (117). b) M e t a l l i c i o n s : A l t e r a t i o n i n t h e m e t a l i o n c o n c e n t r -a t i o n seems t o be one o f t h e most p r o m i s i n g ways f o r m o d u l a t i n g p hase t r a n s i t i o n i n b i o l o g i c a l membrane. A d d i t i o n o f d i v a l e n t + 2 c a t i o n s l i k e Ca t o u n i l a m e l l a r v e s i c l e s o f p h o s p h a t i d y l s e r i n e or p h o g ' p h a t i d y l g l y c e r o l g i v e s r i s e t o l a r g e p l a n a r a r r a y s t t h a t r o l l up t o f o r m c y l i n d e r s ( 5 7 ) , and i n c a r d i o l i p i n v e s i c l e s + 2 Ca c a u s e s c o n v e r s i o n f r o m t h e l a m e l l a r t o t h e h e x a g o n a l (Hjj) p hase ( 1 7 ) . I n c h a r g e d p h o s p h o l i p i d s t h e p h a s e t r a n s i t i o n i s s e n s i t i v e t o t h e i o n i s a t i o n o f t h e p o l a r g r o u p s . T h e r e f o r e t h e t r a n s i t i o n t e m p e r a t u r e s o f p h o s p h o l i p i d s s u c h as p h o s p h a t i d y l -c h o l i n e and p h o s p h a t i d y l e t h a n o l a m i n e were f o u n d t o be pH s ens i t i v e ( 5 2 ) . c) P r o t e i n / l i p i d r a t i o : P a p a h a d j o p o u l o s e_t a_l. ( 6 5 ) h a v e c l a s s i f i e d p r o t e i n s i n t o t h r e e c a t e g o r i e s on t h e b a s i s of how t h e y e f f e c t t h e t h e r m o t r o p i c t r a n s i t i o n i n l i p i d b i l a y e r s . i ) P r o t e i n s w h i c h i n c r e a s e e n t h a l p y , w h i l e t h e t r a n s i t i o n t e m p e r a t u r e i s i n c r e a s e d o r r e m a i n s u n a f f e c t e d e.g. r i b o n u c l e -ase and p o l y m y x i n . i i ) P r o t e i n s w h i c h d r a s t i c a l l y d e c r e a s e b o t h t r a n s i t i o n t e m p e r a t u r e and e n t h a l p y e.g. c y t o c h r o m e C and p r o t e i n s . i i i ) P r o t e i n s w h i c h d i d n o t e f f e c t t h e t r a n s i t i o n t e m p e r a t u r e b u t i n d u c e a l i n e a r d e c r e a s e i n e n t h a l p y e.g. a p o p r o t e i n and g r a m i c i d i n . B o u n d a r y L i p i d And O t h e r L i p i d s S t u d i e s on v a r i o u s membranes i n c l u d i n g t h o s e o f m y c o p l a s m a ( 7 2 ) , E . c o l i . ( 7 3 ) , m i t o c h o n d r i a (72) and s a r c o p l a s m i c r e t i c u l u m ( 5 1 ) , u s i n g a v a r i e t y o f t e c h n i q u e s s u c h as X - r a y s c a t t e r i n g ( 7 5 ) , e l e c t r o n s p i n r e s o n a n c e (ESR) ( 5 1 ) , and f l u o r e s c e n t p r o b e s ( 7 3 ) , have c l e a r l y i n d i c a t e d t h a t 20-30% o f l i p i d s i n t h e membrane a r e i m m o b i l i s e d by i n t e r a c t i o n w i t h p r o t e i n , w h i l e t h e r es t ( 70-80 %) o f t h e l i p i d s - a r e . m o b i l e o r f l u i d . - A n a l y s i s o f ESR s p e c t r a f r o m a s e r i e s o f m i x t u r e s c o n t a i n i n g v a r y i a u s l i p i d : c y t o c h r o m e o x i d a s e r a t i o s h ave been i n t e r p r e t e d i n t e r m s of two d i s t i n c t l i p i d e n v i r o n m e n t s f o r th e s p i n p r o b e ( 6 7 ) . H e s k e t h e_t _ a l . (66) h a v e gone a s t e p f u r t h e r and c l a s s i f i e d t h e b i l a y e r l i p i d s i n t o f o u r z o n e s , + 2 + 2 b a s e d on t h e e x t e n t o f p e r t u r b a t i o n by (Ca -Mg ) A T P a s e . O t h e r e x p e r i m e n t s w i t h c y t o c h r o m e o x i d a s e l e n d s u p p o r t to t h e c o n c e p t of boundary l i p i d . R emoving t h e l i p i d f r o m c y t o c h r o m e o x i d a s e - l i p i d c o m p l e x e s by a c e t o n e e x t r a c t i o n i s r e l a t i v e l y e a s y u n t i l t h e c o m p l e x i s r e d u c e d t o a b o u t 0.2 mg o f l i p i d / m g p r o t e i n . T h i s i s t h e amount o f l i p i d e s t i m a t e d t o be i n t h e b o u d a r y l a y e r ( 6 7 ) . Removing t h i s f i n a l amount of l i p i d , w h i c h . ' i s ' e n r i c h e d i n c a r d i o l i p i n (69) , r e s u l t s i n l o s s o f e n z y m a t i c a c t i v i t y and i n m o r p h o l o g i c a l c h a n g e s ( 7 6 ) . 20 The e s s e n t i a l r e q u i r e m e n t o f c a r d i o l i p i n f o r t h e a c t i v i t y 1 of: c y t o c h r o m e o x i d a s e s u g g e s t e d p r e v i o u s l y (67 ,76) h a s been r e c e n t l y r e f u t e d by W a t t s e _ t ' i L i ' (89) , who were a b l e t o r e t a i n t h e a c t i v i t y e v e n a f t e r s u b s t i t u t i o n o f most o f t h e c a r d i o l i p i n w i t h dimy r i s t o y l p ho s p h a t i d y l c h o l i n e -.- • The " b o u n d a r y l a y e r " o f l i p i d s u r r o u n d i n g t h e ' p r o t e i n b e h a v e s i n many ways a n a l o g o u s t o t h a t o f t h e w a t e r o f h y d r a t i o n ( 7 7 , 1 1 1 ) . The b o u n d a r y l i p i d d o e s n ' t u n d e r g o p h a s e t r a n s i t i o n (73) and i t s v i s c o s i t y i s i n t e r m e d i a t e t o t h a t o f g e l and l i q u i d c r y s t a l l i n e p hase ( 6 7 , 6 8 , 1 1 3 ) . The e q u i l i b r i u m b e t w e e n t h e m o b i l e and i m m o b i l e p h a s e was f o u n d to be t e m p e r a t u r e d e p e n d e n t . The c o n c e p t o f b o u n d a r y . l i p i d a p p e a r s t o be q u i t e a t t r a c t i v e , b u t e x c e p t f o r s t u d i e s w i t h ESR p r o b e s , most o f t h e e v i d e n c e i s i n d i r e c t and c i r c u m s t a n t i a l . A c r i t i c a l a s s u m p t i o n i n ESR s t u d i e s , whose v a l i d i t y c a n be q u e s t i o n e d , i s t h a t t h e b e h a v i o u r o f t h e s p i n p r o b e i s u n a l t e r e d by t h e p r e s e n c e o f p r o t e i n and r e m a i n s t h e same as i n p u r e p h o s p h o l i p i d b i l a y e r s . L i p i d M o d u l a t i o n Of Enzyme A c t i v i t y Many membrane bound enzymes, l i k e c y t o c h r o m e o x i d a s e (67 , 70) , ( N a + - K + ) ATPase ( 6 0 , 6 1 , 6 2 ) , ( C a + 2 - M g + 2 ) A T P a s e ( 5 9 ) , AChE (14 , 1 5 ) and o t h e r s a r e i s o l a t e d i n l i p o p r o t e i n f o r m and h a v e been f o u n d t o r e q u i r e l i p i d s f o r t h e i r e n z y m a t i c a c t i v i t y ( 6 1 ) . A c l a s s i c a l way t o s t u d y t h e l i p i d d e p e n d e n c e o f e n z y m a t i c a c t i v i t y i s t o o b s e r v e t h e l o s s o'f, a c t i v i t y 21 a c c o m p a n y i n g l i p i d d e p l e t i o n f o l l o w e d by r e c o v e r y d u r i n g r e c o n s t i t u t i o n ( 1 3 , 5 9 , 7 0 ) . The l i p i d m o d u l a t i o n o f e n z y m a t i c a c t i v i t y c o u l d a r i s e f r o m any o f t h e f o l l o w i n g m e c h a n i s m s : a) S o l u b i l i s a t i o n o f s u b s t r a t e : I n r e a c t i o n s i n v o l v i n g w a t e r i n s o l u b l e s u b s t r a t e s , l i k e l o n g c h a i n u b i q u i n o n e s o r p l a s m a q v i i n p n e s ' , t h e p r e s e n c e o f l i p i d may be r e q u i r e d f o r s o l u b i l i s a t i o n o f t h e s u b s t r a t e ( 4 9 ) . I n many o f t h o s e c a s e s i t i s p o s s i b l e t o s u b s t i t u t e d e t e r g e n t s f o r l i p i d s . b) P r o t e i n c o n f o r m a t i o n : One o f t h e m a i n f u n c t i o n s o f t h e l i p i d p h a s e i s t o a c t as an a n c h o r and m a i n t a i n t h e enzyme i n an a c t i v e c o n f o r m a t i o n ( 1 0 5 , 1 1 2 ) . T h e r e a r e many i n s t a n c e s where c h a n g e s i n t h e l i p i d b i l a y e r p h a s e i s r e f l e c t e d by t h e m o d i f i c a t i o n o f t h e e n z y m a t i c a c t i v i t y . The r e q u i r e m e n t of a f l u i d b i l a y e r f o r a c t i v i t y has been shown i n t h e c a s e o f + 2 +2 c y t o c h r o m e o x i d a s e (67) and (Ca -Mg ) A T P a s e (77) among o t h e r s . Some membrane t r a n s p o r t i n g p r o t e i n s show maximum a c t i v i t y a t t h e t r a n s i t i o n t e m p e r a t u r e , r a t h e r t h a n above o r b e l o w t r a n s i t i o n ( 7 1 ) . T h i s i s a t t r i b u t e d t o t h e g r e a t e r " p o r o s i t y " o f t h e membrane due t o t h e s i m u l t a n e o u s p r e s e n c e o f g e l and f l u i d p h a s e s ( 1 0 7 ) . c) P r o t e i n d i s t r i b u t i o n and a g g r e g a t i o n : C T P p h o s p h o c h o l i n e c y t i d y l t r a n s f e r a s e f r o m f e t a l r a t l u n g i s a c t i v a t e d by p h o s p h a t i d y l g l y c e r o l i n a c o n c e n t r a t i o n o f 4% by a g g r e g a t i o n o f t h e enzyme t o a h i g h e r m o l e c u l a r w e i g h t f o r m ( 5 8 ) . U s u a l l y most p r o t e i n s a r e r a n d o m l y d i s t r i b u t e d a bove t h e t r a n s i t i o n t e m p e r a t u r e ( 1 0 8 ) . L o w e r i n g o f t e m p e r a t u r e b e l o w t h e t r a n s i t i o n t e m p e r a t u r e r e s u l t s i n " f r e e z i n g o u t " o f t h e p r o t e i n f r o m t h e 22 n e w l y f o r m e d g e l r e g i o n , due t o f a v o u r a b l e p a r t i t i o n i n g o f p r o t e i n s i n t o t h e f l u i d r e g i o n (as s t a t e d e a r l i e r many p r o t e i n s r e q u i r e a l i p i d e n v i r o n m e n t f o r t h e i r a c t i v i t y ) The A r r h e n i u s P l o t : S i g n i f i c a n c e And L i m i t a t i o n s F u n c t i o n a l a c t i v i t i e s o f b i o l o g i c a l o r r e c o n s t i t u t e d enzyme o f t e n show b i o r t r i p h a s i c A r r h e n i u s p l o t s ( 1 1 3 ) . Non l i n e a r i t y i n t h e A r r h e n i u s p l o t c o u l d a r i s e due t o : a) A change i n t h e r a t e d e t e r m i n i n g s t e p of t h e e n z y m a t i c r e a c t i o n . b) A t e m p e r a t u r e d e p e n d e n t s h i f t o f e q u i l i b r i u m b e t w e e n two c o n f o m e r s . . c) Changes i n f a t t y a c y l c h a i n m o b i l i t y due t o p h a s e t r a n s i t i o n . T e m p e r a t u r e change c a n p r o d u c e two t y p e o f e f f e c t s i n t h e l i p i d b i l a y e r : a) I n t e r f a c i a l e f f e c t : L i p i d m o d u l a t i o n o f i s o p r e n o i d p h o s p h o k i n a s e a c t i v i t y h a s b e e n s u g g e s t e d t o be b r o u g h t a b o u t by t h i s m e c h a n i s m ( 7 9 ) . Sandermann (79) f o u n d t h a t l i p i d s w h i c h a r e c a p a b l e o f a c t i v a t i n g t h e above enzyme can' b i n d 3 2.5 t o 5.3 n m oles o f t^O p e r mole o f l i p i d , w h i l e t h e i n a c t i v e 3 c o f a c t o r l i p i d s can o n l y b i n d 0.14 n m oles of ^ O . The number o f w a t e r m o l e c u l e s bound t o t h e p h o s p h o l i p i d h e a d g r o u p i s c r i t i c a l i n d e t e r m i n i n g t h e p r o p e r t y a t t h e l i p o p r o t e i n / w a t e r i n t e r f a c e . b) V i s c o t r o p i c e f f e c t : . . T h e t r a n s i t i o n f r o m g e l t o l i q u i d 2 3 c r y s t a l l i n e phase b r o u g h t about by i n c r e a s e i n t e m p e r a t u r e i s a c c o m p a n i e d by a s i m u l t a n e o u s i n c r e a s e i n t h e m o b i l i t y o f the f a t t y a c y l c h a i n ( 5 2 ) , t h u s a l t e r i n g t h e v i s c o s i t y of the l i p i d p h a s e . V i s c o t r o p i c e f f e c t s have been s t u d i e d u s i n g s p i n l a b e l l e d p r o b e s ( 1 1 4 ) . A r r h e n i u s p l o t s a r e u s u a l l y o b t a i n e d by p l o t t i n g l o g r a t e v e r s u s t h e r e c i p r o c a l of a b s o l u t e t e m p e r a t u r e . d l o g k = - - 2 7 3 R -d(l/T) Most s o l u b l e enzymes w i l l y i e l d a s t r a i g h t l i n e , and i n t h o s e c a s e s a c t i v a t i o n e n e r g y of t h e r e a c t i o n i s c a l c u l a t e d f r o m the s l o p e . Many membrane bound enzymes, i n c l u d i n g a d e n y l a t e c y c l a s e ( 1 1 2 ) , ( C a + 2 - M g + 2 ) A T P a s e ( 6 3 ) , AChE (14,15,126) and ( N a + - K + ) ATPase (61,62,66) y i e l d e d a b i p h a s i c A r r h e n i u s p l o t . The A r r h e n i u s p l o t b r e a k has been a t t r i b u t e d to v a r i o u s c a u s e s , s u c h as phase t r a n s i t i o n ( 6 2 , 8 0 ) , phase s e p a r a t i o n (71,80,81) and c l u s t e r f o r m a t i o n ( 1 7 , 5 5 ) . In e a r l y s t u d i e s (113) t h e b i p h a s i c A r r h e n i u s p l o t was ".almost e x c l u s i v e l y i n t e r p r e t e d i n terms of phase t r a n s i t i o n s , b u t t h i s c a n ' t a c c o u n t f o r the d i f f e r e n c e s i n t h e b r e a k p o i n t i n A r r h e n i u s p l o t o f v a r i o u s enzymes o b t a i n e d f r o m t h e same membrane ( 6 4 ) . A n o t h e r l i m i t a t i o n i s t h a t t h e t r a n s i t i o n d o e s n ' t o c c u r at a s i n g l e t e m p e r a t u r e b u t o v e r a range of t e m p e r a t u r e . + 2 +2 The a c t i v i t y of s a r c o p l a s m i c r e t i c u l u m (Mg -Ca )A T P a s e shows a d i s c o n t i n u i t y i n t h e A r r h e n i u s p l o t a r o u n d 1 8 °C, even though d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y measurments 24 do 'not d e t e c t any p h a s e t r a n s i t i o n a t t h i s t e m p e r a t u r e ( 6 3 ) . S i l v i u s e t i . a l . ( l i s ) h a v e r e c e n t l y r e p o r t e d a t e m p e r a t u r e d e p e n d e n t v a r i a t o n i n b o t h V and K o f ( N a + - K + ) A T P a s e . max m T h i s i n t u r n w i l l g i v e r i s e t o an a r t i f a c t u a l b r e a k i n t h e A r r h e n i u s p l o t ( 1 1 8 ) . 5 ' - N u c l e o t i d a s e (116) i s a n o t h e r e x a m p l e where t h e p r e v i o u s l y c o n s i d e r e d 1 i p i d - d e p e n d e n t . A r r h e n i u s p l o t b r e a k was f o u n d t o be an i n t r i n s i c c h a r a c t e r i s t i c o f t h e p r o t e i n . The s h a r p change i n a c t i v a t i o n e n e r g y o b s e r v e d i n most o f t h e s y s t e m s s h o w i n g a b i p h a s i c A r r h e n i u s p l o t i s n o t a c c o m p a n i e d by a s i g n i f i c a n t change i n t h e r e a c t i o n r a t e (v) ( 7 1 ) . The change t h u s i s i n dV/dT and n o t i n V. T h i s b e h a v i o u r ( c h a n g e i n e n t h a l p y w i t h o u t s i g n i f i c a n t change i n r e a c t i o n r a t e ) i s d i f f i c u l t t o e x p l a i n s i n c e i t r e q u i r e s t h a t t h e change i n a c t i v a t i o n e n t h a l . p y be e x a c t l y c o m p e n s a t e d by a change i n a c t i v a t i o n e n t r o p y ( 7 1 ) . The m o l e c u l a r m e c h a n i s m u n d e r l y i n g b i o r t r i p h a s i c A r r h e n i u s p l o t s h a v e so f a r i n no c a s e been e l u c i d a t e d and t h e r e f o r e any i n t e r p r e t a t i o n o f A r r h e n i u s p l o t breaks has t o be c o n s i d e r e d w i t h r e s e r v a t i o n . 25 MATERIALS AND METHOD M a t e r i a l s : F r e s h b o v i n e b l o o d was o b t a i n e d f r o m 1 4 a l o c a l s l a u g h t e r h o u s e ( I n t e r C o n t i n e n t a l P a c k e r s ) ; ( I - C) A c e t y l c h o l i n e i o d i d e (1-5 C^/mole) was f r o m New E n g l a n d n u c l e a r . A c e t y l c h o l i n e p e r c h l o r a t e was f r o m BDH. S e p h a r o s e 2B was f r o m P h a r m a c i a . B u t a n o l ( s p e c t r a l q u a l i t y ) was f r o m F i s h e r S c i e n t i f i c . A c e t y l c h o l i n e i o d i d e , 5,5 1 - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) , d e c a m e t h o n i u m b r o m i d e , a c r i d i n e , g u a n i d i n e and i o d o m e t h a n e were p u r c h a s e d f r o m S i g m a . The 6 ami n o h . e x a n o y l d e r i v a t i v e o f N - m e t h y l a c r i d i n e and N(6 - a m i n o c a p r o y 1 - m - a m i h o p h e n y 1 ) t r i m e t h y l ammonium b r o m i d e h y d r o b r o m i d e w e r e g i f t s f r o m G e o f f r y Webb, D e p a r t m e n t o f C h e m i s t r y , UBC. A l l i n o r g a n i c s a l t s were o f a n a l y t i c a l r e a g e n t g r a d e . " F l o t a t i o n " t e c h n i q u e f o r c a r d i o l i p i n s e p a r a t i o n : A l l t h e s u c r o s e s o l u t i o n s were p r e p a r e d i n IM s o d i u m b i c a r b o n a t e (pH 8.0) i n t h e p r e s e n c e o r a b s e n c e o f 0.09% T r i t o n X-100. G r a d i e n t s were p r e p a r e d by l a y e r i n g t h e f o l l o w i n g s o l u t i o n s (w/v) o f s u c r o s e : 1 ml o f 60% s u c r o s e ( a c t s as a p l u g ) ; 1 ml of 0.5 mg/ml o f Sigma b o v i n e e r y t h r o c y t e AChE i n 30% s u c r o s e ; 9.5 ml o f 25% s u c r o s e and 1 ml of w a t e r a t t h e t o p . The t u b e s ( 1 2 . 5 ml) were 26 c e n t r i f u g e d f o r 66 h. at lOO.OOOg i n a Beckmann L2-65B u l t r a c e n t r i f u g e . A f t e r c e n t r i f u g a t i o n , 0.287 ml f r a c t i o n s were c o l l e c t e d upwards from t h e b o t t o m o f the tube by t h e use o f a Desaga p e r i s t a l t i c pump and G i l s o n m i c r o f r a c t i o n c o l l e c t o r . The most a c t i v e enzyme f r a c t i o n s were combined a and d i v i d e d i n t o f o u r e q u a l p o r t i o n s . J u s t b e f o r e t h e a s s a y each one of t h e p o r t i o n s was d i l u t e d 200 t i m e s w i t h 0.1M sodium p h o s p h a t e pH 7.4 and A r r h e n i u s p l o t s were d e t e r m i n e d a t t i m e s i n d i c a t e d i n t h e f i g u r e . C a r d i o l i p i n s e p a r a t i o n by 'Sephadex g e l f i l t r a t i o n : A 5x60 cm Sephadex G-200 column was p r e p a r e d and e q u i l i b r a t e d w i t h 1.8M sodium c h l o r i d e , 2 mM sodium p h o s p h a t e pH 7.4 ("high s a l t " ) a t 4°C. A 2 ml sample c o n t a i n i n g a s o l u t i o n of 0.5 mg/ml of Sigma AChE and 2 mg/ml of d e x t r a n b l u e i n " h i g h s a l t " was a p p l i e d to the column. The f l o w r a t e was m a i n t a i n e d a t about 7 ml/h w i t h a Desaga p e r i s t a l t i c pump. F r a c t i o n s (1.2 ml) were c o l l e c t e d . AChE was d e t e r m i n e d by t h e r a d i o m e t r i c method and b l u e d e x t r a n a t ^240" P a r t i a l s p e c i f i c volume: The p a r t i a l s p e c i f i c volume of AChE was d e t e r m i n e d by e q u i l i b r i u m s e d i m e n t a t i o n on a p a r t i a l l y p r e f o r m e d s u c r o s e g r a d i e n t ( 1 5 ) . The p a r t i a l l y p r e f o r m e d s u c r o s e g r a d i e n t was made by l a y e r i n g 1.3 ml of each o f t h e f o l l o w i n g s u c r o s e 27 c o n c e n t r a t i o n s (gm/ml) i n " h i g h s a l t " : 0.906, 0.541, 0.329, 0.213, 0.140, 0.097, 0.069 , '0 .052 , 0 . 037 , 0.010.. R e f r a c t i v e i n d i c e s were d e t e r m i n e d w i t h a F i s h e r r e f r a c t o m e t e r a t 29.2°C, A f f i n i t y c h r o m a t o g r a p h y o f AChE on MAC c o l u m n s S y n t h e s i s and p u r i f i c a t i o n o f MAC: MAC was s y n t h e s i s e d and p u r i f i e d as d e s c r i b e d by M o o s e r e_t • a l _ . ( 9 5 ) . The i o d i d e d e r i v a t i v e o f t h e N - m e t h y l a c r i d i n e , o b t a i n e d by r e a c t i o n of a c r i d i n e w i t h i o d o m e t h a n e , was c o n v e r t e d t o i t s c o r r e s p o n d i n g c h l o r i d e d e r i v a t i v e by p a s s i n g i t t h r o u g h a c o l u m n c o n t a i n i n g a n i o n e x c h a n g e r e s i n Ag 1-X8. A s l i g h t i m p u r i t y c o u l d be d e t e c t e d on TLC u s i n g p r e c o a t e d 60 F^,^^ s i l i c a g e l p l a t e s and 1 - b u t a n o l : a c e t i c a c i d : w a t e r ( 8 : 1 2 : 3 v / v ) as t h e d e v e l o p -i n g s o l v e n t . The i m p u r i t y was removed by p a s s i n g t h e _ N - m e t h y l a c r i d i n i u m c h l o r i d e once t h r o u g h a 1x20 cm S e p h a d e x G-10 c o l u m n and e l u t i n g w i t h w a t e r . F o l l o w i n g l y o p h i l i z a t i o n , t h e y e l l o w c r y s t a l s were r e c r y s t a l l i z e d f r o m acetonitrile,176-179° d e c o m p o s i t i o n ( l i t 17 6-179°) ( 9 5) . C o u p l i n g o f 6 a m i n o h e x a n o y l - d e r i v a t i v e o f MAC t o  S e p h a r o s e 2B: The p r o c e d u r e s f o r t h e c y a n o g e n b r o m i d e a c t i v a t i o n o f S e p h a r o s e , t h e l i g a n d c o u p l i n g t o t h e a c t i v a t e d g e l and w a s h i n g o f t h e c o u p l e d g e l t o remove u n r e a c t e d l i g a n d were t h o s e d e s c r i b e d by M a r c h e_t a_l. ( 1 2 7 ) . The c o u p l i n g r e a c t i o n was c a r r i e d o u t a t room t e m p e r a t u r e r a t h e r t h a n a t 4°C s u g g e s t e d by t h e a u t h o r s . L i g a n d c o n c e n t r a t i o n o f 0.5, 1.0, 2.0 and 2.8 umol e / m l o f S e p h a r o s e w ere o b t a i n e d by v a r y i n g t h e c y a n o g e n b r o m i d e : g e l r a t i o i n t h e a c t i v a t i o n s t e p and t h e l i g a n d : a c t i v a t e d g e l r a t i o i n t h e c o u p l i n g s t e p . C o u p l e d g e l was washed w i t h 0.2M s o d i u m b i c a r b o n a t e b u f f e r pH 9.5. F r e e l i g a n d c o n c e n t r a t i o n was d e t e r m i n e d by m e a s u r i n g a b s o r b a n c e o f t h e wash a t 412 nm. The d i f f e r e n c e i n t h e i n i t i a l l i g a n d c o n c e n t r a t i o n and unbound l i g a n d c o n c e n t r a t i o n was u s e d t o c a l c u l a t e t h e amount o f l i g a n d bound t o t h e g e l . A f f i n i t y c h r o m a t o g r a p h y : One t h o u s a n d u n i t s o f Sigma AChE were d i s s o l v e d i n 200 ml o f 0.IM N a C l , 20mM s o d i u m p h o s p h a t e , pH 7.4 ( b u f f e r L) t o g i v e a s o l u t i o n o f 5 u n i t s Ami . T h i s enzyme c o n c e n t r a t i o n was l o a d e d on t h e c o l u m n . A f f i n i t y c h r o m a t o g r a p h y was p e r f o r m e d i n m i n i a t u r e c o l u m n s c o n s t r u c t e d f r o m 5 ml d i s p o s a b l e s y r i n g e s i n w h i c h t i g h t f i t t i n g d i s c s of p o r o u s p o l y p r o p y l e n e were i n s e r t e d . Columns were p a c k e d w i t h t h e r e s i n and e q u i l i b r a t e d w i t h b u f f e r ( L ) b e f o r e u s e . A m u l t i c h a n n e l p e r i s t a l t i c pump was u s e d t o l o a d , e l u t e and wash t h e f o u r columns s i m u l t a n e o u s l y w i t h a r a t e o f 13 m l / h . E l u t i o n was done w i t h 20mM d e c a m e t h o n i u m i n b u f f e r (L)', and r e g e n e r a t i o n o f t h e co l u m n was a c h i e v e d by w a s h i n g w i t h 5M g u a n i d i n e h y d r o c h l o r i d e i n b u f f e r ( L ) f o l l o w e d by n o r m a l b u f f e r ( L ) w a s h i n g . A l l f r a c t i o n s were c o l l e c t e d i n a G i l s o n m i c r o f r a c t i o n c o l l e c t o r . F r a c t i o n s o b t a i n e d on e l u t i o n o f t h e enzyme w i t h d e c a m e t h o n i u m were d i a l y s e d f o r 16 h a g a i n s t b u f f e r ( L ) , w i t h one change b e f o r e t h e a c t i v i t y o f t h e e l u t e d AChE was d e t e r m i n e d . 29 P r o t e i n e s t i m a t i o n P r o t e i n e s t i m a t i o n was done b o t h by ;.the method o f Murphy and K i e s (82) and Lowry ert a l _ . ( 8 3 ) . F a t t y a c i d f r e e b o v i n e serum a l b u m i n was u s e d as t h e s t a n d a r d . S t a n d a r d c u r v e s by t h e method o f Murphy and K i e s were l i n e a r f r o m 10 t o 75 ug o f p r o t e i n , w h i l e t h a t o f L o w r y was l i n e a r f r o m 25 t o 250 ug p r o t e i n . At t h e l o w e r c o n c e n t r a t i o n s o f p r o t e i n t h e f o r m e r method was f o u n d t o be more s u i t a b l e . R a d i o m e t r i c a s s a y o f AChE The method was e s s e n t i a l l y t h e same as d e s c r i b e d by B e a u r e g a r d and R o u f o g a l i s ( 1 4 ) . 20 u l of t h e enzyme s o l u t i o n was added t o 190 u l o f 0.IM s o d i u m p h o s p h a t e , pH 7.4 i n t h i c k w a l l e d i n c u b a t i o n t u b e s . A f t e r 30 m i n . of p r e i n c u b a t i o n a t t h e r e q u i r e d t e m p e r a t u r e , t h e r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f 20 u l (0 . 0 0 1 mCi) a c e t y l c h o l i n e t o g i v e a f i n a l c o n c e n t r a t i o n o f ImM i n a c e t y l c h o l i n e . The r e a c t i o n was a l l o w e d t o p r o c e e d f o r 5 t o 20 m i n . d e p e n d i n g upon t h e enzyme a c t i v i t y . A t t h e end o f t h e r e a c t i o n p e r i o d 200 u l o f a b s o l u t e a l c o h o l was added t o s t o p t h e r e a c t i o n . U n r e a c t e d a c e t y l c h o l i n e was removed by a d d i n g 1 ml o f 0.87 gm/ml A m b e r l i t e i o n e x c h a n g e r e s i n (CGC 2 4 1 ) . T h i s r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d f o r 20 m i n . d u r i n g w h i c h t h e r e s i n was s e d i m e n t e d . S u p e r n a t a n t (480 u l ) was t r a n s f e r r e d o n t o v i a l s , 10 ml o f a q u a s o l added and t h e r a d i o a c t i v i t y c o u n t e d i n an I s o c a p 30 N u c l e a r C h i c a g o 300 s c i n t i l l a t i o n c o u n t e r . K i n e t i c s t u d i e s a. S p e c t r o p h o t o m e t r i c a s s a y o f AChE: I n h i b i t i o n o f AChE by MAC was d e t e r m i n e d by u s i n g a c e t y 1 t h i o c h o 1 i n e as s u b s t r a t e , by t h e method o f E l l m a n e_t a_l. ( 8 4 ) . The a s s a y c o n d i t i o n s w e r e s i m i l a r t o t h o s e d e s c r i b e d by Wermuth and B r o d b e c k ( 8 5 ) . The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f v a r i o u s c o n c e n t r a t i o n o f a c e t y l t h i o c h o l i n e t o a 3 ml r e a c t i o n m i x t u r e i n a s p e c t r o p h o t o m e t e r c u v e t t e c o n t a i n i n g ( i n a f i n a l c o n c e n t a t i o n ) d i t h i o d i n i t r o b e n z o i c a c i d ( u.125mM), b o v i n e serum a l b u m i n ( 0 . 0 1 % ) and AChE (0.02 u n i t s / 3 ml) i n 25mM s o d i u m p h o s p h a t e pH 7.4. The change i n a b s o r b a n c e a t 412 nm was i m m e d i a t e l y r e c o r d e d on a Beckman m o d e l 25 UV s p e c t r o -p h o t o m e t e r . The i n i t i a l v e l o c i t y was d e t e r m i n e d by m e a s u r i n g t h e s l o p e o f t h e r e c o r d i n g f o r t h e f i r s t 1-2 m i n . b. pH s t a t T i t r i m e t r i c a s s a y : The e f f e c t o f c a l c i u m on MAC i n h i b i t i o n was d e t e r m i n e d by t h e pH s t a t m e t h o d , u s i n g t h e R a d i o m e t e r t i t r i m e t e r TTA31. The r e a c t i o n was c a r r i e d o u t a t 25^C i n a w a t e r j a c k e t e d r e a c t i o n v e s s e l . M A C ~ p u r i f i e d b o v i n e e r y t h r o c y t e AChE (0.5 u n i t s ) and MAC were i n c u b a t e d f o r 5 m i n . , i n a C 0 2 f r e e n i t r o g e n a t m o s p h e r e , i n t h e p r e s e n c e o r a b s e n c e o f 0 . lmM G a C ^ . The pH was a d j u s t e d t o 7.4 w i t h 0.01N s o d i u m h y d r o x i d e and t h e r e a c t i o n s t a r t e d by t h e a d d i t i o n o f 0.4mM a c e t y l c h o l i n e . E x p e r i m e n t s w e re done i n t h e a b s e n c e o f added i o n s o r i n t h e p r e s e n c e o f 40mM s o d i u m c h l o r i d e as i n d i c a t e d i n t h e f i g u r e . 31 I s o l a t i o n and p u r i f i c a t i o n o f AChE f r o m b o v i n e b l o o d : The method f o l l o w e d was s i m i l a r t o t h a t d e s c r i b e d by Cohen and W a r r i n g a (24) w i t h some m o d i f i c a t i o n s ( F i g . 1 . 2 ) . A l l t h e f o l l o w i n g o p e r a t i o n s were c a r r i e d o u t a t 4°C. 1.8 L of b o v i n e b l o o d ( t o w h i c h 0.37% c i t r a t e was added to. p r e v e n t c l o t t i n g ) f r o m f r e s h l y s l a u g h t e r e d a n i m a l s was o b t a i n e d f r o m t h e s l a u g h t e r h o u s e . I t was c e n t r i f u g e d a t 5, OOOg i n 250>iiml b u c k e t s i n t h e IEC B 20A c e n t r i f u g e . The s u p e r n a t a n t and t o p b u f f y la - y e r were removed by s u c t i o n . C o n s i d e r a b l e c a r e was t a k e n t o remove t h e b u f f y c o a t as c o m p l e t e l y as p o s s i b l e , d e s p i t e a c o n s i d e r a b l e l o s s o f b l o o d c e l l s . P a c k e d c e l l (200 ml) were washed t h r e e t i m e s w i t h 4 v o l u m e s o f 0.9% s o d i u m c h l o r i d e , f o l l o w e d by c e n t r i f u g a t i o n and r e m o v a l o f t h e s u p e r n a t a n t , and any r e m a i n i n g b u f f y l a y e r . The p a c k e d c e l l s w e r e h e m o l y s e d o v e r n i g h t i n 15 v o l u m e s o f d i s t i l l e d w a t e r a f t e r a dd i t ion, ; of.a., f ew lump s o f d r y i c e . The s t r o m a w h i c h s e t t l e d was c o l l e c t e d and washed t h r e e t i m e s w i t h 9 t i m e s d i l u t e d T y r o d e ' s s o l u t i o n . A f t e r e a c h w a s h i n g , s u p e r n a t a n t o b t a i n e d a f t e r c e n t r i f u g a t i o n a t 15,000g f o r 15 m i n . was removed by s u c t i o n . The g h o s t s o b t a i n e d f r o m t h e f i n a l w a s h i n g s were s u s p e n d e d i n 350 ml of d i s t i l l e d w a t e r , d i s t r i b u t e d i n t o e q u a l v o l u m e s i n s e v e n s e p a r a t e 2 50 ml b u c k n e r f l a s k s and f r e e z e d r i e d i n a V i r t i s l y o p h i l i z e r . The f r e e z e d r i e d p r o d u c t ( 3 . 1 gm) was s u s p e n d e d i n ' o n e l i t e r o f c o l d d r y b u t a n o l and m i x e d f o r two m i n . i n a w a r i n g b l e n d e r . The b u t a n o l e x t r a c t e d membrane. 32 ("Post b u t a n o l " membrane) was s e p a r a t e d f r o m b u t a n o l on a Whatman number 1 f i l t e r by vacuum f i l t r a t i o n . The r e s i d u e t h u s o b t a i n e d (2.7 gm) was d r i e d i n an e v a c u a t e d d e s s i c a t o r i n t h e p r e s e n c e o f s i l i c a f o r s e v e r a l h o u r s . The d r i e d b u t a n o l e x t r a c t e d membrane was s u s p e n d e d i n 0.IM N a C l , 0.2mM EDTA, lOmM s o d i u m p h o s p h a t e , pH 8 b u f f e r (1 gm/100 ml) and homogen-i z e d i n a S o r v a l l o m n i m i x e r a t spe e d 5, w i t h s i x homogeni?. a t i o n s f o r 10 s e c o n d p e r i o d s , w i t h 20 s e c o n d i n t e r v a l s i n b e t w e e n . The homogenate was c e n t r i f u g e d a t 15,000g f o r 15 m i n . and t h e r e s i d u e d i s c a r d e d . The supernatant was d i v i d e d i n t o two p o r t i o n s ; one p o r t i o n was p u r i f i e d by PTA a f f i n i t y c h r o m a t o g r a p h y and t h e o t h e r by ammonium s u l p h a t e p r e c i p i t a t i o n . AChE p u r i f i c a t i o n on PTA a f f i n i t y c o l u m n : The PTA co l u m n was c o n s t r u c t e d f r o m a 10 ml d i s p o s a b l e h y p o d e r m i c s y r i n g e , i n a manner s i m i l a r t o t h a t d e s c r i b e d f o r t h e MAC c o l u m n . The column was p a c k e d w i t h t h e a f f i n i t y r e s i n , c o n t a i n i n g t h e l i g a n d (N-6-amino c a p r o y l - m - a m i n o p h e n y l ) t r i m e t h y l a m m o n i u m b r o m i d e h y d r o b r o m i d e i n a c o n c e n t r a t i o n o f 4.6 umo l e / m l g e l . A f t e r e q u i l i b r i u m o f t h e c o l u m n , w i t h 0.1M N a C l , 0.2mM EDTA i n lOmM s o d i u m p h o s p h a t e pH 8.0; a p o r t i o n of t h e s u p e r n a t a n t ( o b t a i n e d by h o m o g e n i s a t i o n o f b u t a n o l t r e a t e d membrane i n t h e above b u f f e r ) was l o a d e d o n t o t h e co l u m n a t a r a t e of 13 m l / h . F o l l o w i n g w a s h i n g o f t h e co l u m n w i t h 3 column v o l u m e s o f b u f f e r , t h e enzyme r e t a i n e d on t h e co l u m n was e l u t e d w i t h 20mM d e c a m e t h o n i u m b r o m i d e i n b u f f e r ( L ) ; 1% T r i t o n i n b u f f e r ( L ) ; 1% T r i t o n , 20mM d e c a m e t h o n i u m i n b u f f e r " ( L ) and 50% p o l y e t h y l e n e g l y c o l . F r a c t i o n s e l u t e d f r o m 33 t h e c o l u m n were d i a l y s e d f o r 16 h a g a i n s t b u f f e r (L) b e f o r e a s s a y . AChE a c t i v i t y was a s s a y e d by a R a d i o m e t r i c method i n t h e p r e s e n c e of 0.IM s o d i u m p h o s p h a t e pH 7.4, as d e s c r i b e d a b o v e . Ammonium s u l p h a t e p r e c i p i t a t i o n : A n o t h e r p o r t i o n of s u p e r n a t a n t ( o b t a i n e d by h o m o g e n i s a t i o n o f b u t a n o l t r e a t e d membrane) was t r e a t e d w i t h an e q u a l v o l u m e o f c o l d s a t u r a t e d ammonium s u l p h a t e a t pH 7.0 and k e p t i n t h e r e f e r i g e r a t o r o v e r n i g h t . A f t e r p r e c i p i t a t i o n , t h e p r e c i p i t a t e was t a k e n up i n 150 ml of c o l d d i s t i l l e d w a t e r . C o l d s a t u r a t e d ammonium s u l p h a t e (41.9'.ml) was added and t h e pH a d j u s t e d t o 6.0. A s m a l l p r e c i p i t a t e f o r m e d a f t e r s t i r r i n g and s t a n d i n g f o r 30 m i n . was c e n t r i f u g e d . To t h e s u p e r n a t a n t was added 168 ml of s a t u r a t e d ammonium s u l p h a t e a t pH 6.0. The p r e c i p i t a t e o b t a i n e d a f t e r 2 h of s t a n d i n g and c e n t r i f u ga t l d n ;•. was t a k e n up i n b u f f e r ( L ) . S e d i m e n t a t i o n c o e f f i c i e n t s The s e d i m e n t a t i o n c o e f f i c i e n t o f AChE p u r i f i e d on t h e PTA a f f i n i t y c o l u m n f o l l o w i n g b u t a n o l e x t r a c t i o n was d e t e r m i n e d i n a 5-20% (w/v) c o n t i n u o u s s u c r o s e g r a d i e n t ( 1 5 ) . p r e p a r e d i n d i s t i l l e d w a t e r o r an i s o k i n e t i c g r a d i e n t (28) i n b u f f e r ( L ) . M a r k e r enzymes and AChE were p l a c e d a t t h e t o p of t h e g r a d i e n t and c e n t r i f u g e d i n a Beckman SW 41 T. r o t o r i n a Beckman ° x L2-65B u l t r a c e n t r i f u g e a t 4°C f o r .the t i m e and s p e e d i n d i c a t e d i n t h e f i g u r e l e g e n d . F r a c t i o n s ( 1 8 j d r o p s ) were c o l l e c t e d by c a r e f u l l y l o w e r i n g a c a p i l l a r y i n t o t h e t u b e a t t h e end of 34 t h e r u n and pumping t h e l i q u i d f r o m t h e b o t t o m w i t h a D e s a g a p e r i s t a l t i c pump. AChE a c t i v i t y was d e t e r m i n e d by t h e R a d i o m e t r i c method ( 1 4 ) . B o v i n e serum a l b u m i n and C a t a l a s e were d e t e r m i n e d by m e a s u r i n g a b s o r b a n c e a t 280 and 402 nm, r e s p e c t i -v e l y . - G a l a c t o s i d a s e was a s s a y e d a c c o r d i n g t o a p r e v i o u s l y p u b l i s h e d p r o c e d u r e ( 8 7 ) . P h o s p h o l i p i d e x t r a c t i o n and e s t i m a t i o n : P h o s p h o l i p i d s were e x t r a c t e d by t h e method of B l i g h and D y e r (124) . The p h o s p h o l i p i d e x t r a c t s were s p o t t e d on K i e s e l g e l 60 P254 P r e c o a t e d s i l i c a g e l p l a t e s and t h e p l a t e s were d e v e l o p e d i n c h l o r o f o r m : m e t h a n o 1 : a m m o n i u m h y d r o x i d e i n t h e r a t i o o f 60:30:5. The TLC s p o t s were s c r a p e d o f f t h e p l a t e a f t e r m o i s t e n i n g and t r a n s f e r r e d t o g l a s s c e n t r i f u g e t u b e s . P h o s p h o l i p i d s were e x t r a c t e d t h r e e t i m e s w i t h 2 ml :;. m e t h a n o l . A f t e r e v a p o r a t i o n o f t h e m e t h a n o l , 100::ul w a t e r and 0.5 ml of 10N s u l p h u r i c a c i d were added and t h e m i x t u r e h e a t e d a t 150-160°C f o r 3 h. Once t h e t u b e s were c o o l e d down t o room t e m p e r a t u r e , 3 t o 4 d r o p s o f h y d r o g e n p e r o x i d e were added and t h e m i x t u r e was h e a t e d f o r 2 h a t 150-160°C. A f t e r t h e t u b e s were c o o l e d t o 50°C, 4.6 ml o f ammonium m o l y b d a t e and 80 u l of F i s k and Subbarow r e a g e n t were a d d e d . The s o l u t i o n was m i x e d w e l l and i n c u b a t e d i n b o i l i n g w a t e r f o r 7 m i n . The b l u e c o l o u r d e v e l o p e d was m e a s u r e d a t 830 nm. 35 RESULTS 1. S e p a r a t i o n Of C a r d i o l i p i n From AChE A number o f e x p e r i m e n t s were p e r f o r m e d i n o r d e r t o p h y s i c a l l y s e p a r a t e c a r d i o l i p i n f r o m AChE. These i n c l u d e d g e l f i l t r a t i o n o f t h e " h i g h s a l t " t r e a t e d AChE and s u c r o s e g r a d i e n t c e n t r i f u g a t i o n i n t h e p r e s e n c e o f " h i g h s a l t " . A. P a r t i a l s p e c i f i c volume; o f " h i g h s a l t " t r e a t e d AChE  a f t e r S ephadex G-200 g e l f i l t r a t i o n : F o l l o w i n g t r e a t m e n t o f Sigma AChE w i t h 1.8M N a C l , 2mM s o d i u m p h o s p h a t e , pH 7 .4 , the enzyme was a p p l i e d t o a Sephadex G-200 c o l u m n e q u i l i b r a t e d and r u n i n t h e p r e s e n c e o f t h e same s a l t c o n c e n t r a t i o n . The p r e s e n c e or a b s e n c e o f c a r d i o l i p i n i n AChE o b t a i n e d f r o m t h e S e p h a d e x G-200 g e l f i l t r a t i o n u n d e r " h i g h s a l t " c o n d i t i o n s was d e t e r m i n e d by e q u i l i b r i u m s e d i m e n t a t i o n i n a p r e f o r m e d s u c r o s e g r a d i e n t , as d e s c r i b e d i n t h e m e t h o d s . The peak o f t h e enzyme a c t i v i t y was o b t a i n e d i n t h e 4 t h f r a c t i o n f r o m t h e b o t t o m ( F i g . 3), w h i c h c o r r e s p o n d e d t o a d e n s i t y o f 1.214 g/ml. The r e c i p r o c a l o f t h i s d e n s i t y gave a p a r t i a l s p e c i f i c v o l u m e o f 0. 823.. .ml/g, w h i c h i s h i g h e r t h a n t h e p a r t i a l s p e c i f i c v o l u m e o f 0.725 ml/g e x p e c t e d f r o m t h e am$no a c i d c o m p o s i t i o n o f t h e enzyme i n t h e a b s e n c e o f l i p i d . ( 7 ) . B. C h a r a c t e r i s a t i o n of c a r d i o l i p i n - A C h E i n t e r a c t i o n by A r r h e n i u s p l o t : Sigma AChE was p l a c e d on a s u c r o s e g r a d i e n t i n ' Sedimentation profllr of AChE obtained from 0 a l - filtration in high salt 0 Fraction D e t e r m i n a t i o n o f t h e p a r t i a l s p e c i f i c v o l u m e of AChE o b t a i n e d f r o m Sephadex G-200 g e l f i l t r a t i o n u n d e r " h i g h s a l t " c o n d i t i o n s . t h e p r e s e n c e o f 1.OM s o d i u m b i c a r b o n a t e , pH g . g } d e s i g n e d t o s e p a r a t e c a r d i o l i p i n by " f l o t a t i o n " f r o m t h e d e n s e r AChE p r o t e i n , ( s e e methods f o r d e t a i l ) i f t h e c a r d i o l i p i n was d i s s o c i a t e d f r o m t h e enzyme u n d e r " h i g h s a l t " c o n d i t i o n s . A l t e r n a t i v e l y , i f t h e c a r d i o l i p i n was n o t d i s s o c i a t e d f r o m AChE u n d e r t h e s e c o n d i t i o n s , t h e e x t e n t o f c a r d i o l i p i n - A C h E i n t e r a c t i o n c o u l d be m o n i t o r e d by d e t e r m i n i n g t h e A r r h e n i u s p l o t o f t h e enzyme a t v a r i o u s t i m e i n t e r v a l s f o l l o w i n g t h e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n i n t h e " f l o t a t i o n e x p e r i m e n t The c a r d i o l i p i n d i d n o t a p p e a r t o h a v e been removed f r o m AChE by t h e " h i g h s a l t " t r e a t m e n t and t h e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , as t h e b i p h a s i c A r r h e n i u s p l o t was r e g e n e r a t e d i n t i m e . A s t r a i g h t l i n e was o b t a i n e d i n t h e A r r h e n i u s p l o t 24 h a f t e r r e c o v e r y o f t h e enzyme f r o m t h e s u c r o s e g r a d i e n t ( F i g . 4 A ) . A f t e r 48 h, t h e p o i n t s on t h e A r r h e n i u s p l o t w e r e s c a t t e r e d , t h e r e b y i t was d i f f i c u l t t o d e c i d e b e t w e e n a s t r a i g h t l i n e o r two l i n e s w i t h a b r e a k ; b u t s t o r i n g t h e enzyme f o r a f u r t h e r p e r i o d o f 24 h (72 h a f t e r r e c o v e r y o f t h e enzyme from the gradient) gave an A r r h e n i u s p l o t w i t h a d i s t i n c t b r e a k ( F i g 4 C ) . A f t e r 192 h t h e A r r h e n i u s p l o t showed a d i s t i n c t b r e a k a t 21°C. L i n e s o f b e s t f i t were drawn f r o m l i n e a r r e g r e s s i o n a n a l y s i s . The s u c r o s e g r a d i e n t " f l o t a t i o n " e x p e r i m e n t was t h e n p e r f o r m e d i n t h e p r e s e n c e o f 0.09% T r i t o n X-100 i n an a t t e m p t t o s o l u b i l i s e any c a r d i o l i p i n w h i c h may h a v e d i s s o c i a t e d f r o m t h e enzyme i n t h e p r e s e n c e o f 1.OM s o d i u m b i c a r b o n a t e (pH 8 . 5 ) . The A r r h e n i u s p l o t s were d e t e r m i n e d a t v a r i o u s 38 F i g . 4. A r r h e n i u s p l o t s o f AChE a t v a r i o u s t i m e p e r i o d s f o l l o w i n g s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f AChE i n IM s o d i u m b i c a r b o n a t e , pH 8.0: A) 24 h . , B) 48 h . , C) 72 h . , D) 192 h. X a x i s - 1/T Y a x i s - l o g cpm. 39 time periods a f t e r recovery of the enzyme from the sucrose g r a d i e n t i n "high s a l t " and T r i t o n X-100 ( F i g . 5). The Arrhenius p l o t a f t e r 24 h was int e r m e d i a t e between i.that of a s t r a i g h t l i n e and a d i s t i n c t break. A l d i s t i n c t break at 21°C was observed i n the Arrhenius p l o t a f t e r 58 h. The above r e s u l t suggest that a time dependent r e a s s o c i a t i o n of c a r d i o l i p i n might be taking p l a c e . 40 2. P u r i f i c a t i o n Of AChE By A f f i n i t y Chromatography Attempts were made to p u r i f y s u f f i c i e n t q u a n t i t i e s of AChE f o r f u r t h e r s t u d i e s on the A C h E - c a r d i o l i p i n i n t e r a c t i o n . A. A f f i n i t y chromatography of bovine e r y t h r o c y t e AChE: The r e l a t i v e amounts of bovine e r y t h r o c y t e AChE r e t a i n e d and elu t e d on MAC a f f i n i t y columns of i n c r e a s i n g l i g a n d c o n c e n t r a t i o n are shown i n F i g . 6. As the l i g a n d c o n c e n t r a t i o n i n c r e a s e d , l e s s of the enzyme a c t i v i t y passed through the columns during the lP a d i n g and buffer-wash steps. Very l i t t l e enzyme a c t i v i t y was r e t a i n e d at l i g a n d concentrations of 0.5 and 1.0 umole/ml, as shown by the low e l u t i o n of enzyme a c t i v i t y on a p p l i c a t i o n of 20mM decamethonium. E f f i c i e n t r e t e n t i o n of the enzyme r e q u i r e d a l i g a n d c o n c e n t r a t i o n of 2.0 or 2.8 umole/ml. At these l i g a n d c o n c e n t r a t i o n s l e s s enzyme a c t i v i t y passed through the column during the l o a d i n g and washing steps, and enzyme a c t i v i t y ; ; was eluted by 20mM decamethonium. A s i n g l e a f f i n i t y p u r i f i c a t i o n of the Sigma bovine e r y t h r o c y t e AChE at the highest l i g a n d c o n c e n t r a t i o n of 2.8 umol/ml of g e l y i e l d e d p r e p a r a t i o n s with a s p e c i f i c a c t i v i t y of 60 umole/mg. min ; 41 F i g . 5. C o m p a r i s o n o f A r r h e n i u s p l o t s o b t a i n e d f r o m t h e p r e s e n c e and a b s e n c e o f 0.09% T r i t o n X-100 i n t h e c e n t r i f u g a t i o n m e d i a . A and C i n p r e s e n c e o f T r i t o n a t 24 h and 58 h; B and D i n a b s e n c e of T r i t o n a t 24 h and 48 h. X a x i s - 1/T Y a x i s - l o g cpm. 42 CM A 8 * •* CM (mdo) A 8OT ^ ^ <N • • . <r ^ m <*"> • <n n „ 43 F i g . 6. A f f i n i t y c h r o m a t o g r a p h y o f b o v i n e e r y t h r o c y t e AChE on MAC-Sepharose 2B columns at v a r i o u s l i g a n d c o n c e n t r a t i o n s . The columns were l o a d e d as d e s c r i b e d i n methods. At t h e f i r s t a r row marked " B u f f e r " , B u f f e r (L) was a p p l i e d t o the columns to remove unbound AChE and n o n s p e c i f i c a l l y a b s o r b e d p r o t e i n s . At t h e arrow marked "Deca", 20mM decamethonium was a p p l i e d . F r a c t i o n s (1.4 ml) were a s s a y e d f o r AChE a c t i v i t y by t h e r a d i o m e t r i c a s s a y ( s e e M e t h o d s ) . The l i g a n d c o n c e n t r a t i o n s were 0.49 umole/ml (•) , 0.99 umole/ml (<?), 1.97 umole/ml ( 4 ) , and 2.8 umole/ml ( A ) . 44 t h i s was i n c r e a s e d t o 420 umole/mg.min. a f t e r a s e c o n d p a s s a g e t h r o u g h t h e c o l u m n . B. K i n e t i c s o f MAC i n h i b i t i o n o f b o v i n e e r y t h r o c y t e  AChE: To i n v e s t i g a t e t h e n a t u r e o f t h e i n t e r a c t i o n b e t w e e n MAC and b o v i n e e r y t h r o c y t e AChE, t h e k i n e t i c s o f MAC i n h i b i t i o n were s t u d i e d . L i n e w e a v e r - B u r k p l o t s showed t h a t b o t h K m ( a p p ) a n d Vmax W 6 r e a l t e r e d b Y M A C ( F i S - 7 ) > s u g g e s t i n g m i x e d ( c o m p e t i t i v e / u n c o m p e t i t i v e ) i n h i b i t i o n . R e p l o t s o f t h e s l o p e s and 1 / v - i n t e r c e p t s a g a i n s t MAC c o n c e n t r a t i o n (101) y i e l d e d a K^ v a l u e o f l.OuM f o r t h e c o m p e t i t i v e component i and a K > v a l u e o f 2.QuM f o r t h e uncompetitive component ( F i g . 8 ) . + 2 The e f f e c t o f Ca on t h e i n h i b i t i o n o f b o v i n e e r y t h r o c y t e AChE by MAC i n t h e a b s e n c e of o t h e r added i o n s was a l s o e x a m i n e d . C a C l 2 (O.lmM) s h i f t e d t h e i n h i b i t i o n c u r v e ( V / V q ) t o t h e r i g h t , i n d i c a t i n g t h a t C a C l 2 a n t a g o n i z e d t h e i n h i b i t i o n o f t h e enzyme by MAC ( F i g . 9 A ) . T h i s i s a l s o shown i n D i x o n p l o t s ( F i g . 9B) , where O.lmM C a C l 2 s h i f t e d t h e a p p a r e n t K i f o r MAC i n h i b i t i o n f r o m O.l.uM ( c o m p a r e d t o 0.016 uM i n s i m i l a r i o n i c c o n d i t i o n s f o r e e l AChE ( 7 8 ) ) } t o 0.22 uM. The a n t a g o n i s m by C a C l 2 was f o u n d o n l y i n t h e a b s e n c e o f added i o n s . I n t h e p r e s e n c e o f 40mM s o d i u m c h l o r i d e , 0»ImM C a C l 2 had l i t t l e e f f e c t on t h e i n h i b i t i o n by MAC ( F i g . 1 0 ) . C. R o l e o f t i g h t l y bound c a r d i o l i p i n i n MAC a f f i n i t y : The a f f i n i t y o f b o v i n e e r y t h r o c y t e AChE f o r MAC was 13 f o l d o r more l o w e r t h a n t h a t o f t h e AChE f r o m e l e c t r i c o r g a n of e e l , l i m i t i n g t h e u s e f u l n e s s o f t h i s method f o r t h e F i g . 7. MAC i n h i b i t i o n o f b o v i n e e r y t h r o c y t e A C h E . A c e t y l t h i o c h o l i n e was a s s a y e d by s p e c t r o p h o t o m e t r y method ( s e e M e t h o d s ) . The MAC c o n c e n t r a t i o n was Z e r o (*) , 1 .15 uM ( a ) , 1 . 8 4 uM (•) , 2 . 7 6 uM (c-) , and 3 . 6 8 uM (•) . 46 F i g . 8. S l o p e and i n t e r c e p t r e p l o t s from L i n e w e a v e r - B u r k p l o t s of MAC i n h i b i t i o n o f b o v i n e e r y t h r o c y t e AChE. The s l o p e s (K /V) ( a ) and i n t e r c e p t s (1/v) (*) were o b t a i n e d from F i g . 7. 4 7 MAC (juM) E f f e c t o f C a C ± 2 on t h e i n h i b t i o n o f b o v i n e e r y t h r o c y t e AChE by MAC. A t i t r i m e t r i c a s s a y was u s e d ( s e e M e t h o d s ) i n t h e a b s e n c e o f a d d e d s a l t s (•) and i n t h e p r e s e n c e o f 0.ImM C a C l „ ( 9 ) . The 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 was 0.4mM. A . v / v i s t h e f r a c t i o n o f r e m a i n i n g enzyme a c t i v i t y , where v and V q a r e t h e v e l o c i t i e s i n t h e p r e s e n c e and i n t h e a b s e n c e o f i n h i b i t o r . V q was 0 . 5 0 8 u m o l e / m i n i n t h e a b s e n c e o f C a C l 2 and 0 . 7 2 0 u m o l e / m i n i n t h e p r e s e n c e o f O. lmM C a C l 0 . 4 8 MAC (pM) F i g 9 B D i x o n p l o t o f t h e d a t a i n F i g 9 A . I n t h e a b s e n ^ o f a d d e d s a l t ( «) a n d i n t h e p r e s e n c e o f 0.ImM Ca ( o ) . 49 MAC QJM) F i g . 1 0 . E f f e c t o f C a C l 2 on t h e i n h i b i t i o n o f b o v i n e e r y t h r o c y t e AChE a s s a y e d i n t h e p r e s e n c e of 40mM N a C l i The 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 was 0.4mM i n t h e a b s e n c e ( B ) and i n the p r e s e n c e o f O . lmM C a C l 2 (G). 50 p u r i f i c a t i o n o f t h e b o v i n e e r y t h r o c y t e enzyme. A p o s s i b l e r o l e o f t i g h t l y bound c a r d i o l i p i n i n t h e d i f f e r e n c e i n t h e a f f i n i t y o f b o v i n e e r y t h r o c y t e AChE and e e l e l e c t r i c o r g a n AChE f o r MAC was i n v e s t i g a t e d . The a f f i n i t y of t h e enzyme f o r MAC was d e t e r m i n e d u n d e r two d i f f e r e n t c o n d i t i o n s ; i ) i n t h e p r e s e n c e o f 1.8M s o d i u m . c h l o r i d e , lOmM s o d i u m p h o s p h a t e pH 7.4 ( c a r d i o l -i p i n d i s s o c i a t i n g c o n d i t i o n s ) and i i ) • i n t h e p r e s e n c e o f 1.8M s o d i u m c h l o r i d e a l o n e ( c a r d i o l i p i n - b o u n d t o t h e enzyme) ( 4 4 ) . A s i m i l a r K\ v a l u e o f 3.1 uM f o r MAC ( F i g . 11) was o b s e r v e d u n d e r b o t h t h e above s i t u a t i o n s , s u g g e s t i n g t h a t c o n d i t i o n s (1.8M s o d i u m c h l o r i d e , 2mM s o d i u m p h o s p h a t e ) w h i c h a b o l i s h e d t h e b r e a k i n t h e A r r h e n i u s p l o t d i d n o t a l t e r t h e a f f i n i t y o f t h e MAC f o r t h e enzyme. 3. E x t r a c t i o n o f AChE f r o m B o v i n e E r y t h r o c y t e Membranes A. P h o s p h o l i p i d a n a l y s i s : B u t a n o l t r e a t m e n t o f t h e e r y t h r o c y t e g h o s t s was e x a m i n e d as a method f o r t h e e x t r a c t i o n and p u r i f i c a t i o n o f AChE f r o m e r y t h r o c y t e g h o s t s , b a s e d on a p r e l i m i n a r y s t u d y o f Cohen and W a r r i n g a ( 2 4 ) . The q u a n t i t y o f v a r i o u s p h o s p h o l i p i d s e x t r a c t e d d u r i n g b u t a n o l t r e a t m e n t was e s t i m a t e d f r o m t h e p h o s p h o l i p i d a n a l y s i s o f t h e membrane b e f o r e and a f t e r b u t a n o l t r e a t m e n t . T h i n l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n on p r e - c o a t e d s i l i c a g e l p l a t e s showed t h e p r e s e n c e o f p h o s p h a t i d y l s e r i n e / p h o s p h a t i d y 1 i n o s i t o 1, p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y 1 e t h a n o l a m i n e and an u n i d e n t i f i e d p h o s p h o l i p i d i n b o t h t h e membranes ( F i g . 1 3 ) . 51 F i g . 11. E f f e c t o f e n d o g e n e o u s l y bound c a r d i o l i p i n on MAC a f f i n i t y : The E l l m a n a s s a y was u s e d t o d e t e r m i n e t h e MAC a f f i n i t y f o r t h e enzyme u n d e r two d i f f e r e n t c o n d i t i o n s , r e p r e s e n t a t i n g c a r d i o l i p i n ' a s s o c i a t e d ' and c a r d i o l i p i n ' d i s s o c i a t e d ' f o r m s o f t h e enzyme. A l l s o l u t i o n s were made i n 25mM T r i s b u f f e r i n s t e a d o f 25mM p h o s p h a t e ( a s d e s c r i b e d i n m e t h o d s ) ; t h e r e a c t i o n was c a r r i e d o u t i n a) 1.8M s o d i u m c h l o r i d e (•) and b) 1.8M s o d i u m c h l o r i d e , lOmM p h o s p h a t e (<^ -) . 52 BIOOO ^ 50009 ;15 min-Racked cells Ghosts • hemoiysed i freeze dried Pre butanol membrane 1 butanol ^ extractation Post butanol membrane i 01M NaCI;0-2mM EDTA i m 10 mM Phosphate (PH 8) Solubilised enzyme double ammonium sulphate precipitation affinity purification F i g . 12. F l o w s h e e t f o r t h e p u r i f i c a t i o n o f b o v i n e e r y t h r o c y t e AChE: The method i s e s s e n t i a l l y t h e same as d e s c r i b e d by Cohen and W a r r i n g a (24) w i t h s l i g h t m o d i f i c a t i o n . 53 CL 4 standards . 13. Thi n l a y e r c h romatographic a n a l y s i s of p h o s p h o l i p i d c o m p o s i t i o n b e f o r e and a f t e r the b u t a n o l t r e a t m e n t of the f r e e z e d r i e d e r y t h r o c y t e g h o s t s . PE-phospha-t i d y l e t h a n o l a m i n e ; P S - p h o s p h a t i d y l s e r i n e ; PC-p h o s p h a t i d y l c h o l i n e ; C L - c a r d i o l i p i n and P l - p h o s p h a -t i d y l i n o s i t o l . Spot ' P r e b u t a n o l ' membrane ph o s p h a t e / m g ' P o s t b u t a n o l ' memb r a n e p h o s p h a t e / m g P e r c e n t e x t r a c t e d a 1.95 0.479 75.4 b 1.575 0.455 71.1 c 0.160 0.031 80.6 d 1.4 2 0.29 6 7 9.6 e 0.019 0.000 100.0 T a b l e I I Q u a n t i t a t i v e e s t i m a t i o n of t h e p h o s p h o l i p i d c o n t e n t o f t h e v a r i o u s p h o s p h o l i p i d s p o t s o b t a i n e d on t h i n l a y e r c h r o m a t o g r a p h y ( F i g . 1 3 ) . P h o s p h a t e d e t e r m i n a t i o n o f t h e p h o s p h o l i p i d s on TLC p l a t e s i n d i c a t e d t h a t 70-80% o f v a r i o u s p h o s p h o l i p i d s were e x t r a c t e d by b u t a n o l ( T a b l e 2 ) . T h i s i s i n ag r e e m e n t w i t h p r e v i o u s f i n d i n g s t h a t 70-80% o f membrane l i p i d s a r e m o b i l e ( 7 2 , 7 3 ) , w h i l e t h e r e m a i n i n g l i p i d s a r e i m m o b i l i s e d due t o p r o t e i n : i b i n d i n g . B. R e l e a s e o f AChE f r o m b u t a n o l t r e a t e d b o v i n e  e r y t h r o c y t e membranes: V a r i o u s c o n d i t i o n s f o r t h e r e l e a s e o f AChE f r o m b u t a n o l t r e a t e d f r e e z e d r i e d e r y t h r o c y t e g h o s t s were e x a m i n e d , b e c a u s e lOmM p h o s p h a t e (pH 8.0) u s e d i n t h e o r i g i n a l s t u d y ( 2 4 ) , e x t r a c t e d o n l y 40% of t h e enzyme. The amount o f enzyme r e l e a s e d was i n c r e a s e d t o 60-70% by i n c r e a s -i n g t h e - ^ i o n i c c o n c e n t r a t i o n by a d d i t i o n o f s o d i u m c h l o r i d e ( f i n a l c o n c e n t r a t i o n 0.5M) o r s o d i u m p h o s p h a t e b u f f e r ( f i n a l c o n c e n t r a t i o n lOOmM), A f u r t h e r i n c r e a s e i n t h e amount o f enzyme r e l e a s e d was a c h i e v e d by a d d i n g EDTA ( t o a f i n a l c o n c e n t r a t i o n o f 0.2mM) t o t h e e x t r a c t i o n medium ( T a b l e 3 ) . The i n c r e a s e d r e l e a s e o f t h e enzyme due t o a d d i t i o n o f s o d i u m c h l o r i d e and EDTA were c o n s i s t e n t w i t h p r e v i o u s r e p o r t s i n t h e e x t r a c t i o n o f AChE f r o m e r y t h r o c y t e g h o s t s ( 1 2 , 1 2 9 ) . C. A f f i n i t y p u r i f i c a t i o n o f AChE: The AChE e x t r a c t e d w i t h 0.1M N a C l , 0.2mM EDTA i n lOmM s o d i u m p h o s p h a t e , pH 8.0 f r o m b u t a n o l - t r e a t e d f r e e z e d r i e d e r y t h r o c y t e g h o s t s , was a p p l i e d t o an a f f i n i t y c o l u m n w i t h PTA as t h e a f f i n i t y l i g a n d ( F i g 1 4 ) . Though c o m p l e t e r e t e n t i o n o f t h e enzyme was 56 S y s t e m P e r c e n t e x t r a c t e d W a t e r 4 4 lOmM s o d i u m p h o s p h a t e 41 pH 8.0 0.IM s o d i u m c h l o r i d e i n 41 lOmM s o d i u m p h o s p h a t e pH 8.0 lOOmM s o d i u m p h o s p h a t e 61 pH 8.0 0.5M s o d i u m c h l o r i d e i n 69 lOmM s o d i u m p h o s p h a t e pH 8.0 0.1M s o d i u m c h l o r i d e , 0.2mM 79 EDTA i n lOmM s o d i u m p h o s p h a t e pH 8.0 T a b l e I I I E x t r a c t i o n o f AChE f r o m b u t a n o l t r e a t e d e r y t h r o c y t e g h o s t s w i t h d i f f e r e n t i o n i c s t r e n g t h m e d i a . n 'o - 10 E a < buffer i i 20 mM Decae i n buffer 20mM O e c a . ; \°/a Triton in buffer i i i 50% ethylene glycol in buffer i i i v 100 Fraction number F i g . 14. A f f i n i t y p u r i f i c a t i o n o f AChE e x t r a c t e d f r o m b u t a n o l t r e a t e d e r y t h r o c y t e s g h o s t s w i t h 0.1M N a C l , 0 . 2 m M EDTA, lOmM s o d i u m p h o s p h a t e pH 8.0, on a P T A a f f i n i t y c o l u m n . T h e c o l u m n was e l u t e d w i t h d i f f e r e n t s o l u t i o n s a s d e s c r i b e d u n d e r m e t h o d s . a c h i e v e d d u r i n g c o l u m n l o a d i n g , o n l y 20 and 8 p e r c e n t o f t h e l o a d e d enzyme w e r e e l u t e d w i t h 20mM d e c a m e t h o n i u m i n b u f f e r ( L) and 1% T r i t o n X-100 i n b u f f e r ( L ) j , r e s p e c t i v e l y . The r e m a i n i n g 72% of t h e enzyme c o u l d n o t be e l u t e d , p r e s u m a b l y b e c a u s e i t was t i g h t l y bound t o t h e a f f i n i t y r e s i n . D. S u c r o s e d e n s i t y s e d i m e n t a t i o n : The n a t u r e o f t h e m o l e c u l a r f o r m s o f t h e AChE p r e s e n t u n d e r l o w and h i g h i o n i c s t r e n g t h c o n d i t i o n s was d e t e r m i n e d f r o m t h e s u c r o s e d e n s i t y c e n t r i f u g a t i o n p r o f i l e o f t h e b u t a n o l e x t r a c t e d enzyme. Under l o w i o n i c s t r e n g t h c o n d i t i o n s f o u r d i f f e r e n t f o r m s o f t h e enzyme w i t h s e d i m e n t a t i o n c o e f f i c i e n t s r a n g i n g f r o m 7S t o 20S were o b t a i n e d ( F i g . 1 5 ) . T h i s may be t h e r e s u l t o f a g g r e g a t i o n w h i c h has been r e p o r t e d t o o c c u r a t l o w i o n i c s t r e n g t h c o n d i t i o n s ( 2 6 ) . I n t h e p r e s e n c e o f 0.IM N a C l , 20mM s o d i u m p h o s p h a t e pH 7.4, 70-80 p e r c e n t o f t h e enzyme a p p e a r e d i n a '.peak w i t h a s e d i m e n t a t i o n c o e f f i c i e n t o f 11S ( F i g . 1 6 ) . The p r e s e n c e of a g g r e g a t i o n was n o t e d e v e n a t t h i s i o n i c s t r e n g t h . IQOOf-E a. o y s o o l -o < (A 01 8 n s ft i 10 15 Fraction number 20 Fig 15, S e d i m e n t a t i o n p r o f i l e o f AChE u n d e r l o w i o n i c s t r e n g t h c o n d i t i o n s . The enzyme was l o a d e d on a 5-20% c o n t i n u o u s s u c r o s e g r a d i e n t w i t h c a t a l a s e and b o v i n e serum a l b u m i n as m a r k e r s . G r a d i e n t was c e n t r i f u g e d a t 90,000g f o r 66 h. Enzyme and m a r k e r s were e s t i m a t e d as d e s c r i b e d u n d e r m e t h o d s . vo Fraction number F i g . 16. S e d i m e n t a t i o n p r o f i l e o f AChE i n 0.IM sod i u m c h l o r i d e , 20mM s o d i u m p h o s p h a t e . Enzyme was l o a d e d w i t h m a r k e r enzymes - g a 1 a c t o s i d a s e , c a t a l a s e and b o v i n e serum a l b u m i n on an i s o k i n e t i c s u c r o s e g r a d i e n t p r e p a r e d i n 0.1M s o d i u m c h l o r i d e , 20mM so d i u m p h o s p h a t e . The g r a d i e n t was c e n t r i f u g e d a t 100,000g f o r 9 h. Enzyme and m a r k e r s were e s t i m a t e d as d e s c r i b e d u n d e r methods. o 61 DISCUSSION C a r d i o l i p i n M o d u l a t i o n Of AChE C a t a l y s i s Many p r o t e i n s h a v e been shown t o r e q u i r e l i p i d s f o r t h e i r optimum a c t i v i t y ( 1 3 , 1 4 , 5 9 , 6 0 , 6 1 , 6 2 ) . One o f t h e most common methods f o r s t u d y i n g l i p i d p r o t e i n i n t e r a c t i o n s i s t o o b s e r v e t h e e f f e c t o f v a r i o u s l i p i d s on a " l i p i d f r e e p r o t e i n " s y s t e m ( 1 3 , 8 8 ) . D e t e r g e n t e x t r a c t i o n most commonly u s e d f o r l i p i d d i s p l a c e m e n t s u f f e r s f r o m c e r t a i n d r a w b a c k s . I o n i c d e t e r g e n t s o f t e n d e n a t u r e enzymes ( 1 2 , 1 3 0 ) . W h i l e many non i o n i c d e t e r g e n t s p r e s e r v e enzyme a c t i v i t i e s , i t i s o f t e n d i f f i c u l t t o o b t a i n . , a " d e t e r g e n t f r e e " f o r m o f t h e enzyme, b e c a u s e of t h e t i g h t b i n d i n g o f t h e d e t e r g e n t t o t h e p r o t e i n m o l e c u l e . Two enzymes w h i c h have been shown t o p o s s e s t i g h t l y bound c a r d i l i p i n a r e c y t o c h r o m e o x i d a s e (67) and AChE ( 1 4 , 1 5 ) . C o n t r a r y t o what was b e l i e v e d e a r l i e r , M a r s h e_t a _ l . (89) have r e c e n t l y shown t h a t c a r d i o l i p i n i s n o t e s s e n t i a l f o r c y t o c h r o m e o x i d a s e a c t i v i t y . I n AChE t h e i n v o l v e m e n t of c a r d i o l i p i n i n t h e a c t i v i t y o f AChE i s b a s e d on t h e i n t e r p r e t a t i o n o f A r r h e n i u s p l o t s . B e a u r e g a r d and R o u f o g a l i s (15) o b t a i n e d s o l u b l e and p a r t i c u l a t e f o r m s of t h e enzyme on h y p o - o s m o t i c s h o c k o f 'the > b o v i n e e r y t h r o c y t e g h o s t s . The p a r t i c u l a t e f o r m and a L u b r o l WX 62 s o l u b i l i s e d p r e p a r a t i o n o f t h e p a r t i c u l a t e f o r m gave a b i p h a s i c A r r h e n i u s p l o t , w h i l e t h e s o l u b l e f o r m o f t h e enzyme gave a l i n e a r A r r h e n i u s p l o t . Enzyme p r e p a r a t i o n s s h o w i n g a n o n - l i n e a r A r r h e n i u s p l o t , y i e l d e d a l i n e a r A r r h e n i u s p l o t a f t e r t r e a t m e n t w i t h 1.8M s o d i u m c h l o r i d e , 2mM s o d i u m p h o s p h a t e (pH 7 . 4 ) o r IM s o d i u m b i c a r b o n a t e (pH 8.0-10) o r 0.7M s o d i u m p h o s p h a t e (pH 7 . 4 ) . Under c o n d i t i o n s where t h e b r e a k i n t h e A r r h e n i u s p l o t was a b o l i s h e d i t was p o s s i b l e t o e x t r a c t t h e p h o s p h o l i p i d , c a r d i o l i p i n , by c h l o r o f o r m - m e t h a n o l , w h e r e a s c a r d i o l i p i n c o u l d n o t be e x t r a c t e d b e f o r e t h e " h i g h s a l t " t r e a t m e n t s ( 1 4 , 1 5 ) . From t h e s t u d y o f t h e e f f e c t o f " h i g h s a l t " t r e a t m e n t on t h e v a r i o u s s t e p s o f a c e t y l c h o l i n e h y d r o l y s i s ( 4 3 ) , i t was p o s t u l a t e d t h a t c a r d i o l i p i n m o d u l a t e d a r a t e d e t e r m i n i n g c o n f o r m a t i o n a l change i n t h e e n z y m e - s u b s t r a t e c o m p l e x . I n o r d e r t o o b t a i n . d i r e c t e v i d e n c e f o r t h e i n v o l v e m e n t of c a r d i o l i p i n i n AChE c a t a l y s i s , i t i s e s s e n t i a l t o h a v e a c a r d i o l i p i n - f r e e AChE. The method p r e v i o u s l y u s e d t o remove c a r d i o l i p i n i n v o l v e d " h i g h s a l t " t r e a t m e n t f o l l o w e d by c h l o r o f o r m - m e t h a n o l e x t r a c t i o n , c a u s i n g d e n a t u r a t i o n o f t h e enzyme. Use o f d e t e r g e n t s f o r t h e e x t r a c t i o n o f a f r a c t i o n o f t i g h t l y bound c a r d i o l i p i n h a s b e e n u n s u c c e s s f u l i n t h e c a s e o f c y t o c h r o m e o x i d a s e (90) and AChE ( 1 5 ) . Two p h y s i c a l methods f o r t h e r e m o v a l of c a r d i o l i p i n h a v e been i n v e s t i g a t e d . Sephadex:: g e l f i l t r a t i o n has b een v e r y o f t e n u s e d f o r t h e s e p a r a t i o n o f e x c e s s d e t e r g e n t and l i p i d s f r o m p r o t e i n - d e t e r g e n t c o m p l e x e s ( 1 3 1 ) . I t was h y p o t h e s i z e d t h a t i f u n d e r " h i g h s a l t " c o n d i t i o n s c a r d i o l i p i n d i s s o c i a t e d f r o m t h e enzyme, as s u g g e s t e d by B e a u r e g a r d & R o u f o g a l i s ( 1 4 ) , i t s h o u l d be p o s s i b l e t o s e p a r a t e i t f r o m AChE by S e p a h a d e x g e l c h r o m a t o -g r a p h y , b e c a u s e o f t h e d i f f e r e n c e i n s i z e b e t w e e n c a r d i o l i p i n and AChE. The p a r t i a l s p e c i f i c v o l u m e o f 0.81 ml/g?, ( F i g . 3) o b t a i n e d f o r AChE f o l l o w i n g t r e a t m e n t w i t h " h i g h s a l t " and g e l :;:'f i l t r a t i on on Sephadex G-200 was l a r g e r t h a n 0.72 m l / g c a l c u l a t e d f r o m t h e amino a c i d c o m p o s i t i o n o f t h e p u r e enzyme ( 7 ) , s u g g e s t i n g t h a t bound l i p i d was p r e s e n t i n t h e enzyme e v e n a f t e r " h i g h s a l t " t r e a t m e n t and g e l c h r o m a t o g r a p h y . A g r e a t e r u n d e r s t a n d i n g o f t h e n a t u r e o f t h e AChE- ;• c a r d i o l i p i n i n t e r a c t i o n was o b t a i n e d f r o m a s u c r o s e g r a d i e n t " f l o t a t i o n " e x p e r i m e n t . The a t t e m p t t o s e p a r a t e c a r d i o l i p i n f r o m AChE b y " f l o t a t i o n " i n a s u c r o s e g r a d i e n t was b a s e d on t h e e x p e c t a t i o n t h a t i f " h i g h s a l t " t r e a t m e n t c a u s e d s e p a r a t i o n of c a r d i o l i p i n f r o m t h e enzyme, t h e l e s s dense c a r d i o l i p i n s h o u l d f l o a t t o t h e t o p df t h e g r a d i e n t , w h i l e t h e AChE w o u l d p e n e t r a t e t h e d e n s e r s u c r o s e l a y e r . The f r a c t i o n c o n t a i n i n g AChE a c t i v i t y was a s s a y e d a f t e r v a r i o u s t i m e s :>: f o l l o w i n g r e c o v e r y of t h e enzyme f r o m t h e s u c r o s e g r a d i e n t u s e d i n t h e f l o t a t i o n e x p e r i m e n t . A l i n e a r A r r h e n i u s p l o t was s e e n a f t e r 24 h ( F i g . 4 A ) . H o w e v e r , w i t h t i m e a b r e a k i n t h e A r r h e n i u s p l o t was r e c o v e r e d , w h i c h become v e r y d i s t i n c t i n 5-8 d a y s ( F i g . 4C&4D). I n b e t w e e n t h e two e x t r e m e s a d e g r e e of s c a t t e r was o b s e r v e d i n t h e A r r h e n i u s p l o t s , w h i c h made i t d i f f i c u l t t o d e c i d e b e t w e e n e i t h e r a s i n g l e s t r a i g h t l i n e o r two l i n e s w i t h a b r e a k . R e p e a t i n g t h e above e x p e r i m e n t i n t h e p r e s e n c e o f 0.09% T r i t o n X-100, t o s o l u b i l i s e any c a r d i o l i p i n r e l e a s e d by t h e h i g h s a l t t r e a t m e n t , d i d n o t p r e v e n t t h e r e g e n e r a t i o n o f t h e b r e a k ( F i g . 5 ) . I f a n y t h i n g , t h e r e g e n e r a t i o n seemed t o o c c u r f a s t e r . From t h e s e e x p e r i m e n t s i t i s c l e a r t h a t t h e b r e a k a b o l i s h e d by " h i g h s a l t " t r e a t m e n t r e a p p e a r s w i t h t i m e . T h i s s u g g e s t s t h a t t h e i o n i c d i s r u p t i o n b r o u g h t a b o u t by " h i g h s a l t " t r e a t m e n t was r e v e r s i b l e , as i t u n d e r w e n t a t i m e d e p e n d e n t r ea s s o c i a t i o n .. B e a u r e g a r d and R o u f o g a l i s (44) f o u n d t h a t t h e a d d i t i o n + 2 of Ca w i t h m i l d h o m o g e n i z a t i o n t o " h i g h s a l t " t r e a t e d AChE p r e p a r a t i o n s r e g e n e r a t e d t h e A r r h e n i u s p l o t i b r e a k . H owever, as t h e " h i g h s a l t " t r e a t e d enzyme r e t a i n e d e ndogeneous c a r d i o l i p i n , two mechanisms o f AChE m o d u l a t i o n were p o s s i b l e . + 2 I n t h e f i r s t m echanism b o t h Ca and c a r d i o l i p i n c o o p e r a t e t o r e g u l a t e t h e t e m p e r a t u r e d e p e n d e n t m o d u l a t i o n o f AChE, + 2 w h i l e i n t h e s e c o n d a l t e r n a t i v e Ca a l o n e may be s u f f i c i e n t f o r t h e m o d u l a t i o n p r o c e s s . I f t h e s e c o n d a l t e r n a t i v e i s + 2 c o r r e c t , t h e a b i l i t y o f " h i g h s a l t " i n Ca c h e l a t i n g c o n d i t i o n s t o i n c r e a s e t h e e x t r a c t a b i l i t y o f c a r d i o l i p i n f r o m t h e enzyme by c h l o r o f o r m - m e t h a n o l c o u l d be c o i n c i d e n t a l and u n r e l a t e d t o t h e m o d u l a t i o n m echanism. The f o l l o w i n g e v i d e n c e s u p p o r t s t h e f o r m e r m echanism. The p r e s e n t o b s e r v a t i o n o f t h e r e a p p e a r -a n c e o f t h e A r r h e n i u s p l o t b r e a k w i t h t i m e c a n be b e s t + 2 e x p l a i n e d by t h e s i m u l t a n e o u s i n v o l v e m e n t o f Ca and c a r d i o l i p i n r a t h e r t h a n c a l c i u m a l o n e and s u g g e s t s t h e f o l l o w i n g m o d e l f o r t h e l i p i d - p r o t e i n i n t e r a c t i o n s i n AChE. The i n a c c e s s i b i l i t y o f c a r d i o l i p i n and c a l c i u m t o p h o s p h o l i p a s e s and C,and EDTA, r e p e c t i v e l y , s u g g e s t e d t h a t Ca and c a r d i o l i p i n may be b u r i e d w i t h i n t h e AChE d i m e r ( 4 4 ) . I t i s now p r o p o s e d t h a t c a r d i o l i p i n may be bound t o t h e c o r e o f t h e p r o t e i n by h y d r o p h o b i c as w e l l as i o n i c i n t e r a c t i o n s . The "high'.:..salt" c o n d i t i o n s may d i s r u p t t h e i o n i c i n t e r a c t i o n s , t h e r e b y c a u s i n g a f u n c t i o n a l d i s s o c i a t i o n o f t h e enzyme--p h o p h o l i p i d c o m p l e x , w h i c h may r e s u l t i n a b o l i t i o n •. of t h e A r r h e n i u s p l o t b r e a k . At t h e same t i m e t h e h i g h i o n i c s t r e n g t h t r e a t m e n t w i l l s t r e n g t h e n t h e h y d r o p h o b i c i n t e r a c t i o n ; w h i c h f a c i l i t a t e s t h e r e g e n e r a t i o n o f t h e i o n i c p r o t e i n - l i p i d i n t e r a c t i o n w i t h t i m e . The h y d r o p h o b i c i n t e r a c t i o n and i t s s t r e n g t h e n i n g i n " h i g h s a l t " c o n d i t i o n s may a l s o e x p l a i n t h e l a c k o f " p h y s i c a l s e p a r a t i o n " o f t h e c a r d i o l i p i n f r o m AChE, u n d e r c o n d i t i o n s where " f u n c t i o n a l d i s s o c i a t i o n " has o c c u r r'.e d . T h e r e a r e o t h e r e x a m p l e s where A r r h e n i u s p l o t b r e a k s i n t h e membrane bound e n z y m a t i c a c t i v i t y i s a t t r i b u t e d t o t h e d i r e c t i n v o l v e m e n t o f p h o s p h o l i p i d ( 6 1 , 6 2 , 6 6 ) . R e c e n t l y , + 2 +2 some of t h e s e r e p o r t s h a v e b e e n d i s p u t e d . I n (Mg -Ca ) A T P a s e (63) and 5 ' - n u c e o t i d a s e (116) t h e A r r h e n i u s p l o t b r e a k were a t t r i b u t e d t o p h o s p h o l i p i d i n d e p e n d e n t p r o t e i n c o n f o r m a t i o n a l c h a n g e . I n c a s e s where p h o s p h o l i p i d s a r e i m p l i c a t e d i n m o d u l a t i n g t h e A r r h e n i u s p l o t b e h a v i o u r v a r i o u s m e c h anisms have b e e n p r o p o s e d ; t r a n s i t i o n o f t h e l i p i d f r o m g e l t o l i q u i d c r y s t a l l i n e s t a t e ( 6 2 , 8 0 ) , l i p i d c l u s t e r _ o r r e l a t e d p h a s e c h a n g e s ( 1 7 , 5 5 ) , o r a l t e r e d t e m p e r a t u r e d e p e n d e n t p r o t e i n - l i p i d i n t e r a c t i o n s w i t h t i g h t l y . a s s o c i a t e d 66 l i p i d s ( 9 , 6 6 , 9 0 ) . I n b o v i n e e r y t h r o c y t e AChE, s i m u l t a n e o u s + 2 i n v o l v e m e n t o f b o t h Ca and c a r d i o l i p i n , a l o n g w i t h t h e i r p r e s e n c e i n s i d e t h e p r o t e i n m o l e c u l e , may s u g g e s t t h e f o l l o w i n g + 2 p o s s i b l e mode o f m o d u l a t i o n ; Ca may f o r m a s a l t b r i d g e b e t w e e n t h e a n i o n i c s i t e s o f t h e enzyme and t h e p h o s p h a t e g r o u p o f c a r d i o l i p i n o r i t may be r e q u i r e d t o m a i n t a i n t h e c a r d i o l i p i n i n a p a r t i c u l a r p h a s e c o m p a t i b l e w i t h optimum enzyme a c t i v i t y . C a l c i u m c a r d i o l i p i n e x i s t s i n a c y l i n d r i c a l h e x a g o n a l (H^j) p h a s e (17) i n a 1:1 s t o i c h i o m e t r i c r a t i o + 2 of Ca t o c a r d i o l i p i n . On t h e o t h e r hand t h e s o d i u m - c a r d i o l i p i n c o m p l e x , w h i c h may be p r o d u c e d a f t e r t r e a t m e n t o f AChE i n t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s of s o d i u m s a l t s , e x i s t s p r e d o m i n a n t l y i n t h e b i l a y e r c o n f i g u r a t i o n ( 9 2 ) . Changes i n t e m p e r a t u r e may a l t e r t h e l o c a l t r a n s i t i o n b e t w e e n p h a s e s + 2 of t h e Ca - c a r d i o l i p i n c o m p l e x o r t h e e x t e n t o f p r o t e i n - l i p i d p h a s e i n t e r a c t i o n r e s u l t i n g i n c h a n g e s i n t h e p r o t e i n c o n f o r -m a t i o n , r e f l e c t e d by c h a n g e s i n t h e a c t i v a t i o n e n e r g y o f t h e + 2 enzyme a c t i v i t y . The e x a c t n a t u r e of t h e Ca d e p e n d e n t c a r d i o l i p i n - e n z y m e c h a n g e s t h a t o c c u r a t t h e t r a n s i t i o n i n t h e enzyme a c t i v i t y a t 20°C i s unknown a t p r e s e n t . A c y l c h a i n s of c a r d i o l i p i n do n o t a p p e a r t o u n d e r g o f l u i d i t y c h a n g e s b e t w e e n 4°C and 40°C, when c a r d i o l i p i n i s i n h e x a g o n a l p h a s e (92} a l t h o u g h t h e a d d i t i o n a l i n f l u e n c e o f a d j a c e n t p r o t e i n s o r o t h e r l i p i d s ( 65) i s unknown. C a r d i o l i p i n u n d e r g o e s c l u s t e r f o r m a t i o n around 20°C (93) b u t t h e r e i s no e v i d e n c e o f t h e b i o l i o g i c a l s i g n i f i c a n c e o f t h i s t r a n s i t i o n . F u r t h e r s t u d i e s a r e r e q u i r e d t o c o n f i r m e x a c t l y t h e r o l e o f c a r d i o l i p i n i n AChE as w e l l as t h e m e c h a n i s m of m o d u l a t i o n . 67 A f f i n i t y C h r o m a t o g r a p h y On MAC A f f i n i t y Column The p u r p o s e o f a f f i n i t y c h r o m a t o g r a p h y o f AChE i n t h i s s t u d y was two f o l d a) p u r i f i c a t i o n and b) c a r d i o l i p i n s e p a r a t i o n . To s e p a r a t e c a r d i o l i p i n f r o m AChE, an a f f i n i t y l i g a n d was n e e d e d w h i c h c o u l d r e t a i n t h e enzyme i n t h e p r e s e n c e of h i g h i o n i c s t r e n g t h c o n d i t i o n s s u c h as IM s o d i u m c h l o r i d e . The PTA l i g a n d u s e d p r e v i o u s l y f o r t h e p u r i f i c a t i o n o f e e l (25) and e r y t h r o c y t e AChE (25) e x h i b i t s a l o w a f f i n i t y f o r t h e enzyme u n d e r h i g h i o n i c s t r e n g t h . I n t h e c a s e o f e e l AChE, t h i s p r o b l e m o f l o w a f f i n i t y has b een r e c e n t l y o v e r c o m e by t h e use of N - m e t h y l a c r i d i -n i u m (MAC) as t h e a f f i n i t y l i g a n d ( 2 8 ) . MAC has a l s o b e e n f o u n d t o be t h e most s u i t a b l e l i g a n d f o r t h e p u r i f i c a t i o n o f p i g b r a i n AChE ( 2 9 ) . The a b o v e m e n t i o n e d f a c t o r s , a l o n g , w i t h t h e p o t e n t i a l u s e o f MAC as a f l u o r e s c e n t p r o b e (95 ,96) , has l e d t o t h e s t u d y of MAC as an a f f i n i t y l i g a n d i n t h i s s t u d y . C o m p a r i s o n of t h e e l u t i o n p r o f i l e o f b o v i n e e r y t h r o c y t e AChE w i t h t h a t of t h e e e l AChE ( 2 8 ) , a t d i f f e r e n t l i g a n d c o n c e n t r a t i o n s ' " of MAC, i n d i c a t e d t h a t .they b e h a v e d d i f f e r e n t l y on t h e MAC a f f i n i t y c o l u m n . Af.high s a l t e x t r a c t o f e e l AChE i s o p t i m a l l y r e t a i n e d by t h e a f f i n i t y c o l u m n a t a l i g a n d c o n c e n t r a t i o n of 0.44-0.98 u m o l e / m l g e l ( 2 8 ) . By c o n t r a s t , r e t e n t i o n o f t h e e r y t h r o c y t e enzyme r e q u i r e d a l i g a n d c o n c e n t r a -t i o n o f 2.0-2.8 u m o l e / m l g e l . Under t h e s e c o n d i t i o n s t h e e e l enzyme i s v e r y t i g h t l y r e t a i n e d , and i s e l u t e d i n o n l y p o o r o r z e r o y i e l d by d e c a m e t h o n i u m ( 2 8 ) . V a r i o u s p o s s i b l e r e a s o n f o r t h e d i f f e r e n c e i n b e h a v i o u r b e t w e e n t h e two enzymes h a v e b e e n i n v e s t i g a t e d . E f f e c t of s t r u c t u r e : I t was shown p r e v i o u s l y t h a t d i f f e r e n c e s i n t h e a f f i n i t y c h r o m a t o g r a p h i c r e t e n t i o n of v a r i o u s m o l e c u l a r f o r m s o f e e l AChE was due t o s i m u l t a n e o u s m u l t i p l e l i g a n d i n t e r a c t i o n s w i t h t h e l a r g e r o l i g o m e r i c f o r m s ( 2 8 ) . The " m u l t i s i t e i n t e r a c t i o n " p r o p o s e d a b o v e c a n n o t a c c o u n t f o r t h e r e t e n t i o n d i f f e r e n c e s e e n b e t w e e n t h e d i m e r i c b o v i n e enzyme and t e t r a m e r i c e e l enzyme. T h i s was s u g g e s t e d by t h e f o l l g w i n g o b s e r v a t i o n . T h e 8S d i m e r i c f o r m o f t h e e e l enzyme o b t a i n e d f r o m t h e s o n i c a t i o n o f 14 and 18S f o r m s i n t h e p r e s e n c e o f g u a n i d i n e , c o u l d be o p t i m a l l y r e t a i n e d a t a MAC c o n c e n t r a t i o n o f 0.44 u m o l e / m l g e l ( 9 4 ) . T h e r e f o r e , i t i s c o n c l u d e d t h a t t h e f a c t t h a t b o v i n e e r y t h r o c y t e AChE; i s adim'e.r, i n c o n t r a s t t o t h e 11S e e l AChE w h i c h i s a t e t r a m e r , c a n n o t a c c o u n t f o r the lowr r e t e n t i o n of t h e e r y t h r o c y t e enzyme r e l a t i v e t o t h e e e l enzyme. A f f i n i t y f o r l i g a n d : The i n h i b i t i o n c o n s t a n t , K_, f o r t h e c o m p e t i t i v e component o f MAC i n h i b i t i o n o f b o v i n e e r y t h r o c y t e AChE (1.0 uM) i s 1 3 - f o l d (95) o r more (96) h i g h e r t h a n f o r e e l AChE a t a s i m i l a r i o n i c s t r e n g t h . A t e n - f o l d d e c r e a s e i n b i n d i n g a f f i n i t y of t h e a m i n o h e x a n o y 1 d e r i v a t i v e of PTA w i t h i n c r e a s i n g i o n i c s t r e n g t h c o r r e l a t e s w i t h t h e m arked i o n i c s t r e n g t h d e p e n d e n c e of AChE r e t e n t i o n i n a f f i n i t y c o lumns c o n t a i n i n g t h i s l i g a n d ( 2 8 , 9 7 - 9 9 ) . The d i f f e r e n c e i n MAC b i n d i n g a f f i n i t y b e t w e e n e e l and e r y t h r o c y t e ; A C h E i s e n t i r e l y a d e q u a t e , t h e r e f o r e , t o a c c o u n t f o r t h e o b s e r v e d d i f f e r e n c e s i n a f f i n i t y c h r o m a t o g r a p h i c b e h a v i o u r b e t w e e n t h e two enzymes on MAC a f f i n i t y c o l u m n s . 69 S i t e o f i n h i b i t i o n : The m i x e d e o m p e t i t i v e / u n c o m p e t i t i v e i n h i b i t i o n o f b o v i n e e r y t h r o c y t e AChE by MAC was a l s o f o u n d i n t h e i n h i b i t i o n o f e e l AChE ( 9 5 ) , s u g g e s t i n g t h a t MAC r e a c t s w i t h b o t h enzymes a t s i m i l a r s i t e s . The u n c o m p e t i t v e component has b e e n a t t r i b u t e d t o b i n d i n g o f MAC t o t h e a c e t y l a t e d - e n z y m e i n t e r m e d i a t e o f AChE ( 9 5 ) . A l t e r n a t i v e l y , m i x e d i n h i b i t i o n c o u l d o c c u r as a r e s u l t o f b i n d i n g of MAC t o t h e p e r i p h e r a l a n i o n i c s i t e on AChE ( 1 0 0 ) . I n a p r e v i o u s s t u d y ( 1 00) i t was + 2 shown t h a t t h e i n f l u e n c e o f Ca on t h e i n h i b i t i o n o f AChE i s d i a g n o s t i c o f t h e r e l a t i v e a f f i n i t y o f a l i g a n d f o r + 2 t h e c a t a l y t i c and p e r i p h e r a l a n i o n i c s i t e s . Low Ca c o n c e n t r a -t i o n s enhance t h e i n h i b i t i o n o f t h e enzyme when t h e 1 i g a n d - a f f i n i t y + 2 f o r t h e p e r i p h e r a l s i t e i s h i g h , w h i l e Ca a n t a g o n i s ; e s t h e i n h i b i t i o n when t h e p e r i p h e r a l s i t e - a f f i n i t y i s l o w ( 1 0 0 ) . + 2 The a n t a g o n i s m by Ca a t l o w i o n i c s t r e n g t h , and t h e l o s s of a n t a g o n i s m i n 40mM N a C l i n t h e p r e s e n t r e s u l t s , s u g g e s t t h a t MAC i n h i b i t s b & v i n e e r y t h r o c y t e AChE by b i n d i n g p r e d o m i n a n t l y a t t h e c a t a l y t i c a n i o n i c s i t e ; t h e l i n e a r s l o p e r e p l o t o f t h e L i n e w e a v e r - B u r k p l o t s (101) and t h e l i n e a r D i x o n p l o t (102) s u p p o r t t h i s c o n c l u s i o n . A l t h o u g h a s i m i l a r k i n e t i c s t u d y was n o t made w i t h e e l AChE, f l u o r e s c e n t m e a s u r e m e n t s show t h a t MAC a l s o b i r l d s s e l e c t i v e l y t o t h e c a t a l y t i c a n i o n i c s i t e on t h i s enzyme ( 9 5 . 9 6 ) . Thus t h e d i f f e r e n t b e h a v i o u r o f t h e e r y t h r o c y t e and e e l AChE on MAC a f f i n i t y c o l u m n s c a n n o t be a c c o u n t e d f o r by a d i f f e r e n t s i t e o f i n t e r a c t i o n o f t h e l i g a n d on t h e s i d e - a r m w i t h t h e two enzymes. I n summary, i t a p p e a r s t h a t t h e l o w e r r e t e n t i o n o f b o v i n e e r y t h r o c y t e AChE on M A C - a f f i n i t y c o l u m n s i s a d e q u a t e l y 70 e x p l a i n e d by a 1 3 - f o l d or more l o w e r a f f i n i t y o f t h i s enzyme, compared t o t h e e e l enzyme, f o r t h e s i d e - a r m l i g a n d i n t h e c h r o m a t o g r a p h i c c o n d i t i o n s . The l o w e r a f f i n i t y o f MAC f o r b o v i n e e r y t h r o c y t e AChE a l s o l i m i t s t h e u s e f u l n e s s o f MAC as a f l u o r e s c e n t p r o b e f o r t h i s enzyme, w h i l e i t h a s b e e n u s e d s u c c e s s f u l l y w i t h t h e e e l AChE. ( 95 , 96) . The d i f f e r e n c e ' i n a f f i n i t y b e t w e e n t h e b o v i n e and e e l AChE f o r MAC c o u l d p o s s i b l y be due t o any o f t h e f o l l o w i n g r e a s o n s : a) The t r y p t o p h a n m o i e t y a t t h e a n i o n i c s i t e o f t h e e e l AChE, w h i c h h a s been shown t o b i n d o r g a n i c c a t i o n s (103) , i s - e i t h e r l a c k i n g o r i n a d i f f e r e n t s p a t i a l a r r a n g e m e n t w i t h r e s p e c t t o t h e a n i o n i c s i t e i n t h e b o v i n e e r y t h r o c y t e AChE or b) t h e p r e s e n c e o f a t i g h t l y bound p h o s p h o l i p i d , c a r d i o l i p i n , w h i c h h a s been shown t o be p r e s e n t o n l y i n t h e e r y t h r o c y t e enzyme. A p o s s i b l e p h o s p h o l i p i d r e g u l a t i o n o f t h e p r o p e r t i e s o f c h o l i n o c e p t i v e p r o t e i n s h a s b e e n s u g g e s t e d by Changeux ( 1 2 8 ) . P r e l i m i n a r y e x p e r i m e n t s s u g g e s t e d t h a t t h e c o n d i t i o n s wh 1Qh a b o l i s h e d t h e b r e a k i n t h e A r r h e n i u s p l o t d i d n o t a l t e r t h e a f f i n i t y o f t h e enzyme f o r MAC. However, i n o r d e r t o v e r i f y t h e c o n t r i b u t i o n o f c a r d i o l i p i n t o MAC a f f i n i t y , a c a r d i o l i p i n f r e e enzyme w i l l be r e q u i r e d . I s o l a t i o n And P u r i f i c a t i o n Of B o v i n e E r y t h r o c y t e AChE The p r i m a r y r e q u i r e m e n t f o r t h e use o f t h e d e t e r g e n t e x c h a n g e method (89) i n t h e s u b s t i t u t i o n of t h e endogeneous c a r d i o l i p i n f r o m AChE i s t h e a v a i l b i l i t y o f l a r g e q u a n t i t i e s o f p u r e AChE. The c o m m e r c i a l l y o b t a i n e d enzyme f r o m Sigma c o n t a i n e d two o t h e r p r o t e i n s w i t h l a r g e q u a n t i t i e s .of i n o r g a n i c s a l t , t h e r e b y m a k i n g i t an u n e c o n o m i c a l s o u r c e f o r o b t a i n i n g l a r g e q u a n t i t i e s of p u r e AChE. Most methods r e p o r t e d f o r t h e p u r i f i c a t i o n o f b o v i n e ( 2 5 ) o r human ( 1 3 , 1 9 , 5 7 ) e r y t h r o c y t e AChE use e i t h e r t h e c o m m e r c i a -l l y o b t a i n e d s o l u b l e f o r m of t h e enzyme (13) or d e t e r g e n t s o l u b i l i s e d (19) enzyme f o r a f f i n i t y c h r o m a t o g r a p h y . D e t e r g e n t s s u c h as T r i t o n X-100 a r e c a p a b l e of d i s s o c i a t i n g m u l t i p l e o l i g o m e r i c f o r m s i n v i t r o ( 3 4 ) . P r e s u m i n g t h a t s u c h a phenomenon a l s o e x i s t s i n v i v o , . t h e d-e.t e-r.g'e n t s o l u b i l i s e d f o r m may n o t r e p r e s e n t t h e n a t i v e f o r m o f t h e enzyme. T h i s i s c o n t r a r y t o t h e a l t e r n a t i v e b e l i e f t h a t T r i t o n X-100 may s u b s t i t u t e f o r t h e n a t i v e h y d r o p h o b i c e n v i r o n m e n t of t h e enzyme, t h e r e b y h e l p i n g t o m a i n t a i n t h e n a t i v e i n t a c t s t r u c t u r e of t h e enzyme (123) . A v e r y s m a l l p e r c e n t of AChE c a n be o b t a i n e d i n t h e s o l u b l e f o r m by h e m o l y s i s of b o v i n e e r y t h r o c y t e g h o s t i n h y p o - o s m o t i c p h o s p h a t e b u f f e r ( 1 5 ) , w h i l e t h e r e s t of i t i s r e l e a s e d i n t h e p a r t i c u l a t e f o r m . I n a p r e l i m i n a r y communica-t i o n i n 1 9 5 3 , Cohen and W a r r i n g a (24) h a v e d e s c r i b e d a non d e t e r g e n t method f o r t h e p u r i f i c a t i o n o f AChE f r o m ox e r y t h r o c y t e s . I n t h e p r e s e n t w o r k , v a r i o u s s t e p s i n v o l v e d i n t h e p u r i f i c a t i o n o f b o v i n e e r y t h r o c y t e AChE by t h e above method have b e e n c h a r a c t e r i s e d . B u t a n o l t r e a t m e n t has b e e n p r e v i o u s l y r e p o r t e d t o e x t r a c t p h o s p h o l i p i d s (74) and t h e r e b y f a c i l i t a t e p r o t e i n s o l u b i l i s a t i o n . I n o u r s t u d i e s , by q u a n t i f y i n g t h e p h o p h o l i p i d s b e f o r e and a f t e r t h e b u t a n o l e x t r a c t i o n o f t h e membrane, i t was f o u n d t h a t b u t a n o l t r e a t m e n t e x t r a c t s 70-80% o f v a r i o u s membrane p h o s p h o l i p i d s s u c h as p h o p h a t i d y l s e r i n e , p h o s p h a t i d y l -e t h a n o l a m i n e , p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l i n o s i t o l ( T a b l e 2 ) . T h i s i s i n ag r e e m e n t w i t h f i n d i n g s on v a r i o u s o t h e r membranes ( 7 2 , 7 3 ) , where i t was f o u n d t h a t 70-80% o f p h o s p h o l i p i d s a r e m o b i l e and c o u l d be e x t r a c t e d w h i l e t h e r e s t o f t h e 20-30% o f t h e p h o s p h o l i p i d s a r e i m m o b i l i s e d due t o p r o t e i n b i n d i n g . B u t a n o l e x t r a c t i o n o f t h e m o b i l e p h o s p h o l i p i d s somehow a l t e r s t h e membrane o r g a n i s a t i o n s u c h t h a t t h e e x t r a c t ab i i i t y of AChE i n c r e a s e s f r o m 20-to 8 0 % a r t e r b u t a n o l t r e a t m e n t . B u t a n o l t r e a t m e n t o f t h e f r e e z e d r i e d e l e c t r i c o r g a n of e e l has be e n r e p o r t e d t o e f f e c t t h e r e l e a s e of AChE (125) I n t h e o r i g i n a l c o m m u n i c a t i o n (24), lOmM s o d i u m p h o s p h a t e pH 8.0 was u s e d f o r t h e e x t r a c t i o n of t h e enzyme f r o m t h e b u t a n o l t r e a t e d membrane. As shown i n t a b l e 3 o n l y 40% of t h e enzyme was e x t r a c t e d u n d e r t h e s e c o n d i t i o n s . The e x t r a c t i o n c o u l d be i n c r e a s e d t o 80% by t h e i n c o r p o r a t i o n o f 0.IM N a C l , 0.2mM EDTA. The i n c r e a s e d s o l u b i l i s a t i o n c o u l d be a t t r i b u t e d t o t h e d i s r u p t i o n of i o n i c i n t e r a c t i o n s by 0.IM N a C l (129) and c a l c i u m c h e l a t i o n by 0.2mM EDTA ( 1 2 ) . The a d d i t i o n o f c a l c i u m t o t h e e x t r a c t i o n medium d e c r e a s e s t h e e x t r a c t a b i l i t y o f t h e enzyme ( 1 2 ) . P u r i f i c a t i o n o f t h e s o l u b i l i s e d enzyme by ammonium s u l p h a t e p r e c i p i t a i o n gave a v e r y p o o r y i e l d ( 2 5 % ) . A f f i n i t y p u r i f i c a t i o n by p a s s a g e o f t h e enzyme t h r o u g h a PTA a f f i n i t y c o l u m n , a t a l i g a n d c o n c e n t r a t i o n of 4.4 umo l e / ml g e l , r e s u l t e d i n 100% r e t e n t i o n o f t h e enzyme, b u t o n l y 20 and 8% were r e l e a s e d d u r i n g e l u t i o n w i t h 20mM d e c a m e t h o n i u m and 1% T r i t o n X-100, r e s p e c t i v e l y . 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