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Purification and characterization of chloroplast coupling factor from wheat Moase, Elaine Helen 1980

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PURIFICATION AND CHARACTERIZATION OF CHLOROPLAST COUPLING FACTOR FROM WHEAT by ELAINE HELEN MOASE B . S c , U n i v e r s i t y o f New Brunswick , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department o f Botany We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA June 1980 (5) E l a i n e Helen Moase, 1980 I n 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 a n 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 a n d 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 . D e p a r t m e n t n f Botany 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 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e 20 June 1980 i i ABSTRACT C h l o r o p l a s t c o u p l i n g f a c t o r (CF^) f rom t h y l a k o i d s o f wheat {Triticum aestivum, v a r T h a t c h e r ) was p u r i f i e d by c h l o r o f o r m e x t r a c t i o n and subsequent s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . The wheat enzyme, in common w i t h most c o u p l i n g f a c t o r s , c o n t a i n s f i v e s u b u n i t s , d e s i g n a t e d a, 3, y, 6 and e , o f m o l e c u l a r w e i g h t s 57, 55, 39, 25 and 13 kd r e s p e c t i v e l y . I n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s in a l l s t e p s o f t h e p u r i f i c a t i o n p r e v e n t e d t h e l o s s o f the two s m a l l e s t s u b u n i t s d u r i n g the s u c r o s e g r a d i e n t s t e p . A l t h o u g h p r o t e o l y s i s i n h i b i t o r s d i d not i n t e r f e r e w i t h the e x t r a c t i o n o f CF.j by c h l o r o f o r m , they c o m p l e t e l y p r e v e n t e d CF^ r e l e a s e f rom t h e t h y l a k o i d by the h y p o t o n i c s u c r o s e method o f S t ro tmann e t a l . (1973)-However , CF^ r e l e a s e by h y p o t o n i c s u c r o s e d i d not appear t o r e q u i r e a p r o t e o l y t i c e v e n t in any CF^ s u b u n i t , as no d i f f e r e n c e in a p p a r e n t m o l e c u l a r w e i g h t o f any CF^ s u b u n i t was o b s e r v e d when CF^ was r e l e a s e d by t h i s method . A d o u b l e band o r a p p a r e n t mw's 37 and 39 kd f o r the y s u b u n i t in SDS-PAGE was i d e n t i f i e d as reduced and unreduced forms o f t h e same s u b u n i t , s u g g e s t i n g an" i n t e r n a l d i s u l f i d e b r i d g e in t h e y s u b u n i t o f wheat CF . j . The l a t e n t CaATPase o f wheat CF^ i s a c t i v a t e d by t r y p s i n d i g e s t i o n but not by h e a t , un 1 i ke s p i n a c h C F ^ . Wheat CF^ h y d r o l y z e s GTP at 32% the r a t e o f A T P , but no o t h e r n u c l e o t i d e t r i p h o s p h a t e i s e f f e c t i v e as a s u b s t r a t e . A 1:1 r a t i o o f Ca :ATP gave o p t i m a l ATPase a c t i v i t y . and V were d e t e r m i n e d in the p r e s e n c e o f a l a r g e e x c e s s o f c a l c i u m max r (10 mM) and w i t h an e q u i m o l a r amount o f c a l c i u m and A T P . In the p r e s e n c e o f e x c e s s c a l c i u m , a K M o f 0.125 mM CaATP and a \lmsv o f 18.9 u n i t s / m g i i i p r o t e i n were o b t a i n e d . Under c o n d i t i o n s o f e q u i m o l a r c a l c i u m and A T P , t h e K.. was 0.018 mM CaATP and t h e V was 6.77 u n i t s / m g p r o t e i n . The M max • r o r d e r o f magn i tude d i f f e r e n c e in K ^ ' s i s thought t o be due t o an a l t e r a t i o n o f a f f i n i t y o f t h e enzyme f o r s u b s t r a t e by the p r e s e n c e . o r a b s e n c e o f e x c e s s c a l c i u m . E l e c t r o n m i c r o s c o p y o f n e g a t i v e l y - s t a i n e d wheat CF^ p a r t i c l e s showed a s o l i d hexagona l p a r t i c l e . Escherichia coli gave a s i m i l a r s t r u c t u r e . Markham r o t a t i o n s u p p o r t e d t h e c o n c l u s i o n t h a t wheat CF^ e x h i b i t s a s i x - f o l d symmetry. A n t i b o d i e s to wheat CF^ were g e n e r a t e d by i n j e c t i n g t h e p u r i f i e d enzyme i n t o a r a b b i t . The r e s u l t i n g a n t i s e r u m both p r e c i p i t a t e d wheat CF.j and i n h i b i t e d i t s l a t e n t , t r y p s i n - a c t i v a t e d C a A T P a s e . The serum c r o s s - r e a c t e d w i t h s p i n a c h C F ^ , showing t h a t t h e r e i s c o n s i d e r a b l e a n t i g e n i c s i m i l a r i t y between the two enzymes. C r o s s e d i m m u n o e l e c t r o -p h o r e s i s showed t h a t t h i s a n t i s e r u m r e a c t s s p e c i f i c a l l y w i t h t h e a and £ s u b u n i t s o f wheat C F , . i. V TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 12 M a t e r i a l s 12 P l a n t m a t e r i a l 13 CF.j e x t r a c t i o n and p u r i f i c a t i o n 13 E l e c t r o p h o r e s i s 15 A s s a y s 16 E l e c t r o n m i c r o s c o p y 18 P r e p a r a t i o n and c h a r a c t e r i z a t i o n o f m o n o s p e c i f i c a n t i b o d i e s t o wheat CF^ 19 RESULTS 22 P u r i f i c a t i o n o f wheat CF^ 22 The e f f e c t o f p r o t e o l y s i s i n h i b i t o r s on e x t r a c t i o n o f wheat CF^ 26 The y s u b u n i t o f wheat CF^ c o n t a i n s an i n t e r n a l * ;1 d i s u l f i d e b r i d g e 31 ATPase a c t i v i t y o f wheat CF^ 37 E l e c t r o n m i c r o s c o p y o f wheat CF^ kS Immuno log i ca l s t u d y o f wheat CF^ hS DISCUSSION 5h SUMMARY 64 V LIST OF TABLES Page T a b l e I. P u r i f i c a t i o n o f wheat CaATPase 25 T a b l e II. A p p a r e n t m o l e c u l a r w e i g h t s o f s u b u n i t s o f wheat and s p i n a c h C F ^ ' s 25 T a b l e I I I . H e a t - a c t i v a t e d CaATPase a c t i v i t y o f wheat CF.j t r e a t e d a t v a r i o u s t e m p e r a t u r e s 38 T a b l e IV. S u b s t r a t e s p e c i f i c i t y o f the CaATPase a c t i v i t y o f wheat C F i  v i L IST OF FIGURES Page F i g u r e 1. D iagram o f c r o s s e d I m m u n o e l e c t r o p h o r e s i s g e l 21 F i g u r e 2. P r o f i l e o f s u c r o s e d e n s i t y g r a d i e n t used t o p u r i f y wheat CF.| 23 F i g u r e 3- SDS-PAGE o f c r u d e CF-| sample and p r o t e i n f r a c t i o n s f rom a s u c r o s e d e n s i t y g r a d i e n t 2k F i g u r e k. Compar ison o f c r u d e CF-| e x t r a c t s p r e p a r e d by the c h l o r o f o r m method w i t h t h a t p r e p a r e d by the h y p o t o n i c s u c r o s e method , and the e f f e c t o f i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s upon each 27 F i g u r e 5. E f f e c t o f the i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s d u r i n g CF-| e x t r a c t i o n and p u r i f i c a t i o n upon s u b u n i t c o m p o s i t i o n o f wheat CF^ 28 F i g u r e 6. P r o t e o l y s i s i n h i b i t o r s p r e s e n t d u r i n g s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p r e v e n t l o s s o f the 6 and e s u b u n i t s o f wheat CF^ 30 F i g u r e 7. SDS-PAGE o f wheat and s p i n a c h CF-j , showing a d o u b l e band in the y s u b u n i t p o s i t i o n 32 F i g u r e 8. L i m i t e d p r o t e o l y s i s o f e a c h o f the two bands o f the y s u b u n i t d imer and o f the a and g s u b u n i t s 3k F i g u r e 9- E f f e c t o f r e d u c i n g agent upon the m o b i l i t y o f the y s u b u n i t o f wheat C F 1 in SDS-PAGE 35 F i g u r e 10. E f f e c t o f heat upon the number o f bands found in the y s u b u n i t upon SDS-PAGE 36 F i g u r e 11. T ime c o u r s e o f t r y p s i n a c t i v a t i o n o f wheat CaATPase in the p r e s e n c e and a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s 39 F i g u r e 12. E f f e c t o f the r a t i o o f c a l c i u m t o ATP upon ATPase a c t i v i t y o f t r y p s i n ~ a c t i v a t e d CF^ from wheat k2 F i g u r e 13- M i c h a e l i s - M e n t e n k i n e t i c s o f the t r y p s i n - a c t i v a t e d CaATPase o f wheat CF^ in the p r e s e n c e o f 10 mM C a C ^ . k3 F i g u r e \k. M i c h a e l i s - M e n t e n k i n e t i c s o f the t r y p s i n - a c t i v a t e d CaATPase in the p r e s e n c e o f e q u i m o l a r amounts o f c a l c i u m and ATP kk v i i F i g u r e 1 5 - E l e c t r o n m i c r o g r a p h s o f wheat CF-j , n e g a t i v e l y -s t a i n e d w i t h two d i f f e r e n t s t a i n s 4 6 F i g u r e 1 6 . E l e c t r o n m i c r o g r a p h s o f Escherichia coli F-| , n e g a t i v e l y - s t a i n e d w i t h ammonium m o l y b d a t e 4 7 F i g u r e 1 7 - S e l e c t e d CF.. p a r t i c l e s , showing v a r i o u s d e g r e e s o f hexagonal s t r u c t u r e - 4 8 F i g u r e 1 8 . Markham r o t a t i o n o f t h r e e wheat C F 1 p a r t i c l e s 5 0 F i g u r e 1 9 - O u c h t e r l o n y d o u b l e d i f f u s i o n p l a t e e s t i m a t i o n o f a n t i b o d y t i t e r in serum from a r a b b i t immunized a g a i n s t wheat CF^ 51 F i g u r e 2 0 . C r o s s e d I m m u n o e l e c t r o p h o r e s i s o f the s e p a r a t e d s u b u n i t s o f wheat CF^ a g a i n s t a n t i - w h e a t CF^ s e r u m . . 51 F i g u r e 2 1 . I n h i b i t i o n o f the t r y p s i n - a c t i v a t e d CaATPase a c t i v i t y o f wheat CF^ by a n t i - w h e a t CF^ serum 5 3 F i g u r e 2 2 . P r o p o s e d model f o r the ar rangement o f the s u b u n i t s o f wheat C F n w i t h i n the enzyme complex 6 0 v i i i ACKNOWLEDGEMENTS T h i s r e s e a r c h would not have been p o s s i b l e but f o r the g u i d a n c e and s u p e r v i s i o n o f D r . B e v e r l e y R. G r e e n , and I w i s h t o e x p r e s s my s i n c e r e g r a t i t u d e . Thanks a l s o go to D r . R. S t a c e - S m i t h , Ms. L o i s Johns and Mr . A d i b Rowhani o f the A g r i c u l t u r e Canada l a b o r a t o r i e s in Vancouver f o r t h e i r a s s i s t a n c e in t h e i m m u n i z a t i o n o f r a b b i t s f o r t h i s s t u d y . The a s s i s t a n c e o f Mr . L a s z l o V e t o in a l l a s p e c t s o f the e l e c t r o n m i c r o s c o p y s t u d y i s g r a t e f u l l y a c k n o w l e d g e d . I have had many h e l p f u l d i s c u s s i o n s w i t h D r . P . D . Bragg ( U . B . C . D e p t . o f B i o c h e m i s t r y ) and D r s . A . D . M . G l a s s and G . H . N . Towers ( U . B . C . D e p t . o f B o t a n y ) . S p e c i a l thanks go t o D r . E d i t h Camm f o r her a s s i s t a n c e and e n c o u r a g e m e n t . My thanks to N . S . E . R . C . C f o r f i n a n c i a l a s s i s t a n c e f o r the p e r i o d 1977 t o 1979. 1 INTRODUCTION The "common c u r r e n c y " o f e n e r g y in l i v i n g c e l l s i s the m o l e c u l e a d e n o s i n e t r i p h o s p h a t e ( A T P ) . The p h o s p h o d i e s t e r bond between the S and Y p h o s p h a t e s o f t h i s m o l e c u l e i s o f s u f f i c i e n t energy t h a t i t s c l e a v a g e can p r o v i d e e n e r g y t o d r i v e the many p r o c e s s e s o f the l i v i n g c e l l ( L i p m a n n , 19^6). The mechanism o f ATP s y n t h e s i s and the o r g a n i z a t i o n o f enzymes i n v o l v e d in t h i s p r o c e s s now a p p e a r t o be r e m a r k a b l y s i m i l a r among the many l i v i n g sys tems s t u d i e d . By I960 i t was known t h a t c h l b r o p l a s t s (Arnon e t al . , 195*0 and m i t o c h o n d r i a (Kennedy and L e h n i n g e r , 19^8) a r e t h e s i t e s of p h o t o s y n t h e t i c and o x i d a t i v e p h o s p h o r y l a t i o n of ATP in p l a n t s and a n i m a l s . T h e s e o r g a n e l l e s e a c h c o n t a i n membrane-bound compartments and an e l e c t r o n t r a n s p o r t c h a i n . A t c e r t a i n p o i n t s in the e l e c t r o n t r a n s p o r t c h a i n s , the p a s s a g e of e l e c t r o n s i s t i e d t o ATP s y n t h e s i s . It was p o s t u l a t e d t h a t a h i g h e n e r g y c h e m i c a l i n t e r m e d i a t e p roduced at t h e s e s i t e s dona ted i t s energy f o r t h e s y n t h e s i s o f ATP ( S l a t e r , 1953). In 1961, the E n g l i s h b i o c h e m i s t P e t e r M i t c h e l l p r o p o s e d a r a d i c a l l y d i f f e r e n t scheme f o r the s y n t h e s i s o f A T P . In the c h e m i o s m o t i c h y p o t h e s i s ( M i t c h e l l , 1961), he p o s t u l a t e d t h a t the f l o w o f e l e c t r o n s t h r o u g h the e l e c t r o n t r a n s p o r t c h a i n c a r r i e d w i t h i t p r o t o n s , d e l i v e r i n g them t o one s i d e o f a membrane. T h i s r e s u l t s in the a c c u m u l a t i o n o f p r o t o n s on one s i d e o f t h e membrane, and the f o r m a t i o n o f an e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t a c r o s s the membrane. The d i s s i p a t i o n o f t h i s g r a d i e n t by the l e a k a g e o f p r o t o n s back a c r o s s the membrane was p r o p o s e d t o be the d r i v i n g f o r c e f o r ATP s y n t h e s i s . C r i t i c a l t o t h i s h y p o t h e s i s i s t h e membrane a c r o s s which the e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t i s g e n e r a t e d , and the a s s y m -2 m e t r i c a l a r rangement o f components o f the e l e c t r o n t r a n s p o r t c h a i n , r e s u l t i n g in the a c c u m u l a t i o n o f p r o t o n s on one s i d e o f the membrane. In c h l o r o p l a s t s , the f i r s t e v i d e n c e f o r a p r o t o n g r a d i e n t d r i v i n g ATP s y n t h e s i s came in 1966 f rom the work o f J a e g e n d o r f and U r i b e . C h l o r o p l a s t s , t r a n s f e r r e d f rom h i g h pH t o low pH s o l u t i o n s in the d a r k , s y n t h e s i z e d ATP when ADP and i n o r g a n i c p h o s p h a t e were p r o v i d e d . A pH g r a d i e n t a c r o s s the t h y l a k o i d was s u f f i c i e n t d r i v i n g f o r c e f o r ATP g e n e r a t i o n . Over the p a s t twenty y e a r s , much e v i d e n c e has been a c c u m u l a t e d to s u p p o r t t h e c h e m i o s m o t i c h y p o t h e s i s o f ATP s y n t h e s i s , a l t h o u g h the mechanism by wh ich ATP i s made in c o n j u c t i o n w i t h the d i s s i p a t i o n o f the e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t i s s t i l l i n c o m p l e t e l y u n d e r s t o o d . For s i x v iews on t h i s s u b j e c t by s i x s c i e n t i s t s a c t i v e in t h i s f i e l d , see t h e m u l t i a u t h o r e d r e v i e w by Boyer e t a l . ( 1 9 7 7 ) • The c o u p l i n g o f ATP s y n t h e s i s to the e l e c t r o n t r a n s p o r t - g e n e r a t e d e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t i s t h r o u g h an enzyme complex wh ich i s dubbed " c o u p l i n g f a c t o r " o r " F ^ " . In both c h l o r o p l a s t s and m i t o c h o n d r i a , i t has been shown t h a t e x t r a c t i o n w i t h an EDTA s o l u t i o n removes a p r o t e i n component , r e s u l t i n g in u n c o u p l i n g ( J a e g e n d o r f and S m i t h , 1 9 6 1 ; P e n e f s k y e t a l . , 1 9 6 0 ) . When t h i s c o u p l i n g f a c t o r i s added back t o the d e f i c i e n t membranes in the p r e s e n c e o f an a p p r o p r i a t e d i v a l e n t c a t i o n , e l e c t r o n t r a n s p o r t - d e p e n d e n t ATP s y n t h e s i s i s resumed ( A v r o n , 1 9 6 3 ; P e n e f s k y e t a l . , I 9 6 0 ) under a p p r o p r i a t e c o n d i t i o n s . The s o l u b i l i z e d c o u p l i n g f a c t o r w i l l c a t a l y z e the r e v e r s e r e a c t i o n t o ATP s y n t h e s i s , t h a t i s , ATP h y d r o l y s i s . For the m i t o c h o n d r i a l F^ , ATPase a c t i v i t y i s m a n i f e s t in the s o l u b l e enzyme (Pu l lman e t a l . , I 9 6 0 ) , but the a c t i v i t y o f the c h l o r o p l a s t enzyme i s e x h i b i t e d o n l y a f t e r a s h o r t d i g e s t i o n 3 w i t h t r y p s i n , o r by h e a t i n g t o 65*C f o r two m i n u t e s (Vambutas and R a c k e r , 1965). A number o f d i f f e r e n t ATPase a c t i v i t i e s a r e a s s o c i a t e d w i t h c h l o r o -p l a s t c o u p l i n g f a c t o r ( C F ^ ) , as d e m o n s t r a t e d by t h e f a c t t h a t s e p a r a t i o n of the a c t i v i t y f rom the t h y l a k o i d r e s u l t s in a l o s s o f c o u p l i n g , and r e a s s o c i a t i o n o f t h i s p r o t e i n w i t h the t h y l a k o i d r e s t o r e s i t . B a 1 t s c h e f f s k y (1959) and W e s s e l s and B a l t s c h e f f s k y ( i960) f i r s t showed t h a t a Mg-dependent ATPase a c t i v i t y was a s s o c i a t e d w i t h t h y l a k o i d s . A v r o n (1962, 1963) d e m o n s t r a t e d a MgATPase e x t r a c t e d f rom t h y l a k o i d s by EDTA s o l u t i o n . Vambutus and Racker (1965) p r e p a r e d a s t a b l e p r o t e i n f r a c t i o n wh ich showed a CaATPase a c t i v i t y a f t e r e i t h e r a s h o r t t r y p s i n d i g e s t i o n , o r two m i n u t e s h e a t i n g at 6 5 ° C . F a r r o n (1970) and F a r r o n and Racker (1970) c h a r a c t e r i z e d the p r o p e r t i e s o f t h e h e a t - a c t i v a t e d C a A T P a s e . By heat a c t i v i a t i o n a t pH 6 .0 in m a l e a t e b u f f e r and 8 mM M g C ^ , N e l s o n e t a 1. (1972a) o b t a i n e d a MgATPase a c t i v i t y f rom s o l u b l e C F ^ . in vivo, CF.j i s a t t a c h e d t o the o u t e r s u r f a c e o f the t h y l a k o i d membrane. It has been i d e n t i f i e d as t h e k n o b - l i k e p r o j e c t i o n s seen on the s u r f a c e o f t h y l a k o i d s t h a t have been n e g a t i v e l y s t a i n e d ( L e i n and R a c k e r , 1971a). T h e s e knobs can be removed by a number o f t r e a t m e n t s known t o u n c o u p l e c h l o r o p l a s t s , s u c h as EDTA wash (Howel l and M o u d r i a n a k i s , 1967) o r h y p o t o n i c s u c r o s e wash ( M i l l e r and S t a e h e l i n , 1976). M i l l e r and S t a e h e l i n (1976) d e m o n s t r a t e d , by f r e e z e - f r a c t u r e t e c h n i q u e s , t h a t CF^ p r o j e c t i o n s on the t h y l a k o i d s u r f a c e a r e l i m i t e d to t h o s e r e g i o n s wh ich a r e not s t a c k e d . In the m i t o c h o n d r i o n , F.j has a l s o been i d e n t i f i e d w i t h k n o b - l i k e p r o j e c t i o n s t h a t can be removed by p r o c e d u r e s known t o remove F^ . In the n a t i v e c o n f o r m a t i o n o f m i t o -c h o n d r i a l membranes, F^ i s a t t a c h e d t o the i n n e r s u r f a c e o f the i n n e r m i t o c h o n d r i a l membrane, p r o j e c t i n g i n t o the m i t o c h o n d r i a l m a t r i x . P r o t o n s a r e pumped by the e l e c t r o n t r a n s p o r t c h a i n i n t o the i n t r a t h y l a k o i d s p a c e o f c h l o r o p l a s t s , w h i l e in m i t o c h o n d r i a , p r o t o n s a r e pumped out o f the m a t r i x . -The d i f f e r e n c e in d i r e c t i o n o f p r o t o n pumping e x p l a i n s the d i f f e r e n t o r i e n t a t i o n o f t h e s e two c o u p l i n g f a c t o r c o m p l e x e s . The c o u p l i n g f a c t o r complex b i n d s to an i n t r i n s i c membrane c o m p l e x , d e n o t e d F^. T o g e t h e r F Q " ^ c o m p r i s e s a complex which c o n t a i n s bo th the p r o t o n channe l by wh ich the c r o s s - m e m b r a n e p r o t o n g r a d i e n t i s d i s s i p a t e d in a c o n t r o l l e d manner , and the enzyme wh ich u t i l i z e s the e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t t o s y n t h e s i z e A T P . C F ^ has been p u r i f i e d and r e c o n s t i -t u t e d i n t o p h o s p h o l i p i d v e s i c l e s . When CF^ was added t o t h e s e v e s i c l e s , ATP was s y n t h e s i z e d i n ' r e s p o n s e t o an a c i d - t o - b a s e t r a n s i t i o n ( P i c k and R a c k e r , 1979). When the p r o t o n - t r a n s l o c a t i n g c h r o m a t o p h o r e b a c t e r i o r h o d o p s i n was i n c o r p o r a t e d i n t o C F g - c o n t a i n i n g v e s i c l e s , CF^ b i n d i n g gave v e s i c l e s wh ich c a t a l y z e d l i g h t - d e p e n d e n t ATP f o r m a t i o n ( Y o u n i s and W i n g e t , 1977). The number o f p o l y p e p t i d e s in CFg i s u n c l e a r . T h r e e or f o u r CFQ p o l y -p e p t i d e s have been r e p o r t e d ( Y o u n i s and W i n g e t , 1977; P i c k and R a c k e r , 1979; E l l e n s o n e t a l . , 1978), a l t h o u g h one p o l y p e p t i d e , o f mw 8300, when r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s , f u n c t i o n e d as a p r o t o n pore t h a t c o u l d be b l o c k e d by the u n c o u p l e r d i c y c l o h e x y l c a r b o d i i m i d e (DCCD) (•Nelson e t a 1 . ,,1(977) . The o t h e r p o l y p e p t i d e s in the C F Q complex may a c t as r e g u l a t o r y o r C F ^ - a t t a c h m e n t c o m p o n e n t s . The CF^ complex i s r e l e a s e d f rom the t h y l a k o i d by a number o f d i f f e r e n t t r e a t m e n t s , i n c l u d i n g wash ing in EDTA ( L i e n and R a c k e r , 1971b), h y p o t o n i c s u c r o s e (S t ro tmann e t a l . , 1973)> o r s i 1 i c o t u n g s t i c a c i d ( L i e n and R a c k e r , 1971a), and e m u l s i f y i n g a t h y l a k o i d s u s p e n s i o n in c h l o r o f o r m ( Y o u n i s e t a l . , 1977). Once r e l e a s e d f rom the t h y l a k o i d , CF^ i s c o l d l a b i l e u n l e s s c r y o p r o t e c t e d in a number o f ways . L a b i l i t y i s c h a r a c t e r i z e d by a l o s s o f 5 ' C a A T P a s e a c t i v i t y , due t o d i s s o c i a t i o n o f t h e c o m p l e x . CF^ c a n be s t o r e d a t k° C o n l y i n a s a t u r a t e d s o l u t i on' o f amnion i u m ' s u l f a t e . Upon f r e e z i n g , CF.j i s c o l d - l a b i l e u n l e s s s t o r e d i n t h e p r e s e n c e o f 2 mM ATP and s u c r o s e o r g l y c e r o l ( L i e n e t a l . , 1 9 7 2 ) . R e a s s o c i a t i o n o f CF^ w i t h t h e t h y l a k o i d t o g i v e r e s t o r a t i o n o f p h o t o p h o s p h o r y l a t i o n r e q u i r e s d i v a l e n t c a t i o n s , and i t a p p e a r s t h a t b o t h EDTA and h y p o t o n i c s u c r o s e w a s h m e t h o d s f o r CF^ e x t r a c t i o n a c t by r e m o v i n g t h o s e c r i t i c a l c a t i o n s r e q u i r e d f o r t h e b i n d i n g o f CF.| t o t h e t h y l a k o i d . S i 1 i c o t u n g s t i c a c i d i s a c h a o t r o p e , and i t p r o b a b l y a c t s by a l t e r i n g 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 b e t w e e n CF^ a n d i t s m e m b r a n e - a s s o c i a t e d l i n k , p e r m i t t i n g t h e s o l u b i l i z a t i o n o f t h e c o m p l e x . A new m e t h o d f o r t h e s o l u b i 1 i z a t i o n o f F^  by c h l o r o f o r m t r e a t m e n t h a s p r o v e n u s e f u l . B e e c h e y e t a l . ( 1 9 7 5 ) f o u n d t h a t F^  c o u l d be e x t r a c t e d f r o m b o v i ne h e a r t mi t o c h o n d r i a by e m u l s i f y i n g them i n c h l o r o f o r m . P r e s u m a b l y t h i s a c t s a s an o r g a n i c s o l v e n t , n e u t r a l i z i n g f o r c e s i n v o l v e d i n a t t a c h m e n t o f F^  t o t h e membrane. F^  p r e p a r e d by c h l o r o f o r m e x t r a c t i o n a p p e a r s t o h a v e much t h e same p r o p e r t i e s as F^  p r e p a r e d by o t h e r m e t h o d s e x c e p t t h a t i t i s no l o n g e r c o l d l a b i l e ( S p i t s b e r g and B l a i r , 1977; P e n i n e t a 1 . , 1 9 7 9 ) . Y o u n i s e t a l . ( 1 9 7 7 ) m o d i f i e d t h e c h l o r o f o r m m e t h o d t o e x t r a c t s p i n a c h C F ^ . The p r o p e r t i e s o f CF^ n o t e d h e r e a r e c h a r a c t e r i s t i c o f t h e enzyme c o m p l e x f r o m s p i n a c h . S i n c e s p i n a c h i s an e a s y p l a n t w i t h w h i c h t o w o r k , i t h a s b e e n t h e p l a n t o f c h o i c e f o r t h e s t u d y o f C F ^ , a n d i n d e e d h a s s e r v e d a s a m o d e l o r g a n i s m f o r t h e s t u d y o f c o u p l i n g f a c t o r s i n g e n e r a l . T h e p o l y p e p t i d e c o m p o s i t i o n o f C F ^ , and many o t h e r c o u p l i n g f a c t o r c o m p l e x e s , h a s b e e n w e l l s t u d i e d . CF^ i s a l a r g e c o m p l e x , w i t h a m o l e c u l a r w e i g h t i n t h e r a n g e o f 3 5 0 , 0 0 0 . I t i s composed o f f i v e s u b u n i t s , d e n o t e d a , 3, Y» ? ^nd e i n o r d e r o f d e c r e a s i n g m o l e c u l a r w e i g h t ( R a c k e r 6 et a l . , 1971; L ien et a l . , 1972). The observed pa t te rn of po lypep t ides ob ta ined in SDS-PAGE has proven to be c h a r a c t e r i s t i c o f coup l ing f a c t o r s , having been found f o r the coup l i ng f a c t o r s of mi tochondr ia (Brooks and S e n i o r , 1971; C a t t e r a l l and Pederson, 1971), Escherichia c o l i (Wilson and Smi th , 1977), Streptococcus f a e c a l i s (Abrams et a l . , 1976), Micrococcus lysodeikticus (Huberman and Sa1 t o n , 1979) , the t he rmoph i l i c bacter ium PS3 (Ypshida et a l . , 1975), and the thermophi1 ic b lue-green a lga Mastigocladus laminosus (B inder and Bachofen, 1979)• A l t h o u g h t h e number o f d i f f e r e n t po lypep t ides compr is ing coup l i ng f a c t o r complexes i s g e n e r a l l y accepted as f i v e , the s t o i ch i ome t r y of these subun i ts i s by no means c l e a r . S to i ch iome t r ies o f 3a: 3 3 : l y : 1<$: 1e ( S e n i o r and Brooks , 1971; Yoshida et a l . , 1979), 2 a : 23 : 2y : ?<5: 2e (Sen i o r , -1975) and 2 a : 2g : 1y: 16: 2e (Binder et a 1. , 1978) have been repor ted f o r CF^ and o ther coup l i ng f a c t o r s . Excep t ions e x i s t in the l i t e r a t u r e . There are severa l repor ts o f p r o t o n - t r a n s l o c a t i n g ATPases that have a sma l le r number of p o l y p e p t i d e s , such as the two po lypep t ide F ^ 1 s o f Bacillus magaterium (Mirsky and Bar low, 1973) and B a c i l l u s s u b t i l i s (Ser rah ima-Ze iger and M o n t e i l , 1978), and the three po lypep t ide F^ o f Clostridium pasteurianum (C la rke et a l . , 1979). Roles in photophosphory la t ion have been ass igned to some of the subun i ts o f CF^ . Removal o f the 6 subuni t prevents b ind ing o f the complex to the t h y l a k o i d (Younis et a l . , 1977), but though coup l i ng i s p reven ted , t h i s d e f i c i e n t complex s t i l l has ATPase a c t i v i t y . Coupl ing a c t i v i t y can be res to red by a d d i t i o n of a minor subuni t f r a c t i o n c o n t a i n i n g the <5 and e s u b u n i t s . Recent ly Cox e t a 1. , (1978) demonstrated that e x t r a c t i o n o f E. coli F^ by a low i o n i c s t reng th wash was prevented by an i n h i b i t o r o f p r o t e o l y s i s , p-aminobenzamidine. Bragg and Hou (1979) found that the 6 7 s u b u n i t o f ECF^ undergoes a p r o t e o l y t i c c l e a v a g e d u r i n g r e l e a s e f rom the b a c t e r i a l membrane. I n h i b i t i o n o f t h i s p r o t e o l y s i s p r e v e n t s r e l e a s e o f ECF.J . T h i s e v i d e n c e i n d i c a t e s t h a t the 6 s u b u n i t o f c o u p l i n g f a c t o r s i s i n v o l v e d i n t h e a t t a c h m e n t o f t h e complex t o t h e membrane. The e s u b u n i t a c t s as an i n h i b i t o r o f the l a t e n t , h e a t - a c t i v a t e d ATPase o f s p i n a c h CF^ (Ne lson e t a 1., 1972b). I ts i n h i b i t o r y a c t i v i t y i s l o s t upon d i g e s t i o n by t r y p s i n ( N e l s o n e t a l . , 1975)- It has been p r o p o s e d t h a t t h e e s u b u n i t p r e v e n t s ATP h y d r o l y s i s by CF^ when i t i s a t t a c h e d t o the membrane, as w e l l as b e i n g r e s p o n s i b l e f o r the l a t e n c y o f ATPase a c t i v i t y o f the s o l u b l e C F 1 ( B a k k e r - G r u n w a l d and Van Dam, 1974; N e l s o n e t a l . , 1975). A c t i v a t i o n o f CF^ p r o b a b l y p r o c e e d s by removal o r d i g e s t i o n o f the e s u b u n i t . The a c t u a l c a t a l y t i c s i t e o f CF^ has been d i f f i c u l t to l o c a t e , as CF^ d e f i c i e n t in one o r more s u b u n i t s does not g i v e c o u p l i n g o f p h o s p h o r y l a t i o n to e l e c t r o n t r a n s p o r t . In s t u d y i n g the l o c a t i o n o f the a c t i v e s i t e o f ATPase a c t i v i t y in the s o l u b l e , a c t i v a t e d enzyme, one makes the a s s u m p t i o n t h a t ATP s y n t h e s i s and ATP h y d r o l y s i s o c c u r a t t h e same s i t e . F a r r o n and Racker (1970) o b s e r v e d an i n c r e a s e in the number o f t i t r a b l e s u l f h y d r y l g r o u p s on the n a t i v e CF^ a f t e r heat a c t i v a t i o n o f the l a t e n t A T P a s e . T h e s e n e w l y - e x p o s e d s u l f h y d r y l g roups c o u l d be o x i d i z e d w i t h o -i o d o s o b e n z o a t e t o form two new d i s u l f i d e b r i d g e s , one e a c h in t h e 8 and y s u b u n i t s (Andreo e t a l . , 1979). S i m i l a r e x p e r i m e n t s w i t h the membrane-bound GF^ showed an i n c r e a s e in ; N - e t h y l m a l e i m i d e b i n d i n g t o the y s u b u n i t upon i l l u m i n a t i o n (McCar ty and F a g e n , 1973). These o b s e r v a t i o n s s u g g e s t a c o n f o r m a t i o n a l change i n t h e y s u b u n i t o f CF^ upon s t i m u l a t i o n o f p h o t o p h o s -p h o r y l a t i o n by i l l u m i n a t i o n o r upon heat a c t i v a t i o n o f the l a t e n t C a A T P a s e . N e l s o n e t a l . (1973) a t t empted to d e t e r m i n e which s u b u n i t s o f CF^ were i n v o l v e d in ATP s y n t h e s i s and h y d r o l y s i s by r e a c t i n g C F 1 w i t h a n t i -8 b o d i e s g e n e r a t e d a g a i n s t each o f the s u b u n i t s . No s i n g l e a n t i s e r u m was a b l e t o i n h i b i t the ATPase a c t i v i t y o f i s o l a t e d C F ^ . E i t h e r a n t i - a o r a n t i - y serum i n h i b i t e d p h o t o p h o s p h o r y l a t i o n and the l i g h t - t r i g g e r e d MgATPase a c t i v i t y in i s o l a t e d c h l o r o p 1 a s t s . A l t h o u g h the a u t h o r s c o n c l u d e d t h a t the a and y s u b u n i t s a r e i n v o l v e d in p h o t o p h o s p h o r y l a t i o n , K o e n i g and c o - w o r k e r s (1978) o b t a i n e d i n h i b i t i o n o f ATPase a c t i v i t y w i t h a n t i s e r a a g a i n s t any o f the s u b u n i t s o f C F ^ . The d i f f e r e n c e in r e s u l t s i s most l i k e l y due t o d i f f e r e n t a n t i g e n i c s i t e s between a n t i b o d y p r e p a r a t i o n s . T h i s d e m o n s t r a t e d t h a t one s h o u l d be e x t r e m e l y c a u t i o u s about making c o n c l u s i o n s about a c t i v e s i t e s s o l e l y upon the e v i d e n c e o f m o n o s p e c i f i c a n t i body i n h i b i t ! o n o f : e n z y m e a c t i v i t y . . P h o t o c h e m i c a l l a b e l l i n g o f h e a t - a c t i v a t e d CF^ d e m o n s t r a t e d ATP b i n d i n g s i t e s on the 3 s u b u n i t , and ADP s i t e s on both the a and 3 s u b u n i t s ( C a r l i e r e t . a l . , 1 9 7 9 ) • However , i t i s d i f f i c u l t t o say whether t h e s e s i t e s a r e a l l c a t a l y t i c , o r a l l r e g u l a t o r y in c h a r a c t e r , o r a m i x t u r e o f b o t h . N u c l e o t i d e b i n d i n g a n a l y s e s tend t o g i v e more i n f o r m a t i o n on the mechanism o f p h o s p h o r y l a t i o n than on the f u n c t i o n of the component p o l y p e p t i d e s . F o r an o v e r v i e w o f i n f o r m a t i o n on n u c l e o t i d e b i n d i n g s i t e s on c o u p l i n g f a c t o r s , see the r e v i e w by H a r r i s (1978). The r e c o n s t r u c t i o n e x p e r i m e n t s o f Y o s h i d a e t al.(1977) have p r o v i d e d some v e r y s u g g e s t i v e i n f o r m a t i o n on the r o l e o f i n d i v i d u a l s u b u n i t s in ATPase a c t i v i t y . I n d i v i d u a l s u b u n i t s o f F^ from the t h e r m o p h i l i c b a c t e r i u m PS3 were added back t o g e t h e r and the r e c o n s t i t u t e d complex was a s s a y e d f o r the a b i l i t y t o h y d r o l y z e A T P . No ATPase a c t i v i t y was o b t a i n e d u n l e s the 3 s u b u n i t was i n c l u d e d in the r e c o n s t i t u t e d c o m p l e x , and a l 1 ' c o m b i n a t i o n s o f s u b u n i t s c o n t a i n i n g the 3 and y s u b u n i t s e x h i b i t e d i d e n t i c a l c a t a l y t i c p r o p e r t i e s and amount o f ATPase a c t i v i t y t o t h o s e o f the n a t i v e PS3 F n . 9 However , r e c o n s t i t u t i o n o f the c o u p l i n g f a c t o r s f rom o r g a n e l l e s and m e s o p h i l i c b a c t e r i a seems t o be more d i f f i c u l t , p r o b a b l y because the amino a c i d s e q u e n c e s o f t h e i r p o l y p e p t i d e s make them l e s s r e s i s t a n t to the d i s s o c i a t i n g a g e n t s and the m a n i p u l a t i o n n e c e s s a r y t o r e c o n s t r u c t the complex (Kagawa, 1978). A n o t h e r a p p r o a c h to d e t e r m i n i n g the s u b u n i t o r s u b u n i t s n e c e s s a r y f o r ATPase a c t i v i t y has been t o remove s u b u n i t s f rom F^ by t r y p s i n d i g e s t i o n , as was done w i t h the enzyme f rom Micrococcus lysodeikticus ( M o l l i n e d o e t a l . , I98O). T r y p s i n removes c o n s i d e r a b l e amounts o f the a and y s u b u n i t s , but no 3 s u b u n i t . A form o f the ATPase w i t h a s u b u n i t r a t i o o f 1.67a: 33: 0.17y e x h i b i t e d more than t w i c e the a c t i v i t y o f the n a t i v e enzyme, but removal o f more a and y gave an i n a c t i v e A T P a s e . Thus i t a p p e a r s t h a t the 3 s u b u n i t in M. lysodeikticus F^ i s a b s o l u t e l y r e q u i r e d f o r ATPase a c t i v i t y , but a and y may be n e c e s s a r y in l e s s e r amounts as r e g u l a t o r s o r e f f e c t o r s . The e v i d e n c e f rom s t u d i e s o f o t h e r c o u p l i n g f a c t o r complexes i s t h a t the 3 s u b u n i t i s p r o b a b l y the a c t i v e s i t e o f ADP p h o s p h o r y l a t i o n and ATPase a c t i v i t y . . It i s t e m p t i n g to compare c a t a l y t i c f u n c t i o n s o f the s u b u n i t s o f t h e above ment ioned c o u p l i n g f a c t o r s w i t h t h o s e o f C F ^ , but o n l y f u r t h e r r e s e a r c h w i l l p r o v e whether t h i s i s a v a l i d c o m p a r i s o n . The o b j e c t o f t h i s r e s e a r c h was to c h a r a c t e r i z e some o f the p r o p e r t i e s o f the CFj f rom the wheat c h l o r o p l a s t . To t h i s e n d , a method was d e v i s e d f o r the p u r i f i c a t i o n o f wheat CF^ c o n t a i n i n g a l l i t s s u b u n i t s . , E l e c t r o -p h o r e t i c and e l e c t r o n m i c r o s c o p i c a n a l y s e s gave i n f o r m a t i o n about t h e s t r u c t u r e o f wheat C F ^ . F u n c t i o n a l d a t a was o b t a i n e d f rom e n z y m a t i c a n a l y s i s o f the l a t e n t A T P a s e . The v a l u e o f such a s t u d y i s t w o - f o l d . The s t r u c t u r a l and e n z y m a t i c p r o p e r t i e s o f C F 1 f rom a m o n o c o t y l e d o n have 10 never been c h a r a c t e r i z e d . One c a n n o t assume t h a t an enzyme f rom a mono-c o t y l e d o n has i d e n t i c a l p r o p e r t i e s t o t h a t f rom d i c o t y l e d o n s such as s p i n a c h , l e t t u c e o r s n a p d r a g o n . In a d d i t i o n , any i n f o r m a t i o n on the s t r u c t u r e and f u n c t i o n o f a c o u p l i n g f a c t o r f rom an u n s t u d i e d s p e c i e s w i l l add t o the b u l k o f i n f o r m a t i o n f rom wh ich t o draw when f o r m u l a t i n g a u n i f y i n g model about s t r u c t u r e and f u n c t i o n o f e n e r g y - t r a n s d u c i n g c o u p l i n g f a c t o r s . T h i s r e s e a r c h p r o j e c t o r i g i n a l l y i n c l u d e d a s t u d y o f t h e b i o s y n t h e s i s o f wheat c h l o r o p l a s t c o u p l i n g f a c t o r . It was d e c i d e d to a p p r o a c h t h i s p r o b l e m by i s o l a t i n g wheat RNA, t r a n s l a t i n g i t in an in vitro p r o t e i n s y n t h e s i z i n g s y s t e m , then i s o l a t i n g components o f CF^ t r a n s l a t e d in such a s y s t e m by immunoprec ip i t a t i o n , u s i n g m o n o s p e c i f i c a n t i b o d i e s t o the s u b u n i t s o f wheat C F ^ . Of s p e c i a l i n t e r e s t a r e the s u b u n i t s o f CF^ s y n t h e s i z e d o u t s i d e the c h l o r o p l a s t and t r a n s p o r t e d p o s t - t r a n s l a t i o n a l l y i n t o the o r g a n e l l e . M e n d i o l a - M o r g e n t h a l e r e t a l . (1976) showed t h a t the i s o l a t e d i n t a c t c h l o r o p l a s t s f rom s p i n a c h s y n t h e s i z e o n l y the a, 6 and e s u b u n i t s o f CF.j . From t h i s they c o n c l u d e d t h a t the y and 6 s u b u n i t s a r e s y n t h e s i z e d in the c y t o p l a s m . T h e s e f i n d i n g s were c o n f i r m e d by two o t h e r g roups ( E l l i s , 1977; B o u t h y e t t e and J a e g e n d o r f , 1978). R e c e n t l y , N e l s o n e t a l . (1980) found e v i d e n c e t h a t the y s u b u n i t , as w e l l as a, 6 and e a r e s y n t h e s i z e d in i s o l a t e d i n t a c t s p i n a c h c h l o r o p l a s t s . They r e p o r t e d t h a t the 6 s u b u n i t i s s y n t h e s i z e d as a s l i g h t l y l a r g e r p r e c u r s o r p o l y p e p t i d e in the c y t o p l a s m . S t u d i e s on the c y t o p l a s m i c a l l y - s y n t h e s i z e d s u b u n i t s o f y e a s t m i t o c h o n d r i a l F.j ( M a e c c e c c h i n i e t a l . , 1979) and the sma l l s u b u n i t o f c h l o r o p l a s t r i b u l o s e - 1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e ( H i g h f i e l d and E l l i s , 1978; Cashmore e t a l . , 1979; Chua and S c h m i d t , 1978) show t h a t t h e s e p o l y p e p t i d e s a r e o r i g i n a l l y made as l o n g e r p o l y p e p t i d e s . At some p o i n t d u r i n g t h e i r t r a n s p o r t i n t o t h e o r g a n e l l e , the p o l y p e p t i d e s a r e tr immed down t o t h e i r f i n a l s i z e , 11 p r o b a b l y by s p e c i f i c enzymes . Time d i d not p e r m i t the c o m p l e t i o n o f such a s t u d y o f t h e wheat CF ^ s u b u n i t s . P r e s e n t e d here a r e the r e s u l t s o f the i m m u n i z a t i o n o f one r a b b i t a g a i n s t wheat C F ^ , and the c h a r a c t e r i z a t i o n o f the a n t i g e n i c p r o p e r t i e s o f the wheat a n t i b o d i e s p r o d u c e d . 12 MATERIALS AND METHODS The f o l l o w i n g a b b r e v i a t i o n s a r e used t h r o u g h o u t t h i s t e x t : SDS, sodium d o d e c y l s u l f a t e ; S D S - P A G E , SDS p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s ; T r i s , t r i s ( h y d r o x y m e t h y l ) aminomethane; T r i c i n e , N - t r i s (hydroxymethy l ) m e t h y l g l y c i n e ; EDTA, e t h y l e n e d i a m i n e - t e t r a a c e t a t e a c i d d i s o d i u m s a l t ; A T P , a d e n o s i n e t r i p h o s p h a t e ; ADP, a d e n o s i n e d i p h o s p h a t e ; DTT, d i t h i o t h r e i t o l ; , c o u p l i n g f a c t o r ; C F ^ , c h l o r o p l a s t c o u p l i n g f a c t o r ; ECF^ , Escherichia coli c o u p l i n g f a c t o r ; F^ , an i n t r i n s i c membrane p r o t e i n c o m p l e x , the s i t e o f a t t a c h m e n t o f F^ t o t h e membrane; A T P a s e , a d e n o s i n e t r i p h o s p h a t a s e ; C a A T P a s e , c a l c i u m - d e p e n d e n t A T P a s e ; TEMED, N , N , N 1 , N 1 - t e t r a m e t h y l e t h y l e n e -d i a m i n e ; T C A , t r i c h l o r o a c e t i c a c i d ; PMSF, p h e n y l m e t h y l s u l f o n y l f 1 u o r i d e ; k d , k i l o d a l t o n ; mw, m o l e c u l a r w e i g h t . C o u p l i n g f a c t o r and ATPase a r e used i n t e r c h a n g e a b l y in r e f e r r i n g t o the two a c t i v i t i e s o f the same enzyme c o m p l e x . A u n i t o f ATPase a c t i v i t y i s the amount o f enzyme t h a t w i l l c a t a l y z e the r e l e a s e o f one u m o l e . o f i n o r g a n i c p h o s p h a t e per m i n u t e . " P r o t e o l y s i s i n h i b i t o r s " i s used t o s i g n i f y the i n c l u s i o n o f 6 mM p - a m i n o b e n z a m i d i n e d i - H C l , hO mM e - a m i n o - n - c a p r o i c a c i d and 0.1 mM PMSF. Mater i a 1s M i r a c l o t h (Chi;copee M i l l s , I n c . ) was s u p p l i e d by C a l b i o c h e m . Sigma p r o v i d e d e - a m r n o - n - c a p r o i c a c i d , PMSF, a g a r o s e ( low EEO) , L u b r o l PX , p o l y e t h y l e n e g l y c o l ( P E G ) , t r y p s i n ( D P C C - t r e a t e d ) and soybean t r y p s i n i n h i b i t o r . M o l e c u l a r w e i g h t s t a n d a r d s f o r SDS-PAGE were f rom P h a r m a c i a (ova lbumin) o r Sigma ( p y r u v a t e k i n a s e , c a r b o n i c a n h y d r a s e and r i b o n u c l e a s e A ) . A l d r i c h s u p p l i e d p - a m i n o b e n z a m i d i n e d i - H C l . The c o l o u r r e a g e n t f o r t h e B r a d f o r d (1976) p r o t e i n a s s a y was p u r c h a s e d f rom B i o - R a d . F r e u n d ' s Comple te A d j u v a n t was s u p p l i e d by D i f c o . Sodium d e o x y c h o l a t e came f rom S c h w a r z / M a n n . A l l o t h e r c h e m i c a l s were o f r e a g e n t g r a d e . 13 P l a n t M a t e r i a l Young wheat s e e d l i n g s (Triticum aestivum, v a r T h a t c h e r ) were grown f rom seed s u p p l i e d by B u c k e r f i e l d 1 s L t d . , V a n c o u v e r , B . C . o r by A l b e r t a Wheat P o o l , C a l g a r y , A l t a . Seeds were vacuum i n f i l t r a t e d w i t h "*>% h y p o -c h l o r i t e s o l u t i o n f o r f i v e m i n u t e s , soaked in the same f o r 30 m i n u t e s , washed t h o r o u g h l y and g e r m i n a t e d by bubbl ing a i r t h r o u g h t h e seeds suspended in d i s t i l l e d wate r o v e r n i g h t . G e r m i n a t e d seed was sown in v e r m i c u l i t e and grown i n a g r e e n h o u s e f o r 8 t o 15 d a y s , w i t h 16 L : 8 D l i g h t supp lement as n e c e s s a r y . S p i n a c h l e a v e s were c u t f rom f o u r - w e e k - o l d s e e d l i n g s (Spinacea oleracea, v a r L o n g s t a n d i n g B l o o m s d a l e ) grown in s o i l in a g r e e n h o u s e . CF,j E x t r a c t i o n and P u r i f i c a t i o n A l l s t e p s up to the e x t r a c t i o n o f CF^ from t h y l a k o i d s were done o at k C. The b l a d e s o f young wheat s e e d l i n g s were removed , washed in tap w a t e r , then d i s t i l l e d w a t e r , chopped w i t h s c i s s o r s , , and b l e n d e d in 10 mg/ g f r e s h w e i g h t in c o l d I s o l a t i o n i .Medium (0.3 M m a n n i t o l , 50 mM T r i c i n e , 1 mM EDTA, 1 mM 8 - m e r c a p t o e t h a n o l , pH 8.0) sin a c h i l l e d b l e n d o r f o r ten s e c o n d s . The b r e i was f i l t e r e d t h r o u g h f o u r l a y e r s o f M i r a c l o t h and the c h l o r o p l a s t s p e l l e t e d by c e n t r i f u g i n g a t 6,000 x g f o r ten s e c o n d s . The c r u d e c h l o r o p l a s t p e M e t was r e s u s p e n d e d in 1.5 volumes o f I s o l a t i o n Medium and p e l l e t e d as b e f o r e . S t roma l r i b u l o s e - 1 , 5 " b i s p h o s p h a t e c a r b o x l y a s e was removed by s t i r r i n g f o r 30 m inu tes in about 1 m l / g f r e s h we igh t c o l d 10 mM sodium p y r o p h o s p h a t e pH 7.k (S t rotmann e t : a l . , 1973) to w h i c h was added p r o t e o l y s i s i n h i b i t o r s (6 mM p - a m i n o b e n z a m i d i n e d i - H C l , kO mM e - a m i n o - n - c a p r o i c a c i d , 0.1 mM PMSF) . T h i s wash was r e p e a t e d f o r a t o t a l o f f o u r t imes a t H ° C , the t h y l a k o i d s b e i n g pe l 1eted a t 10,000 x g between w a s h e s . D u r i n g the f i n a l w a s h , the c h l o r o p h y l l 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 the method o f Arnon (19^ *9) • 14 Subsequent work was p e r f o r m e d a t room t e m p e r a t u r e . C h l o r o f o r m e x t r a c t i o n was per fo rmed v i a a m o d i f i c a t i o n o f the method o f Y o u n i s e t a l . ( 1 9 7 7 ) . The t h y l a k o i d s were suspended a t 2.0 t o 2 . 5 mg ch lorophy 1 1 /m l in 0 . 2 5 M s u c r o s e , 10 mM T r i s / S O ^ pH 7 - 6 , 1 mM EDTA, 2 mM A T P , 5 mM DTT p l u s p r o t e o l y s i s i n h i b i t o r s . To t h i s s u s p e n s i o n , i volume o f c h l o r o f o r m was added and the m i x t u r e was e m u l s i f i e d by s t i r r i n g w i t h a m a g n e t i c s t i r bar f o r 15 s e c o n d s . The e m u l s i o n was broken by low speed c e n t r i f u g a t i o n and the aqueous phase was r e c e n t r i f u g e d a t 48,000 x g 30 m i n . The s u p e r n a t a n t was c o n c e n t r a t e d t o 1 t o 3 ml w i t h a M i l l i p o r e Immers ib le CX M o l e c u l a r S e p a r a t o r . T h i s sample i s d e s i g n a t e d " c r u d e C F ^ " . T o f u r t h e r p u r i f y CF^, the c r u d e sample was loaded o n t o a 37 ml s u c r o s e g r a d i e n t (8 t o 25% s u c r o s e in 10 mM T r i s / S O ^ pH 7 - 6 , 1 mM EDTA, 2 mM A T P , 5 mM DTT, p l u s p r o t e o l y s i s i n h i b i t o r s ) and c e n t r i f u g e d a t 26,000 rpm in a Beckman SW27 r o t o r f o r 28 h o u r s . The g r a d i e n t was f r a c t i o n a t e d by pumping d e n s e r s u c r o s e i n t o the bottom and c o l l e c t i n g 30 -drop f r a c t i o n s o f f the t o p . S u c r o s e c o n t e n t o f g r a d i e n t f r a c t i o n s was d e t e r m i n e d u s i n g a r e f T a c t o m e t e r . P r o t e i n was d e t e r m i n e d by the d y e - b i n d i n g method o f B r a d f o r d (1976). The C F 1 thus p r e p a r e d was g r e a t e r than 98% p u r e , as d e t e r m i n e d by SDS-PAGE. S p i n a c h CF^ was p r e p a r e d in a s i m i l a r way e x c e p t t h a t the r a t i o o f I s o l a t i o n Medium t o f r e s h we igh t o f young l e a v e s was 3 : 1 . In v a r i o u s e x p e r i m e n t s , p r o t e o l y s i s i n h i b i t o r s were o m i t t e d a t c e r t a i n s t a g e s o f the p r o c e d u r e o u t l i n e d a b o v e . T h e s e changes a r e noted in the t e x t and f i g u r e s as they o c c u r . A second method f o r the p r e p a r a t i o n o f a c r u d e CF^ e x t r a c t , a c c o r d i n g t o the p r o c e d u r e o f S t ro tmann e t a l . (1973) was used f o r c o m p a r a t i v e p u r p o s e s . T h y l a k o i d s were washed f o u r t imes in c o l d 10 mM sodium 15 p y r o p h o s p h a t e pH 7 -4 w i t h o u t p r o t e o l y s i s i n h i b i t o r s . CF^ was then r e l e a s e d by wash ing in a h y p o t o n i c s u c r o s e s o l u t i o n ( 0 . 3 M s u c r o s e , 2 mM T r i c i n e - K O H pH 7 -8 ) c o n t a i n i n g no p r o t e o l y s i s i n h i b i t o r s . T h i s c r u d e CF^ e x t r a c t was p u r i f i e d t o homogene i ty by s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n as a b o v e . E l e c t r o p h o r e s i s S D S - P o l y a c r y 1 amide ge l e l e c t r o p h o r e s i s was per fo rmed a c c o r d i n g to the method o f K i r c h a n s k i and Park ( 1 9 7 6 ) . The r e s o l v i n g ge l c o n t a i n e d 10% (w/v) a c r y l a m i d e , 0 .27% (w/v) b i s a c r y l a m i d e , 1 . 1347 M T r i s - H C I , pH 9 - 8 , 0 .1% (w/v) SDS, 0 . 4 7 ( v / v ) TEMED, and 0 .1% (w/v) ammonium p e r s u l f a t e . T h i s l a s t compo-nent was added a f t e r d e - a e r a t i o n , t o i n i t i a t e p o l y m e r i z a t i o n . The s t a c k i n g ge l c o n t a i n e d 5% (w/v) a c r y l a m i d e , 0.14% (w/v) b i s a c r y l a m i d e , 0 .1 M T r i s - H C 1 pH 6 . 1 , 0 .1% (w/v) SDS, 0.16% ( v / v ) TEMED, and 0 .025% (w/v) ammonium p e r s u l -f a t e . The r u n n i n g b u f f e r c o n s i s t e d o f 0 . 1 9 2 M g l y c i n e , 0 . 0 2 5 M T r i s and 0 .1 (w/v) SDS. P r o t e i n samples were d i s s o c i a t e d in 65mM T r i s - H C 1 pH 6 . 8 , 10% ( v / v ) g l y c e r o l , 2% (w/v) SDS, and e i t h e r 0.2% ( v / v ) g - m e r c a p t o e t h a n o l o r 10 mM DTT. S l a b g e l s (2 mM t h i c k ) were used t h r o u g h o u t . E l e c t r o p h o r e s i s was r o u t i n e i y run a t 15 t o 20 mA per s l a b f o r f o u r t o s i x hours a t room t e m p e r a t u r e . At the end o f e l e c t r o p h o r e s i s , p o l y a c r y l a m i d e s l a b s were s t a i n e d f o r 30 m i n u t e s in a s o l u t i o n o f 0.2% (w/v) Coomass ie B l u e R, 50% ( v / v ) m e t h a n o l , 7% ( v / v ) a c e t i c a c i d , and d e s t a i n e d in s e v e r a l changes o f 20% ( v / v ) m e t h a n o l , 7% ( v / v ) a c e t i c a c i d . A t u f t o f u n b l e a c h e d wool was added d u r i n g d e s t a i n i n g t o speed up the p r o c e s s . M o l e c u l a r we ight a n a l y s i s o f p o l y p e p t i d e bands in SDS-PAGE was done by the method o f Webber and Osborne ( 1 9 6 9 ) , u s i n g as s t a n d a r d s p y r u v a t e k i n a s e (mw 57 k d ) , o v a l b u m i n (mw 45 k d ) , c a r b o n i c a n h y d r a s e (mw 30 kd) and r i b o n u c l e a s e A (mw 13 -7 k d ) . .16 I n - g e l l i m i t e d p r o t e o l y s i s was p e r f o r m e d a c c o r d i n g t o t h e m e t h o d o f C l e v e l a n d e t a l . (1977). P r o t e i n b a n d s s e p a r a t e d by SDS-PAGE w e r e v i s u a l i z e d by s t a i n i n g f o r f i v e m i n u t e s , t h e n d e s t a i n i n g t o t h e p o i n t w h e r e b a n d s w e r e v i s i b l e . T h e s e w e r e e x c i s e d w i t h a r a z o r b l a d e and s o a k e d f o r 30 m i n u t e s i n 10 ml o f s o a k i n g b u f f e r (0.125 M T r i s - H C 1 pH 6.8, 0 . 1 % S D S , 1mM E D T A ) . A 15% SDS-PAGE s l a b g e l ( a s d e s c r i b e d a b o v e , b u t c o n t a i n -i n g 1 mM EDTA i n a d d i t i o n ) w i t h a f o u r cm l o n g s t a c k i n g g e l c o n t a i n i n g 3% a c r y l a m i d e (no EDTA) was m a d e . I n t o e a c h s l o t on t h e s l a b was p l a c e d t h e e x c i s e d s o a k e d band and 10 u l o f 20% g l y c e r o l i n s o a k i n g b u f f e r . O v e r t h i s , 10 u l o f 50 u g / m l s o l u t i o n o f Staphylococcus aureus V8 p r o t e a s e i n 10% g l y c e r o l , 0 . 1 % B r o m p h e n o l b l u e and s o a k i n g b u f f e r was o v e r l a i d . A c u r r e n t o f 15 mA was r u n u n t i l t h e s a m p l e and t r a c k i n g d y e had s t a c k e d i n t h e m i d d l e o f t h e 3% g e l . The c u r r e n t was t u r n e d o f f f o r h0 m i n u t e s t o a l l o w d i g e s t i o n t o o c c u r . The c u r r e n t was t u r n e d b a c k on a t t h e end o f t h i s p e r i o d and e l e c t r o p h o r e s i s p r o c e e d e d a t 20 mA u n t i l t h e t r a c k i n g d y e had r u n o f f t h e end o f t h e 15% g e l . S t a i n i n g and d e s t a i n i n g w e r e d o n e t h e same a s f o r o t h e r SDS g e l s . ASSAYS T h e l a t e n t C a A T P a s e o f CF^ was a c t i v a t e d by t r y p s i n d i g e s t i o n and a s s a y e d a c c o r d i n g t o t h e m e t h o d o f L i e n and R a c k e r ( 1 9 7 1 b ) . B r i e f l y , t o a s a m p l e c o n t a i n i n g 50 ug p r o t e i n was a d d e d 0 . 0 1 ml ATP ( 0 . 1 M pH 7 s t o c k ) , 0 . 0 2 ml EDTA ( 0 . 2 M pH 7-6 s t o c k ) , 0 . 0 3 ml T r i c i n e - K O H (1 M pH 8 s t o c k ) , and t h e v o l u m e was b r o u g h t t o 0 . 9 3 ml w i t h d i s t i l l e d w a t e r . T r y p s i n ( 0 . 0 2 ml o f a 5 mg/ml s t o c k p r e p a r e d i n 1 mM H^SO^) was a d d e d and d i g e s t i o n p r o c e -e d e d f o r 6 m i n u t e s a t room t e m p e r a t u r e . D i g e s t i o n was h a l t e d by t h e a d d i t i o n o f 0 . 0 5 ml s o y b e a n t r y p s i n i n h i b i t o r (5 mg/ml i n 5mM T r i c i n e - K O H pH 7 ) . 17 A l i q u o t s o f t h e a c t i v a t e d enzyme c o n t a i n i n g 2 . 5 ug CF^ w e r e b r o u g h t ) t o 0 . 2 ml w i t h 20 mM T r i c i n e - K O H pH 8. T o t h i s was a d d e d 0.8 ml o f s u b s t r a t e m i x c o n t a i n i n g kO mM T r i c i n e - K O H pH 8, 1 mM A T P , and 10 mM C a C I ^ . A f t e r a 10 m i n u t e i n c u b a t i o n a t 37 ° C , t h e r e a c t i o n was h a l t e d by t h e a d d i t i o n o f 1 ml 0 . 5 M c o l d T C A . S a m p l e s w e r e a n a l y z e d f o r i n o r g a n i c p h o s p h a t e (Chen e t a l . , 1 9 5 6 ) . In a l l a s s a y s f o r A T P a s e a c t i v i t y , z e r o t i m e i n o r g a n i c p h o s p h a t e was d e t e r m i n e d , and a c o r r e c t i o n was made f o r A T P a s e a c t i v i t y o f t h e u n a c t i v a t e d e n z y m e . ATP was o m i t t e d f r o m t h e t r y p s i n d i g e s t i o n s t e p i n e x p e r i m e n t s f o r d e t e r m i n a t i o n o f K^ and V m a x > i n o r d e r t o a s s a y A T P a s e a c t i v i t y a t v e r y l o w c o n c e n t r a t i o n s o f s u b s t r a t e . P r i o r t o enzyme a c t i v a t i o n w i t h t r y p s i n , p r o t e o l y s i s i n h i b i t o r s w e r e removed by d i a l y s i s a g a i n s t t w o c h a n g e s o f 10 mM T r i s - S O ^ pH l.k, 1 mM EDTA, 1 mM A T P , 5 mM DDT. The l a t e n t , h e a t a c t i v a t e d C a A T P a s e o f CF^ was a c t i v a t e d and a s s a y e d a c c o r d i n g t o t h e m e t h o d o f L i e n and R a c k e r ( 1 9 7 1 b ) . To a 0 . 2 ml a l i q u o t o f p r o t e i n c o n t a i n i n g 150 t o 200 ug C F 1 , 0 . 0 5 ml ATP ( 0 . 2 M pH7) , 0 . 0 1 5 ml DTT ( O . t M) and 0 . 0 3 ml T r i c i n e - K O H (1 M pH 8) was a d d e d . T h e m i x t u r e was h e a t e d i n a 6 0 ° C w a t e r b a t h f o r f o u r m i n u t e s , and t h e n 1 . 7 ml T r i c i n e - K O H -EDTA b u f f e r (40 mM T r i c i n e K0H pH 8 . 0 , 2 mM EDTA) a t room t e m p e r a t u r e was a d d e d . A T P a s e a c t i v i t y was a s s a y e d a s d e s c r i b e d a b o v e . N u c l e o t i d e s p e c i f i c i t y o f t h e t r y p s i n - a c t i v a t e d A T P a s e was a s s a y e d by s u b s t i t u t i n g 1 mM o f t h e v a r i o u s s u b s t r a t e s f o r A T P , and a c t i v a t i n g i n t h e a b s e n c e o f A T P . F o r d e t e r m i n a t i o n o f K u and V g r a p h i c a l l y , t h e v e l o c i t y o f t h e M . max r ' ' enzyme r e a c t i o n was d e t e r m i n e d by s a m p l i n g a r e a c t i o n m i x t u r e a t one m i n u t e i n t e r v a l s o v e r a t h r e e m i n u t e r e a c t i o n . V e l o c i t y was p l o t t e d a g a i n s t t h e 18 2+ c o n c e n t r a t i o n o f Ca ATP c o m p l e x , r a t h e r t h a n t h e c o n c e n t r a t i o n o f A T P . 2+ The c o n c e n t r a t i o n o f Ca ATP was d e t e r m i n e d u s i n g t h e f o l l o w i n g e q u a t i o n o f A h l e r s e t a l . (1975) : [ C a 2 + A T P ] = [ C a ] t + f A T P ] t + K ^ ^ l + J£J j [ C a ] t + [ATP] t . + K C a A T p ^1 + [ H ^ [ C a ] t [ATP^ (1) w i t h K C a A - j - p = 0.725 mM ( N a n n i n g a , 1957; o b t a i n e d f r o m S i l l e n and M a r t e l 1 , 1964) and K, = 3 x 10"7 ( S i l l e n and M a r t e l1, 1964). [ c a ] t > [ A T P ] t . a n d [ H + ] t r e f e r t0 t h e t o t a l c o n c e n t r a t i o n o f e a c h o f t h e s e c o m p o n e n t s . E l e c t r o n M i c r o s c o p y A l i g h t c o a t i n g o f c a r b o n was e v a p o r a t e d o n t o c o l l o d i o n - c o a t e d 200 mesh e l e c t r o n m i c r o s c o p y g r i d s , and t h e p l a s t i c b a c k i n g was removed by g e n t l y d i p p i n g t h e c f r i d i n t o a c e t o n e f o r f i v e s e c o n d s . CF^ s a m p l e s w e r e d i a l y z e d o v e r n i g h t a g a i n s t 2 mM T r i s - H C l pH 7-5 t o remove s a l t s p r i o r t o n e g a t i v e s t a i n i n g . S a m p l e s o f w h e a t CF^ ( g r e a t e r t h a n SS% p u r i t y a s d e t e r m i n e d by SDS-PAGE) o r Escherichia coli F^  ( k i n d l y p r o v i d e d by D r . P . D . B r a g g , UBC D e p a r t m e n t o f B i o c h e m i s t r y ) w i t h a b o u t 0.1 t o 0.2 mg p r o t e i n / m l w e r e m i x e d w i t h an e q u a l v o l u m e o f n e g a t i v e s t a i n f o r 30 s e c o n d s , and s p r a y e d o n t o t h e c a r b o n f i l m w i t h t h e s p r a y gun a p p a r a t u s d e s c r i b e d by B a c k u s a n d W i l l i a m s (1950). N e g a t i v e s t a i n s u s e d w e r e 2% ammonium m o l y b d a t e i n 12 mM s o d i u m o x a l a t e pH 7.2 o r 2% p h o s p h o t u n g s t i c a c i d - K O H pH 7.0. U r a n y l a c e t a t e (2% s o l u t i o n , pH 4.3) was n o t a s a t i s f a c t o r y 19 s t a i n . N e g a t i v e s t a i n e d CF^ and ECF^ w e r e e x a m i n e d u s i n g a Z e i s s EM-10 e l e c t r o n m i c r o s c o p e , a t m a g n i f i c a t i o n s o f 4 0 , 0 0 0 X o r g r e a t e r , w i t h a v o l t a g e s e t t i n g o f - 8 0 * k V . CF^ d i a m e t e r s w e r e m e a s u r e d on p h o t o g r a p h i c p r i n t s and t h e a c t u a l s i z e o f t h e p a r t i c l e was c a l c u l a t e d f r o m t h e m a g n i f i c a t i o n o f t h e p r i n t and t h e n e g a t i v e . F o r t h e m a g n i f i c a t i o n s u s e d , c a l i b r a t i o n o f t h e EM-10 g a v e m a g n i f i c a t i o n s t h a t w e r e w i t h i n 0 . 8 t o 2 . 2 % o f t h e i n s t r u m e n t s e t t i n g s . Image e n h a n c e m e n t o f e l e c t r o n m i c r o g r a p h s was d o n e by t h e m e t h o d o f Markham e t a l . ( 1 9 6 3 ) . P r e p a r a t i o n and C h a r a c t e r i z a t i o n o f M o n o s p e c i f i c A n t i b o d i e s t o Wheat CF^ 200 ug o f CF.| ( g r e a t e r t h a n 98% p u r e ) was e m u l s i f i e d i n an e q u a l v o l u m e o f F r e u n d ' s C o m p l e t e A d j u v a n t and i n j e c t e d i n t r a m u s c u l a r l y i n t o t h e h i n d l e g o f a New Z e a l a n d w h i t e r a b b i t . A t t e n d a y i n t e r v a l s , s e c o n d and t h i r d i n j e c t i o n s w e r e g i v e n and two w e e k s l a t e r t h e r a b b i t was b l e d f r o m t h e e a r v e i n . A f t e r c l o t t i n g , s e r u m was s e p a r a t e d f r o m t h e b l o o d c e l l s by c e n t r i f u g a t i o n . P r e s e r v a t i v e ( 0 . 1 % s o d i u m a z i d e ) was a d d e d t o a l l s e r a and t h e y w e r e s t o r e d a t 4 ° C . A n t i b o d y t i t e r was d e t e r m i n e d by t h e d o u b l e d i f f u s i o n m e t h o d o f O u c h t e r l o n y - ( 1 9 6 8 ) . C r o s s e d i m m u n o e l e c t r o p h o r e s i s was p e r f o r m e d a c c o r d i n g t o t h e m e t h o d o f Chua and B l o m b e r g (1979) e x c e p t t h a t w h o l e s e r u m , r a t h e r t h a n p u r i f i e d I g G , was u s e d . T h e f o l l o w i n g s t o c k s o l u t i o n s w e r e p r e p a r e d : (a) b u f f e r A : 90 mM T r i s pH 8 . 6 w i t h a c e t i c a c i d , hO mM s o d i u m a c e t a t e , 1 mM EDTA; (b) 20% ( v / v ) L u b r o l PX i n d i s t i l l e d w a t e r ; ( c ) 10% (w/v) s o d i u m d e o x y c h o l a t e i n d i s t i l l e d w a t e r ; (d) 50% PEG (w/v) i n b u f f e r A ; (e) 1.2% (w/v) a g a r o s e i n b u f f e r A . The g e l was c a s t b e t w e e n two g l a s s p l a t e s w i t h 20 3 mm t h i c k s p a c e r b a r s on t h e s i d e s and b o t t o m . The a p p a r a t u s was c l a m p e d t o g e t h e r i n a v e r t i c a l p o s i t i o n d u r i n g c a s t i n g , and i n d i v i d u a l l a y e r s w e r e c a s t f r o m b o t t o m t o t o p a s d i a g r a m m e d i n f i g . 1 . S o l u t i o n (e) was d i s s o l v e d by h e a t i n g i n a b o i l i n g w a t e r b a t h , and a l l s o l u t i o n s w e r e k e p t i n a 65 C w a t e r b a t h p r i o r t o c a s t i n g . The a n o d a l g e l was p r e p a r e d by m i x i n g 2 . 9 ml o f s o l u t i o n (b) w i t h 3 5 - 1 ml o f s o l u t i o n ( e ) . M i x i n g was a c c o m p l i s h e d by g e n t l e s w i r l i n g i n a p r e - w a r m e d f l a s k . T h e g e l was t h e n c a s t by p o u r i n g t h e warm m i x t u r e b e t w e e n t h e p l a t e s , b e i n g c a r e f u l n o t t o make b u b b l e s . A f t e r t h i s l a y e r had s o l i d i f i e d , s u c c e s s i v e l a y e r s w e r e p o u r e d on t o p i n t h e same m a n n e r . To p r e p a r e t h e a n t i b o d y g e l , 1 . 3 ml o f s o l u t i o n (d) and 1 8 . 7 ml o f s o l u t i o n (e) w e r e m i x e d , and a d d e d t o 1 ml o f a n t i s e r u m t h a t had b e e n p r e w a r m e d . The s h o r t i n t e r m e d i a t e g e l was c a s t f r o m e x t r a a n o d a l g e l m i x t u r e . An u n s t a i n e d , u n f i x e d l a n e f r o m a S D S -PAGE g e l was e x c i s e d , r i n s e d i n d i s t i l l e d w a t e r , and p l a c e d a b o v e t h e i n t e r -m e d i a t e g e l a s d i a g r a m m e d . The c a t h o d a l g e l , c o m p o s e d o f 1 ml o f s o l u t i o n ( c ) and 9 ml o f s o l u t i o n ( e ) , was c a s t a r o u n d t h e SDS g e l . The b o t t o m s p a c e r b a r was r e m o v e d and J - C l o t h ( J o h n s o n and J o h n s o n ) w i c k s w e r e f u s e d t o t h e c a t h o d a l a n d a n o d a l e n d s o f t h e g e l u s i n g warm s o l u t i o n (e) a s a b i n d e r . The c o m p o s i t e g e l was t h e n p l a c e d h o r i z o n t a l l y a c r o s s t h e s u p p o r t s o f a Gelman f l a t b e d e l e c t r o p h o r e s i s c h a m b e r . E a c h r e s e r v i o r was f i l l e d w i t h a b o u t hOO ml o f b u f f e r A . E l e c t r o p h o r e s i s was r u n a t a c o n s t a n t v o l t a g e o f 100 V f o r 18 t o 20 h . T h e s t a r t i n g c u r r e n t was a b o u t 20 mA. I m m u n o p r e c i p i t a t e s i n b o t h d o u b l e d i f f u s i o n p l a t e s and c r o s s e d Immuno-e l e c t r o p h o r e s i s g e l s w e r e v i s u a l i z e d by s t a i n i n g w i t h C o o m a s s i e B l u e a f t e r s o a k i n g t h e g e l i n 0 . 1 5 M N a C l f o r o n e d a y and i n d i s t i l l e d w a t e r f o r two d a y s . Due t o t h e f r a g i l i t y o f a g a r o s e g e l s , s t a i n i n g and d e s t a i n i n g w e r e d o n e w i t h o u t s h a k i n g . 21 F i g u r e 1 : D i a g r a m o f c r o s s e d I m m u n o e l e c t r o p h o r e s i s g e l , a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . 21a Cathodal Gel SDS Gel Intermediate Gel T 25 mm 1 8 mm T 50 mm Antibody Gel Anodal Gel 25 mm A 22 RESULTS P u r i f i c a t i o n o f Wheat CF.^  A s a t i s f a c t o r y method was d e v e l o p e d f o r o b t a i n i n g homogeneous w h e a t C F . j , u s i n g a m o d i f i c a t i o n o f t h e c h l o r o f o r m e x t r a c t i o n m e t h o d o f Y o u n i s e t a l . (1977) . C F 1 , f r o m t h y l a k o i d s s t r i p p e d o f t h e c h l o r o p l a s t e n v e l o p e and s t r o m a l p r o t e i n s , was r e l e a s e d i n t o t h e a q u e o u s medium by e m u l s i f y i n g t h e t h y l a k o i d s u s p e n s i o n i n c h l o r o f o r m . The e x t r a c t was p u r i f i e d t o g r e a t e r t h a n 38% h o m o g e n e i t y on a l i n e a r 8 t o 25% s u c r o s e g r a d i e n t . P r o t e o l y s i s i n h i b i t o r s w e r e i n c l u d e d i n a l l s o l u t i o n s e x c e p t t h e i n i t i a l g r i n d i n g m e d i u m . F r a c t i o n a t i o n o f a t y p i c a l s u c r o s e g r a d i e n t g a v e t h e p r o f i l e i l l u s t r a t e d i n f i g . 2. CF^ b a n d s a t 20 t o 21% s u c r o s e . A n a l y s i s o f t h e m a j o r p r o t e i n p e a k i n t h e s u c r o s e g r a d i e n t by SDS-PAGE r e v e a l e d t h a t a l l f i v e s u b u n i t s o f C F 1 w e r e p r e s e n t ( f i g . 3). T h e s m a l l e r p r o t e i n p e a k a t t h e b o t t o m o f t h e s u c r o s e g r a d i e n t c o n t a i n s p o l y p e p t i d e s c o r r e s p o n d i n g i n a p p a r e n t m o l e c u l a r w e i g h t t o t h e l a r g e and s m a l l s u b u n i t s o f r i b u l o s e - 1 , 5 " b i s p h o s p h a t e c a r b o x y l a s e (EC 4.1.1.39), t h e m a j o r p r o t e i n i n t h e c h l o r o p l a s t s t r o m a ( l a n e 6, f i g . 3). As c a n be s e e n i n l a n e 1 o f f i g . 3, t h e " c r u d e C F ^ " i s v e r y n e a r l y h o m o g e n e o u s , s o t h a t f u r t h e r p u r i f i c a t i o n by 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 r e s u l t e d i n o n l y a 1.5 t o 2 - f o l d i n c r e a s e i n C a A T P a s e a c t i v i t y a t b e s t . R e s u l t s o f a t y p i c a l e x p e r i m e n t a r e g i v e n i n T a b l e I. 23 F i g u r e 2 : P r o f i l e o f s u c r o s e d e n s i t y g r a d i e n t u s e d t o p u r i f y w h e a t CF.| . A c r u d e c h l o r o f o r m e x t r a c t o f w h e a t t h y l a k o i d s c o n t a i n i n g 4 . 9 mg p r o t e i n was l o a d e d o n t o a 34 ml g r a d i e n t (8 t o 25% s u c r o s e i n b u f f e r c o n t a i n i n g p r o t e o l y s i s i n h i b i t o r s ) w i t h a 50% s u c r o s e c u s h i o n , and t h e g r a d i e n t was c e n t r i f u g e d 28 h a t 2 6 , 0 0 0 rpm i n a Beckman SW 27 r o t o r . The g r a d i e n t was f r a c t i o n a t e d i n t o 30 d r o p a l i q u o t s . S u c r o s e c o n t e n t , p r o t e i n c o n c e n t r a t i o n and t r y p s i n -a c t i v a t e d A T P a s e a c t i v i t y w e r e a s s a y e d a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . • • % SUCROSE n • ACTIVITY (units-mg protein"1) 2k F i g u r e 3' SDS-PAGE o f c r u d e CF^ s a m p l e and p r o t e i n f r a c t i o n s f r o m a s u c r o s e d e n s i t y g r a d i e n t s i m i l a r t o t h e o n e shown i n F i g u r e 2. L a n e 1, 20 ug c r u d e c h l o r o f o r m e x t r a c t ; l a n e 2, 19 ug o f g r a d i e n t f r a c t i o n 16; l a n e 3, 35 ug o f g r a d i e n t f r a c t i o n 17; l a n e k, 33 ug o f g r a d i e n t f r a c t i o n 18; l a n e 5, 18 ug o f g r a d i e n t f r a c t i o n 19; l a n e 6, 9.6 ug o f g r a d i e n t f r a c t i o n 23. LS a n d SS r e f e r t o t h e l a r g e and s m a l l s u b u n i t s o f r i b u l o s e - 1 , 5 " " b i s p h o s p h a t e c a r b o x y l a s e . 25 T a b l e I . P u r i f i c a t i o n o f w h e a t C a A T P a s e S a m p l e C o n e . T o t a l T o t a l S p e c i f i c T o t a l D e g r e e o f P r o t e i n , V o l u m e , P r o t e i n , A c t i v i t y * A c t i v i t y , P u r i f i c a -mg/ml ml mg u n i t s t i o n C r u d e C F 1 2.96 4.6 13.62 5.42 71.4 1 S u c r o s e g r a d i e n t 0.69 1 . 3 0.90 10.42 9.4 1.99 p e a k " u n i t s / m g p r o t e i n S p i n a c h CF^ was p r e p a r e d by t h e same p r o c e d u r e f o r c o m p a r a t i v e p u r p o s e s . I t was f o u n d t o h a v e t h e same s e d i m e n t a t i o n v e l o c i t y a s w h e a t CF.j i n s u c r o s e g r a d i e n t s , and t h e same p o l y p e p t i d e c o m p o s i t i o n , a l t h o u g h t h e a p p a r e n t m o l e c u l a r w e i g h t s o f t h e s u b u n i t s d i f f e r somewhat ( T a b l e I I ) . T a b l e I I . A p p a r e n t m o l e c u l a r w e i g h t s o f s u b u n i t s o f w h e a t and s p i n a c h CF.j , a s d e t e r m i n e d by t h e m e t h o d o f Webber and O s b o r n e (1969). S u b u n i t A p p a r e n t m o l e c u l a r w e i g h t ( k d ) Wheat C F t S p i n a c h C F 1 a 57 58 6 55 54 Y 37-39 36-37 <5 25 19 e 13 13 26 The E f f e c t o f P r o t e o l y s i s I n h i b i t o r s on E x t r a c t i o n o f Wheat CF^ In i n i t i a l CF^ e x t r a c t i o n s , p r o t e o l y s i s i n h i b i t o r s w e r e n o t i n c l u d e d i n s o l u t i o n s u s e d f o r CF^ r e l e a s e and p u r i f i c a t i o n . CF^ e x t r a c t e d i n t h i s manner c o n t a i n e d o n l y t h e t h r e e l a r g e s t s u b u n i t s . When p r o t e o l y s i s i n h i b i t o r s w e r e a d d e d t o a l l s o l u t i o n s u s e d i n 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 w h e a t C F ^ , a l l f i v e s u b u n i t s c o - p u r i f i e d . To t e s t a t w h a t s t a g e t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s i s c r i t i c a l f o r t h e p r e s e r v a t i o n o f t h e i n t a c t enzyme c o m p l e x , w h e a t CF^ was p r e p a r e d by c h l o r o f o r m e x t r a c t i o n w i t h and w i t h o u t 6 mM p - a m i n o b e n z a m i d i n e , kO mM e - a m i n o - n - c a p r o i c a c i d and 0.1 mM p h e n y l m e t h y l s u l f o n y l f 1 u o r i d e . The c r u d e c h l o r o f o r m e x t r a c t s w e r e i d e n t i c a l i n p o l y p e p t i d e c o m p o s i t i o n ( f i g . k, l a n e s A and B ) , b u t when t h e c r u d e CF^ p r e p a r e d w i t h o u t p r o t e o l y s i s i n h i b i t o r s was p u r i f i e d on a s u c r o s e g r a d i e n t a l s o w i t h o u t p r o t e o l y s i s i n h i b i t o r s , t h e CF^ p e a k i n t h e g r a d i e n t l a c k e d t h e <5 and e s u b u n i t s ( f i g . 5). I t a p p e a r s t h a t p r o t e o l y s i s i n h i b i t o r s p r e v e n t t h e l o s s o f t h e s e m i n o r s u b u n i t s f r o m t h e enzyme c o m p l e x d u r i n g t h e p r o l o n g e d room t e m p e r a t u r e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . In a d d i t i o n t o c h l o r o f o r m e x t r a c t i o n , t h e r e a r e s e v e r a l o t h e r m e t h o d s f o r r e l e a s i n g CF^ f r o m t h e t h y l a k o i d , a s m e n t i o n e d i n t h e I n t r o d u c t i o n . I t was d e c i d e d t o c o m p a r e CF^ e x t r a c t e d by t h e h y p o t o n i c s u c r o s e m e t h o d ( S t r o t m a n n e t a l . , 1973) w i t h t h e m e t h o d d e s c r i b e d h e r e . A d d i t i o n o f p r o t e o l y s i s i n h i b i t o r s t o t h e h y p o t o n i c s u c r o s e e x t r a c t i o n medium c o m p l e t e l y p r e v e n t e d CF^ r e l e a s e ( f i g . h, l a n e s C and D ) . E x t r a c t i o n o f CF.j w i t h h y p o t o n i c s u c r o s e m i n u s p r o t e o l y s i s i n h i b i t o r s r e l e a s e d many more p o l y p e p t i d e s t h a n a r e r e l e a s e d by c h l o r o f o r m t r e a t m e n t ( c o m p a r e i n f i g . k, l a n e A w i t h l a n e D ) . C h l o r o f o r m t h u s seems t o be a much more s p e c i f i c m e t h o d f o r C F , e x t r a c t i o n t h a n t h e s u c r o s e m e t h o d . 27 F i g u r e k: C o m p a r i s o n o f c r u d e CF^ p r e p a r e d by t h e c h l o r o f o r m m e t h o d w i t h t h a t p r e p a r e d by t h e h y p o t o n i c s u c r o s e m e t h o d , and t h e e f f e c t o f t h e i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s upon e a c h . A . C r u d e c h l o r o f o r m e x t r a c t , p r e p a r e d i n t h e p r e s e n c e o f a l l p r o t e o l y s i s i n h i b i t o r s . 25 ug p r o t e i n . B . C r u d e c h l o r o f o r m e x t r a c t , f o r w h i c h a l l p r o t e o l y s i s i n h i b i t o r s w e r e o m i t t e d f r o m b o t h t h e s o d i u m p y r o p h o s p h a t e w a s h and t h e c h l o r o f o r m e x t r a c t i o n m e d i u m . 25 ug p r o t e i n . C . H y p o t o n i c s u c r o s e wash i n w h i c h p r o t e o l y s i s i n h i b i t o r s w e r e i n c l u d e d . The s m a l l amount o f a and B s e e n i n t h i s l a n e was w a s h e d o v e r f r o m t h e a d j a c e n t l a n e . 6.25 ug p r o t e i n . D. H y p o t o n i c s u c r o s e w a s h o f t h y l a k o i d s , p r e p a r e d i n t h e a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s . 73 ug p r o t e i n . 27a 28 F i g u r e 5- E f f e c t o f t h e i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s d u r i n g CF.j e x t r a c t i o n and p u r i f i c a t i o n upon s u b u n i t c o m p o s i t i o n o f w h e a t C F ^ . A . CF^ e x t r a c t e d by t h e c h l o r o f o r m m e t h o d and p u r i f i e d , a l l i n t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s . B. CF^ e x t r a c t e d by t h e c h l o r o f o r m m e t h o d and p u r i f i e d i n t h e a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s . 28a 29 T h e p r e v e n t i o n o f CF^ r e l e a s e by h y p o t o n i c s u c r o s e when p r o t e o l y s i s i n h i b i t o r s a r e i n c l u d e d r a i s e s t h e q u e s t i o n o f w h e t h e r a p r o t e o l y t i c e v e n t i s r e q u i r e d f o r CF^ r e l e a s e by t h i s m e t h o d . S u c h a p r o t e o l y t i c e v e n t h a s b e e n r e p o r t e d f o r ECF^ (Cox e t a l . , 1978, B r a g g and H o u , 1979). B r a g g and Hou (1979) c o m p a r e d ECF.^ r e l e a s e d by c h l o r o f o r m i n t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s w i t h t h a t r e l e a s e d by l o w i o n i c s t r e n g t h b u f f e r i n t h e a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s . T h e y f o u n d a s l i g h t l y l a r g e r 6 s u b u n i t i s r e l e a s e d by c h l o r o f o r m , and showed t h a t t h e c h l o r o f o r m -r e l e a s e d ECF.J r e b i n d s t o E .coli membranes t o a l e s s e r e x t e n t t h a n ECF^ r e l e a s e d by a l o w i o n i c s t r e n g t h w a s h . T h e y p r o p o s e d t h a t p r o t e o l y s i s o f t h e 6 s u b u n i t i s r e q u i r e d f o r t h e r e l e a s e o f A T P a s e f r o m E.:coli membranes w i t h l o w i o n i c s t r e n g t h b u f f e r . T o t e s t w h e t h e r t h e i n h i b i t i o n o f a p r o t e o l y t i c c l e a v a g e i n one o f t h e CF.j s u b u n i t s a c c o u n t s f o r t h e i n h i b i t i o n o f CF^ r e l e a s e by h y p o t o n i c s u c r o s e when p r o t e o l y s i s i n h i b i t o r s a r e i n c l u d e d , t w o m e t h o d s o f CF^ r e l e a s e w e r e c o m p a r e d . T h y l a k o i d s f r o m t h e same b a t c h o f w h e a t p l a n t s w e r e u s e d f o r t h i s c o m p a r i s o n . In t h e f i r s t c a s e , CF^ was r e l e a s e d f r o m t h y l a k o i d s by t h e c h l o r o f o r m m e t h o d , i n t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s . In t h e s e c o n d m e t h o d , n o p r o t e o l y s i s i n h i b i t o r s w e r e p r e s e n t , i n e i t h e r t h e s o d i u m p y r o p h o s p h a t e w a s h e s , o r i n t h e e x t r a c t i o n o f CF^ w i t h h y p o t o n i c s u c r o s e . B o t h c r u d e CF^ e x t r a c t s t h u s o b t a i n e d w e r e p u r i f i e d on s u c r o s e g r a d i e n t s c o n t a i n i n g a l l p r o t e o l y s i s i n h i b i t o r s . When t h e two s a m p l e s o f p u r i f i e d CF^ w e r e c o m p a r e d ( f i g . 6 ) , no d i f f e r e n c e i n a p p a r e n t m o l e c u l a r w e i g h t i n any o f t h e s u b u n i t s was e v i d e n t , and t h e 6 and e s u b u n i t s w e r e p r e s e r v e d i n b o t h . T h u s i s seems t h a t t h e r e l e a s e o f CF^ f r o m t h e t h y l a k o i d by h y p o t o n i c s u c r o s e d o e s n o t i n v o l v e any d e t e c t a b l e p r o t e o l y s i s o f C F ^ , a l t h o u g h t h e s e r e s u l t s do n o t r u l e o u t a p r o t e o l y t i c e v e n t a t t h e CF 30 F i g u r e 6 : P r o t e o l y s i s i n h i b i t o r s p r e s e n t d u r i n g s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p r e v e n t t h e l o s s o f t h e 6 and e s u b u n i t s o f w h e a t C F ^ . A . T h y l a k o i d s w e r e p r e p a r e d w i t h o u t p r o t e o l y s i s i n h i b i t o r s , w a s h e d i n s o d i u m p y r o p h o s p h a t e w i t h o u t p r o t e o l y s i s i n h i b i t o r s , a n d a c r u d e CF^ e x t r a c t was p r e p a r e d by w a s h i n g i n h y p o t o n i c s u c r o s e c o n t a i n i n g no p r o t e o l y s i s i n h i b i t o r s . CF^ w a s p u r i f i e d by s u c r o s e d e n s i t y g r a d i e n t 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 c o n t a i n i n g a l l p r o t e o l y s i s i n h i b i t o r s . 62 ug p r o t e i n . B . Wheat CF^ e x t r a c t e d by t h e c h l o r o f o r m m e t h o d i n t h e p r e s e n c e o f a l l p r o t e o l y s i s i n h i b i t o r s and p u r i f i e d on a s u c r o s e d e n s i t y g r a d i e n t c o n t a i n i n g a l l p r o t e o l y s i s i n h i b i t o r s , kk ug p r o t e i n . 30a M I G R A T I O N 31 a t t a c h m e n t s i t e on C F ^ . I t may be t h a t some o t h e r f a c t o r , s u c h as o s m o t i c s t r e n g t h , i s t h e r e a s o n why CF^ i s n o t r e l e a s e d i n t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s w i t h t h e h y p o t o n i c s u c r o s e m e t h o d . H o w e v e r , t h e l o s s o f 8 and e i n t h e s u c r o s e g r a d i e n t s r u n a t 18 C f o r 28 h o u r s w i t h o u t p r o t e o l y s i s i n h i b i t o r s d o e s s u g g e s t t h a t enzyme p r e p a r e d by b o t h m e t h o d s c o n t a i n s t r a c e s o f p r o t e a s e a c t i v i t y . The y S u b u n i t o f Wheat CF^ C o n t a i n s an I n t e r n a l D i s u l f i d e B r i d g e In m a n y , b u t n o t a l l CF^ p r e p a r a t i o n s , a p a i r o f b a n d s o f a b o u t 2 kd d i f f e r e n c e i n a p p a r e n t mw was s e e n i n t h e r e g i o n o f t h e y s u b u n i t o f b o t h w h e a t and s p i n a c h CF^ upon SDS-PAGE ( f i g . 7 ) I t was f i r s t t h o u g h t t h a t o n e o f t h e s e t w o b a n d s m i g h t be t h e r e s u l t o f p r o t e o l y s i s o f t h e a o r 3 s u b u n i t , a s f o u n d by R y r i e and G a l l a g h e r (1979) i n t h e y e a s t m i t o c h o n d r i a l F^. W i t h t h a t e n z y m e , t h e a u t h o r s f o u n d t h a t a kO kd p o l y p e p t i d e p u r i f y i n g w i t h F^  i s a c t u a l l y a p r o t e o l y t i c f r a g m e n t o f t h e a s u b u n i t . P r o t e o l y s i s o f t h e a s u b u n i t was d u e t o a l a t e n t , S D S - a c t i v a t e d p r o t e a s e c o n t a m i n a t i n g t h e i r F^  p r e p a r a t i o n i n s m a l l q u a n t i t i e s . To t e s t w h e t h e r p r o t e o l y s i s o c c u r s d u r i n g t h e p r e p a r a t i o n o f w h e a t C F ^ , p r o t e o l y s i s i n h i b i t o r s w e r e a d d e d t o a l l s o l u t i o n u s e d t o p r e p a r e w h e a t C F ^ . B o t h o f t h e y s u b u n i t p o l y p e p t i d e s w e r e s t i l l p r e s e n t o n S D S - P A G E . In o r d e r t o d e t e r m i n e w h e t h e r o n e o f t h e b a n d s was a b r e a k d o w n p r o d u c t o f a o r 3, p e p t i d e a n a l y s i s was p e r f o r m e d a c c o r d i n g t o t h e m e t h o d o f C l e v e l a n d e t a 1. (1977). W i t h Staphylococcus aureus V8 p r o t e a s e , a c o m p a r i s o n o f t h e l i m i t e d p r o t e o l y s i s f r a g m e n t s o f t h e y d o u b l e t w i t h t h o s e o f t h e a and 3 s u b u n i t s showed no c l e a r s i m i l a r i t i e s b e t w e e n any o f t h e two l a r g e s t s u b u n i t s and t h e d i m e r . H o w e v e r , when t h e two b a n d s o f t h e d o u b l e t w e r e s e p a r a t e l y s u b j e c t e d t o t h e same p r o c e d u r e , t h e r e s u l t i n g p e p t i d e f i n g e r p r i n t shows 32 F i g u r e 7- SDS-PAGE o f w h e a t and s p i n a c h C F ^ , s h o w i n g a d o u b l e band i n t h e y s u b u n i t p o s i t i o n . A . S p i n a c h C F ^ , d i s s o c i a t e d i n 65 mM T r i s - H C 1 pH 6.8, 2% S D S , 10% g l y c e r o l and 0 . 2 % 0 - m e r c a p t o e t h a n o l , 50 ug p r o t e i n . B. Wheat C F ^ , d i s s o c i a t e d i n 65 mM T r i s - H C 1 pH 6.8, 2% S D S , 10% g l y c e r o l and 2 . 5 mM DTT, 38 ug p r o t e i n . 33 t h a t t h e u p p e r and l o w e r y b a n d s c o n t a i n i d e n t i c a l f r a g m e n t s ( f i g . 8 ) . I f t h e r e had b e e n a 2 k d mw d i f f e r e n c e b e t w e e n t h e t w o b a n d s , i t s h o u l d h a v e shown up as a t l e a s t o n e d i f f e r e n t band i n t h e l i m i t e d p r o t e o l y s i s . T h i s s t r o n g l y s u g g e s t s t h e t w o p o l y p e p t i d e s a t t h e y p o s i t i o n a r e i d e n t i c a l . E l e c t r o p h o r e s i s up t o t h i s p o i n t had been p e r f o r m e d upon s a m p l e s d i s s o c i a t e d i n a b u f f e r c o n t a i n i n g 65mM T r i s - H C 1 pH 6 . 8 , 2% S D S , 10% g l y c e r o l , and 0 . 2 % p - m e r c a p t o e t h a n o l . In o r d e r t o d e t e r m i n e i f t h e d i f f e r e n c e i n m o b i l i t i e s was due t o i n c o m p l e t e d i s u l f i d e bond r e d u c t i o n o f t h e y s u b u n i t w i t h g - m e r c a p t o e t h a n o l , s a m p l e s w e r e d i s s o c i a t e d i n i n c r e a s i n g amounts o f a s t r o n g e r r e d u c i n g a g e n t , DTT. F i g u r e 9 shows t h a t w i t h i n c r e a s i n g a m o u n t s o f DTT t h e l o w e r band o f t h e y d o u b l e t i s d e c r e a s e d w h i l e t h e u p p e r band i s i n c r e a s e d . T h i s s u g g e s t s t h a t t h e l o w e r band o f t h e d o u b l e t m i g r a t e s f a s t e r t h a n t h e u p p e r o n e b e c a u s e o n e o r m o r e d i s u l f i d e b o n d s a r e n o t r e d u c e d , m a k i n g a s h o r t e r e f f e c t i v e l e n g t h p o l y p e p t i d e . I t was c o n c l u d e d t h a t c o m p l e t e r e d u c t i o n was o b t a i n e d w i t h 33 n m o l e s DTT/mg CF^ s i n c e no f u r t h e r i n c r e a s e i n a p p a r e n t mw was o b s e r v e d when CF^ was d i s s o c i a t e d i n a h i g h e r r a t i o o f DTT t o p r o t e i n . A n d r e o e t a 1. (1979) showed t h a t new d i s u l f i d e b r i d g e s c a n be f o r m e d i n t h e 3 and y s u b u n i t s o f s p i n a c h CF^ upon h e a t a c t i v a t i o n o f A T P a s e a c t i v i t y i n t h e p r e s e n c e o f an o x i d i z i n g a g e n t , o - i o d o s o b e n z o a t e . T o t e s t i f t h e l o w e r a p p a r e n t m o l e c u l a r w e i g h t p o l y p e p t i d e o f t h e d o u b l e t i s an a r t e f a c t o f t h e h e a t i n g s t e p u s e d t o d i s s o c i a t e p o l y p e p t i d e s f o r e l e c t r o p h o r e s i s , CF^ was d i s s o c i a t e d w i t h o u t h e a t i n g , i n t h e p r e s e n c e o r a b s e n c e of r e d u c i n g a g e n t s ( f i g . 1 0 ) . CF^ d i s s o c i a t e d i n 2% SDS w i t h o u t h e a t g i v e s t h e same two b a n d s f o r t h e y s u b u n i t a s t h a t h e a t e d i n S D S . A d d i t i o n o f DTT t o CF.J d u r i n g d i s s o c i a t i o n y i e l d s o n l y o n e band f o r t h e y s u b u n i t ( f i g . 1 0 , l a n e s b and c ) i n d e p e n d e n t o f h e a t i n g t h e s a m p l e . T h u s i t 3h F i g u r e 8 : L i m i t e d p r o t e o l y s i s o f e a c h o f t h e two b a n d s o f t h e y s u b u n i t d i m e r and o f t h e a and 8 s u b u n i t s . A s a m p l e o f p u r i f i e d CF^ c o n t a i n i n g hk ug p r o t e i n was d i s s o c i a t e d i n b u f f e r c o n t a i n i n g 65 mM T r i s - H C 1 pH 6 . 8 , 10% g l y c e r o l , 2% SDS and 0 . 2 % 8 - m e r c a p t o e t h a n o l and t h e s u b u n i t s w e r e s e p a r a t e d by S D S - P A G E . The g e l was s t a i n e d f o r f i v e m i n u t e s , and d e s t a i n e d t o t h e p o i n t w h e r e b a n d s w e r e v i s i b l e . T h e s e w e r e e x c i s e d and s u b j e c t e d t o l i m i t e d p r o t e o l y s i s a c c o r d i n g t o t h e i n - g e l m e t h o d o f C l e v e l a n d e t a l . (1977) a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . A . P r o t e o l y t i c p a t t e r n g e n e r a t e d f r o m t h e u p p e r p o l y p e p t i d e o f t h e y d i m e r . B. P a t t e r n f r o m t h e l o w e r p o l y p e p t i d e o f t h e y d i m e r . C . P a t t e r n f r o m a c o m b i n e d s a m p l e o f t h e a and 8 s u b u n i t s . 34a M I G R A T I O N > 35 F i g u r e 9: E f f e c t o f r e d u c i n g a g e n t upon t h e m o b i l i t y o f t h e Y s u b u n i t o f w h e a t CF^ i n S D S - P A G E . A l l l a n e s c o n t a i n 38 ug p r o t e i n . A . CF^ d i s s o c i a t e d i n a 2% SDS s o l u t i o n c o n t a i n i n g 11 n m o l e s DTT/mg C F ^ . B . 15.4 n m o l e s DTT/ mg C F , . C . 33 n m o l e s DTT/mg C F , . 35a M I G R A T I O N > 36 F i g u r e 1 0 : E f f e c t o f h e a t upon t h e number o f b a n d s f o u n d i n t h e y s u b u n i t i n S D S - P A G E . A l l l a n e s c o n t a i n 3 7 . 5 ug p r o t e i n . a . CF.j d i s s o c i a t e d i n 2% S D S , 2 . 5 mM DTT, no h e a t i n g . b . C F 1 d i s s o c i a t e d i n 2% S D S , 1 2 . 5 mM DTT, and h e a t e d 60 s e c a t 100 C . c . C F . d i s s o c i a t e d i n 2% S D S , 1 2 . 5 mM DTT, no h e a t i n g . 36a a b c 37 a p p e a r s t h a t h e a t d o e s n o t a r t i f i c i a l l y i n t r o d u c e a d i s u l f i d e b r i d g e i n t o t h e y s u b u n i t o f w h e a t C F ^ . S i n c e two d i s c r e t e b a n d s a r e o b t a i n e d , o n e c a n s u g g e s t t h a t t h e r e i s a t l e a s t o n e i n t e r n a l d i s u l f i d e b r i d g e i n t h i s s u b u n i t . T h e two b a n d s c o u l d be t h e c o m p l e t e l y r e d u c e d s u b u n i t and t h e s u b u n i t w i t h i t s i n t e r n a l c r o s s l i n k ( s ) . A T P a s e A c t i v i t y o f Wheat CF. ] S p i n a c h c h l o r o p l a s t c o u p l i n g f a c t o r , t h e b e s t c h a r a c t e r i z e d o f t h e 2+ p l a n t C F ^ ' s , e x h i b i t s a l a t e n t Ca - d e p e n d e n t A T P a s e a c t i v i t y t h a t c a n be a c t i v a t e d by a s h o r t t r y p s i n d i g e s t i o n ( V a m b u t a s and R a c k e r , 1965) o r by h e a t t r e a t m e n t i n t h e p r e s e n c e o f a l o w c o n c e n t r a t i o n o f DTT ( F a r r o n , 1970). In t h e i n i t i a l w h e a t CF^ p r e p a r a t i o n s f o r t h i s s t u d y , C a A T P a s e a c t i v i t y was r o u t i n e l y unmasked by t r y p s i n i z a t i o n , b u t when i t was d i s c o v e r e d t h a t t h e i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s i m p r o v e d t h e i s o l a t i o n o f i n t a c t CF^ c o m p l e x , i t was d e c i d e d t o d e t e r m i n e w h e t h e r t h e w h e a t CF^ h a s a h e a t a c t i v a t e d C a A T P a s e a c t i v i t y a l s o . T a b l e I I I g i v e s t h e r e s u l t o f an e x p e r i m e n t t o i n d u c e a C a A T P a s e a c t i v i t y by h e a t i n g a t v a r i o u s t e m p e r a t u r e s . 38 TABLE I I I . H e a t a c t i v a t e d C a A T P a s e a c t i v i t y o f w h e a t C F 1 t r e a t e d a t v a r i o u s t e m p e r a t u r e s and a s s a y e d a c c o r d i n g t o t h e m e t h o d o f L i e n and R a c k e r ( 1 9 7 1 b ) . 2+ L e n g t h o f t i m e Ca - d e p e n d e n t A T P a s e a c t i v i t y o f w h e a t CF^ h e a t e d ( m i n u t e s ) a c t i v a t e d a t t h e f o l l o w i n g t e m p e r a t u r e s ( u n i t s / m g p r o t e i n ) 4 9 ° C 5 6 ° C 6 1 ° C 0 0 0 0 0 . 5 0 . 1 0 0 0 1 0 . 2 2 0 0 2 0 . 4 6 0 0 3 0 . 4 8 0 0 4 0 . 4 0 0 0 6 0 . 3 0 0 0 In c o m p a r i s o n , a c t i v a t i o n by t h e t r y p s i n m e t h o d r o u t i n e l y g i v e s 7 t o 20 u n i t s / m g p r o t e i n o f C a A T P a s e a c t i v i t y . T h u s t h e m a x i m a l h e a t - a c t i v a t e d a c t i v i t y o b t a i n e d ( 0 . 4 8 u n i t s / m g p r o t e i n ) c a n be c o n s i d e r e d n e g l i g b l e . The i n a b i l i t y t o h e a t a c t i v a t e i s a p r o p e r t y o f t h e w h e a t CF^ c o m p l e x , s i n c e s p i n a c h CF^ a c t i v a t e d by h e a t i n g f o r 4 m i n a t 6 0 ° C showed 10 u n i t s / mg p r o t e i n - C a A T P a s e a c t i v i t y . On t h e b a s i s o f t h e s e r e s u l t s i t was d e c i d e d t o r e m a i n w i t h t h e t r y p s i n m e t h o d f o r a c t i v a t i n g t h e l a t e n t C a A T P a s e o f w h e a t C F 1 . S i n c e p r o t e o l y s i s i n h i b i t o r s i n c l u d e d i n t h e w h e a t CF^ p r e p a r a t i o n s o l u t i o n s ( p - a m i n o b e n z a m i d i n e , e - a m i n o - n - c a p r o i c a c i d and PMSF) a r e s p e c i f i c i n h i b i t o r s o f s e r i n e p r o t e a s e s , i n c l u d i n g t r y p s i n , i t was n e c e s s a r y t o d e t e r m i n e w h a t e f f e c t t h e p r e s e n c e of t h e s e i n h i b i t o r s m i g h t h a v e upon t h e a c t i v a t i o n o f C a A T P a s e a c t i v i t y . F i g u r e 11 shows t h a t a l o n g e r t r y p s i n 39 F i g u r e 1 1 : T i m e c o u r s e o f t r y p s i n a c t i v a t i o n o f w h e a t C a A T P a s e i n t h e p r e s e n c e and a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s . # CF.j s a m p l e d i a l y z e d t o remove p r o t e o l y s i s i n h i b i t o r s b e f o r e t r y p s i n a c t i v a t i o n . A T r y p s i n a c t i v a t i o n o f CF^ i n t h e p r e s e n c e o f p r o t e o l y s i s i n h i b i t o r s . T r y p s i n a c t i v a t i o n was d o n e i n a s a m p l e o f p r o t e i n w i t h a f i n a l c o n c e n t r a t i o n o f 2 . 9 mM e - a m i n o - n - c a p r o i c a c i d , 0 .H3 mM p - a m i n o b e n z a m i d i n e a n d 0 . 0 0 7 2 mM P M S F . 39a TIME ACTIVATED (min) ko d i g e s t i o n i s r e q u i r e d t o r e a c h m a x i m a l a c t i v i t y i n t h e p r e s e n c e o f t h e s e p r o t e o l y s i s i n h i b i t o r s t h a n i n t h e i r a b s e n c e . In e i t h e r t h e p r e s e n c e o r a b s e n c e o f i n h i b i t o r s , t h e s i x m i n u t e s t r y p s i n d i g e s t i o n r o u t i n e u s e d was a d e q u a t e t o a c t i v a t e t h e l a t e n t C a A T P a s e o f w h e a t CF^ i n t h e e x p e r i m e n t s h o w n . H o w e v e r , d e p e n d i n g upon t h e v o l u m e o f enzyme added t o t h e a c t i v a -t i o n m i x , t h e c o n c e n t r a t i o n o f p r o t e o l y s i s i n h i b i t o r s i n a n y o n e enzyme a c t i v a t i o n w i l l v a r y g r e a t l y . T h u s i t was d e c i d e d t o r o u t i n e l y d i a l y z e o u t p r o t e o l y s i s i n h i b i t o r s p r i o r t o c r i t i c a l enzyme a s s a y s , and r e m a i n w i t h t h e s i x m i n u t e s d i g e s t i o n . S u b s t r a t e s p e c i f i c i t y o f w h e a t C a A T P a s e was d e t e r m i n e d , and t h e r e s u l t s a r e g i v e n i n T a b l e I V . A b o u t o n e - t h i r d o f t h e n u c l e o t i d e t r i p h o s p h a t a s e a c t i v i t y w i t h ATP as s u b s t r a t e was o b t a i n e d w i t h G T P , t h e o t h e r p u r i n e n u c l e o t i d e t r i p h o s p h a t e . N e g l i g i b l e a c t i v i t y was o b t a i n e d w i t h C T P , UTP o r TTP a s s u b s t r a t e s . Wheat C a A T P a s e i s t h u s v e r y s u b s t r a t e s p e c i f i c , a c c e p t i n g o n l y p u r i n e b a s e n u c l e o t i d e t r i p h o s p h a t e s i n t o t h e enzyme a c t i v e s i t e ( s ) , b u t c l e a v i n g GTP t o a l e s s e r e x t e n t t h a n A T P . T a b l e I V . S u b s t r a t e s p e c i f i c i t y o f t h e C a A T P a s e a c t i v i t y o f w h e a t C F ^ . L a t e n t A T P a s e a c t i v i t y was a c t i v a t e d by t r y p s i n d i g e s t i o n i n t h e a b s e n c e o f A T P . A l l s u b s t r a t e s w e r e 1mM n u c l e o t i d e t r i p h o s p h a t e i n kO mM T r i c i n e - K O H pH 8 . 0 , 10 mM C a C 1 _ . S u b s t r a t e % o f a c t i v i t y w i t h ATP ATP 100 GTP 32 UTP 0 CTP ].k TTP 0 41 F o r a s s a y i n g t h e l a t e n t C a A T P a s e a c t i v i t y o f w h e a t C F ^ , 1 mM ATP was u s e d a s s u b s t r a t e . To t e s t w h e t h e r t h e w h e a t A T P a s e i s s t r i c t l y c a l c i u m -d e p e n d e n t , t h e c a l c i u m c o n c e n t r a t i o n was v a r i e d , h o l d i n g t h e ATP c o n c e n t r a t i o n c o n s t a n t a t 1 mM. As shown i n f i g . 1 2 , o p t i m a l a c t i v i t y i s o b t a i n e d w i t h a 1 :1 r a t i o o f c a l c i u m t o A T P , i m p l y i n g t h a t t h e c a t i o n - A T P c o m p l e x i s t h e t r u e s u b s t r a t e f o r t h e A T P a s e r e a c t i o n . E x c e s s c a l c i u m 2+ (10 Ca : 1 ATP) d o e s i n h i b i t enzyme a c t i v i t y a b o u t 2 5 % . K.. a n d V f o r t h e C a A T P a s e o f w h e a t C F , w e r e d e t e r m i n e d u n d e r M max 1 two d i f f e r e n t s e t s o f c o n d i t i o n s . In t h e f i r s t , i n i t i a l v e l o c i t i e s o f t h e 2+ r e a c t i o n w e r e d e t e r m i n e d a t v a r i o u s c o n c e n t r a t i o n s o f ATP w i t h Ca i n e x c e s s , a t a c o n s t a n t c o n c e n t r a t i o n o f 10 mM. S u b s t r a t e was p l o t t e d a s t h e c o n c e n t r a t i o n o f CaATP c o m p l e x , a s c a l c u l a t e d u s i n g e q u a t i o n ( 1 ) . The r e s u l t o f o n e s u c h e x p e r i m e n t i s shown i n f i g . 1 3 . E a d i e - H o f s t e e a n a l y s i s o f t h i s d a t a g i v e s a K., o f 0 . 1 2 5 mM CaATP a n d a V o f 1 8 . 9 u n i t s / mg M max p r o t e i n . ^ m a x w a s f o u n d t o v a r y g r e a t l y f r o m enzyme p r e p a r a t i o n t o 2+ enzyme p r e p a r a t i o n , b u t u n d e r t h e same c o n d i t i o n s o f e x c e s s Ca , was f o u n d t o be i n t h e r a n g e o f 0 . 1 t o 0 . 3 mM C a A T P . In t h e s e c o n d s e t o f c o n d i t i o n s , t h e c a l c i u m l e v e l was k e p t e q u i m o l a r t o t h a t o f A T P . U s i n g e q u a t i o n (1) t o c a l c u l a t e t h e c o n c e n t r a t i o n o f CaATP c o m -p l e x , a K.. o f 18 uM CaATP a n d a V o f 6.77 u n i t s / m g p r o t e i n w e r e o b t a i n e d . M max 3 r ( f i g . 1 4 ) . I t was o b s e r v e d t h a t t h e m a x i m a l enzyme a c t i v i t y , b u t n o t t h e K^, v a r i e s f r o m enzyme p r e p a r a t i o n t o enzyme p r e p a r a t i o n . The enzyme u s e d i n t h i s d e t e r m i n a t i o n was f r o m a d i f f e r e n t p r e p a r a t i o n a n d a d i f f e r e n t b a t c h o f s e e d , t h a n t h a t u s e d f o r t h e e x p e r i m e n t shown i n f i g . 1 3 . s o V v a l u e s a r e n o t r • ' max c o m p a r a b l e . H o w e v e r , t h e i n t h e c a s e o f e q u i m o l a r c a l c i u m and ATP i s an o r d e r o f m a g n i t u d e l o w e r t h a n t h a t o b t a i n e d w i t h a l a r g e e x c e s s o f c a l c i u m , and a p p e a r s t o be a d i r e c t e f f e c t o f t h e d i f f e r e n t l e v e l s o f c a l c i u m . F i g u r e 1 2 : E f f e c t o f t h e r a t i o o f c a l c i u m t o ATP upon A T P a s e a c t i v i t y o f t h e t r y p s i n - a c t i v a t e d CF^ f r o m w h e a t . The ATP c o n c e n t r a t i o n i n t h e a s s a y m i x was h e l d c o n s t a n t a t 1 mM a n d t h e c a l c i u m c o n c e n t r a t i o n was v a r i e d . 42a 43 F i g u r e 1 3 : M i c h a e 1 i s - M e n t e n k i n e t i c s o f t h e t r y p s i n - a c t i v a t e d C a A T P a s e o f w h e a t CF^ i n t h e p r e s e n c e o f 10 mM C a C I ^ . S u b s t r a t e c o n c e n t r a t i o n (S) was d e t e r m i n e d by c a l c u l a t i n g CaATP a c c o r d i n g t o e q u a t i o n ( 1 ) . I n s e t i s an E a d i e - H o f s t e e p l o t o f t h i s d a t a , u s e d f o r t h e d e t e r m i n a t i o n of K.. and V M max T h i s l i n e has a c o r r e l a t i o n c o e f f i c i e n t of 0 . 9 3 -43a kk F i g u r e Ik: M i c h a e l i s - M e n t e n k i n e t i c s o f t h e t r y p s i n - a c t i v a t e d C a A T P a s e o f w h e a t C F ^ . C a l c i u m and ATP w e r e a d d e d i n e q u i m o l a r a m o u n t s t o t h e a s s a y m i x , and s u b s t r a t e c o n c e n t r a t i o n was c a l c u l a t e d a c c o r d i n g t o e q u a t i o n ( 1 ) . I n s e t i s an E a d i e - H o f s t e e p l o t o f t h i s d a t a , u s e d f o r t h e d e t e r m i n a t i o n o f K.. a n d V . T h i s l i n e M max h a s a c o r r e l a t i o n c o e f f i c i e n t o f 0 . 8 7 -45 P r e l i m i n a r y e x p e r i m e n t s t o t e s t t h e e f f e c t o f ADP upon C a A T P a s e a c t i v i t y showed t h a t ADP was i n d e e d i n h i b i t o r y . H o w e v e r , t h e s e r e s u l t s do n o t g i v e a c l e a r p i c t u r e a s t o w h e t h e r ADP i s a c o m p e t i t i v e i n h i b i t o r o r a c t s by some a l l o s t e r i c m e c h a n i s m . F u r t h e r m o r e , t h e s e e x p e r i m e n t s w e r e d o n e b e f o r e t h e e f f e c t o f h i g h c o n c e n t r a t i o n o f c a l c i u m was o b s e r v e d , and p a r t o f t h e i n h i b i t i o n s e e n may h a v e b e e n d u e t o e x c e s s c a l c i u m r a t h e r t h a n t o t h e p r e s e n c e o f ADP. E l e c t r o n M i c r o s c o p y o f Wheat CF^ N e g a t i v e s t a i n i n g o f w h e a t C F ^ , w i t h e i t h e r ammonium m o l y b d a t e o r p h o s p h o t u n g s t i c a c i d , g a v e i d e n t i c a l p a r t i c l e s when v i e w e d i n t h e e l e c t r o n m i c r o s c o p e ( f i g . 15)- P a r t i c l e d i a m e t e r s w e r e e s t i m a t e d t o be 108 A + 9-5 A\ w i t h ammonium m o l y b d a t e s t a i n , and 110 A + 6.3 A w i t h p h o s p h o -t u n g s t i c a c i d . P h o s p h o t u n g s t i c a c i d g a v e s l i g h t l y b e t t e r r e s o l u t i o n , and more c o n s i s t e n t r e s u l t s t h a n d i d ammonium m o l y b d a t e . U r a n y l a c e t a t e s t a i n c a u s e d CF^ t o d i s s o c i a t e i n t o i t s s u b u n i t s . E. coli F^  shows a v e r y s i m i l a r s t r u c t u r e t o CF^ when s t a i n e d by t h e same p r o c e d u r e ( f i g . 16). ECF^ h a s a p a r t i c l e d i a m e t e r o f 100 A" + 5.1 A when p a r t i c l e s a r e n e g a t i v e l y s t a i n e d w i t h ammonium m o l y b d a t e . P a r t i c l e s o f CF^ show many o r i e n t a t i o n when n e g a t i v e l y s t a i n e d , a s t h e y s e t t l e o n t o t h e e l e c t r o n m i c r o s c o p y g r i d a t r a n d o m . I t , i s p o s s i b l e t o p i c k o u t p a r t i c l e s w h i c h h a v e a h e x a g o n a l s t r u c t u r e , w i t h a c e n t r a l c o r e and w h a t may be s i x s u b u n i t s a t t h e v e r t i c i e s ( f i g . 17). Many t i m e s s u c h an o r i e n t a t i o n seems t o be t i l t e d o u t o f t h e p l a n e , g i v i n g p a r t i c l e s w i t h a c e n t r a l d e p r e s s i o n . H o w e v e r , a t s u c h h i g h m a g n i f i c a t i o n s , i t i s d i f f i c u l t t o o b t a i n g o o d r e s o l u t i o n o f enzyme p a r t i c l e s b e c a u s e o f t h e l a r g e b a c k g r o u n d g r a i n s i z e . 46 F i g u r e 1 5 : E l e c t r o n m i c r o g r a p h s o f w h e a t C F ^ , n e g a t i v e l y - s t a i n e d w i t h two d i f f e r e n t s t a i n s . A . 2% p h o s p h o t u n g s t i c a c i d KOH pH 7 - 0 . B. 2% ammonium m o l y b d a t e i n 12 mM s o d i u m o x a l a t e pH 7 - 2 . M a g n i f i c a t i o n i n b o t h m i c r o g r a p h s i s 5 1 6 , 0 0 0 x . 46a hi F i g u r e 16: E l e c t r o n m i c r o g r a p h s o f E. c o l i F^, n e g a t i v e l y - s t a i n e d w i t h 2% ammonium m o l y b d a t e i n 12 mM s o d i u m o x a l a t e pH 7 . 2 . M a g n i f i c a t i o n i s 4 1 3 , 0 0 0 x . 47a 48 F i g u r e 17: S e l e c t e d CF^ p a r t i c l e s , n e g a t i v e l y s t a i n e d w i t h 2% p h o s p h o t u n g s t i c a c i d - K O H pH 1.2, s h o w i n g v a r i o u s d e g r e e s o f h e x a g o n a l s t r u c t u r e . M a g n i f i c a t i o n i s 739,000 x . F o r t h i s r e a s o n , i t was d e c i d e d t o t r y image e n h a n c e m e n t o f t h e m o s t s y m m e t r i c a l m o l e c u l e s p r i n t e d t o b r i n g o u t a n y i n h e r e n t s t r u c t u r e . CF^ p a r t i c l e s e x h i b i t i n g w h a t a p p e a r e d t o be a s i x - f o l d s y m m e t r y w e r e b l o w n up p h o t o g r a p h i c a l l y a n d image a n a l y s i s was p e r f o r m e d by Markham r o t a t i o n (Markham et- a l . , 1963). F i g u r e 18 shows t h e r e s u l t o f r o t a t i o n o f t h r e e s u c h p a r t i c l e s . From t h e o r i g i n a l image i t i s d i f f i c u l t t o d i f f e r e n t i a t e b e t w e e n f i v e - , s i x - , and s e v e n - f o l d s y m m e t r y , b u t f o r t h e m o s t p a r t f i g . 18 shows t h e b e s t r e s o l u t i o n o f a p i c i e s w i t h a s i x - f o l d r o t a t i o n , w i t h t w o - a n d t h r e e - f o l d r o t a t i o n s a l s o g i v i n g h e x a g o n a l p a r t i c l e s . I m m u n o l o g i c a l S t u d y o f Wheat CF^ A n t i - w h e a t CF^ s e r u m , w i t h an a n t i b o d y t i t e r o f 1/80, was o b t a i n e d f r o m a r a b b i t a f t e r t h r e e i n j e c t i o n s o f 0.2 mg p u r i f i e d CF^ ( f i g 19). Serum t a k e n f r o m t h e r a b b i t p r i o r t o i m m u n i z a t i o n d i d n o t b i n d t o C F ^ , s i n c e i t g a v e no p r e c i p i t i n l i n e a g a i n s t w h e a t CF^ i n d o u b l e d i f f u s i o n p l a t e s . Wheat CF^ a n t i s e r u m a l s o p r e c i p i t a t e d p u r i f i e d s p i n a c h CF^ , i n d i c a t i n g s i m i l a r a n t i g e n i c s i t e s on t h e CF^ c o m p l e x e s f r o m t h e two p l a n t s . C o m p l e t e CF^ was i n j e c t e d i n t o t h e r a b b i t t o g e n e r a t e a n t i b o d i e s . In o r d e r t o d e t e r m i n e a g a i n s t w h i c h CF^ s u b u n i t o r s u b u n i t s t h e a n t i b o d i e s r e a c t e d , two d i m e n s i o n a l i m m u n o e l e c t r o p h o r e s i s was p e r f o r m e d ( f i g . 20). A p r e c i p i t i n r o c k e t d e v e l o p e d a g a i n s t t h e a and 8 s u b u n i t s , b u t n o t a g a i n s t t h e t h r e e s m a l l e r s u b u n i t s . I t i s i m p o s s i b l e t o a s c e r t a i n w h e t h e r t h e a n t i - C F ^ s e r u m r e a c t s a g a i n s t b o t h t h e a and 8 s u b u n i t s , o r o n l y o n e s u b u n i t , b e c a u s e t h e s e two s u b u n i t s a r e n o t w e l l r e s o l v e d i n a 10% SDS-PAGE g e l . H o w e v e r , t h e i m m u n o p r e c i p i t a t i o n r o c k e t i s s y m m e t r i c a l , s o i t p r o b a b l y r e a c t s w i t h b o t h o f t h e l a r g e s t s u b u n i t s o f w h e a t C F , . T h u s t h i s a n t i s e r u m w o u l d 50 F i g u r e 1 8 : Markham r o t a t i o n o f t h r e e w h e a t CF^ p a r t i c l e s . T h e n u m b e r s d e n o t e t h e d i v i s i o n s o f a c i r c l e t h r o u g h w h i c h t h e p h o t o g r a p h i c p a p e r was r o t a t e d i n m a k i n g t h e m u l t i p l e e x p o s u r e s . 50a 51 F i g u r e 19: O u c h t e r l o n y d o u b l e d i f f u s i o n p l a t e e s t i m a t i o n o f a n t i b o d y t i t e r i n s e r u m f r o m r a b b i t i m m u n i z e d a g a i n s t w h e a t C F ^ . W h o l e s e r u m was d i l u t e d a s i n d i c a t e d i n t h e p e r i p h e r a l w e l l s , and p r e c i p i t i n l i n e s w e r e d e v e l o p e d a g a i n s t CF^ o v e r n i g h t . T h e s e p r e c i p i t i n l i n e s w e r e t h e n v i s u a l i z e d by s t a i n i n g w i t h C o o m a s s i e b l u e a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . F i g u r e 20: C r o s s e d I m m u n o e l e c t r o p h o r e s i s o f t h e s e p a r a t e d s u b u n i t s o f w h e a t CF^ a g a i n s t a n t i - w h e a t CF^ s e r u m , a c c o r d i n g t o t h e m e t h o d o f Chua and B l o m b e r g (1979). a . Wheat C F 1 s u b u n i t s , s e p a r a t e d by SDS-PAGE on a 10% g e l . The s a m p l e c o n t a i n e d k~J ug p r o t e i n . The l a n e shown h e r e i s a l a n e a d j a c e n t t o t h e o n e e l e c t r o p h o r e s e d i n t o t h e a n t i b o d y g e l . b . A n t i b o d y g e l , s h o w i n g i m m u n o p r e c i p i t i n r o c k e t w h i c h h a s d e v e l o p e d a g a i n s t t h e a and 3 s u b u n i t s o f w h e a t CF.j . The a n t i b o d y g e l c o n t a i n e d 1 ml o f u n f r a c t i o n a t e d r a b b i t a n t i s e r u m . P r e c i p i t i n l i n e s w e r e v i s u a l i z e d by s t a i n i n g w i t h C o o m a s s i e b l u e a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . 51a 52 be u s e f u l i n p r e c i p i t a t i n g t h e a and $ s u b u n i t s i n a c e l l - f r e e t r a n s l a t i o n o f w h o l e w h e a t RNA. The a n t i s e r u m o b t a i n e d a l s o i n h i b i t s t h e l a t e n t C a A T P a s e o f t r y p s i n - a c t i v a t e d C F 1 ( f i g . 2 1 ) . V e r y s m a l l a m o u n t s (15 u l ) o f a n t i s e r u m c o m p l e t e l y i n h i b i t A T P a s e a c t i v i t y . 53 F i g u r e 2 1 : I n h i b i t i o n o f t h e t r y p s i n - a c t i v a t e d C a A T P a s e a c t i v i t y o f w h e a t CF^ by a n t i - w h e a t CF^ s e r u m . The l a t e n t C a A T P a s e was a c t i v a t e d by t r y p s i n d i g e s t i o n , and a n t i -s e r u m was a d d e d t o 2 . 5 ug a l i q u o t s o f t h e a c t i v a t e d e n z y m e . C a A T P a s e was a s s a y e d i m m e d i a t e l y , a c c o r d i n g t o t h e m e t h o d o f L i e n and R a c k e r ( 1 9 7 1 b ) . 1 % INITIAL ATPase ACTIVITY 5k DISCUSS ION In t h e p r o c e d u r e d e v e l o p e d h e r e , w h e a t c h l o r o p l a s t c o u p l i n g f a c t o r was removed f r o m t h e s u r f a c e o f t h e t h y l a k o i d and p u r i f i e d t o h o m o g e n e i t y by a s i m p l e t w o - s t e p p r o c e d u r e c o n s i s t i n g o f c h l o r o f o r m e x t r a c t i o n and s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . B e t t e r p r e s e r v a t i o n o f a CF^ c o m p l e x c o n t a i n i n g a l l s u b u n i t s was o b t a i n e d by t h e i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s d u r i n g i s o l a t i o n a n d p u r i f i c a t i o n . In e x p e r i m e n t s i n w h i c h w h e a t CF.| was i s o l a t e d i n t h e a b s e n c e o f p r o t e o l y s i s i n h i b i t o r s , b o t h t h e 6 a n d e s u b u n i t s w e r e l o s t d u r i n g t h e s u c r o s e g r a d i e n t s t e p . I n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s y i e l d e d a CF^ w i t h a l l f i v e s u b u n i t s p r e s e n t . T h i s c o u l d be d u e t o a number o f f a c t o r s . S e v e r a l a u t h o r s h a v e n o t e d t h e t e n d e n c y o f t h i s enzyme t o l o s e s u b u n i t s o n c e i t h a s b e e n s e p a r a t e d f r o m t h e t h y l a k o i d . B i n d e r e t a l . (1978) f o u n d t h a t t h e 6 s u b u n i t o f s p i n a c h CF^ was o f t e n l o s t i n p r e p a r a t i o n s w h e r e CF^ was c h l o r o f o r m - r e l e a s e d f r o m l a r g e a m o u n t s o f t h y l a k o i d s . P a s s a g e o f s p i n a c h CF^ t h r o u g h a DEAE-S e p h a d e x A50 c o l u m n a l s o c a u s e d t h i s c o m p l e x t o l o s e t h e 6 s u b u n i t ( Y o u n i s e t a l . , 1976). I t seems t h a t t h i s c o m p l e x i s i n h e r e n t l y f r a g i l e , a b o v e and b e y o n d i t s e o T d l a b i l i t y p r o p e r t i e s . W h e t h e r t h e p r o t e c t i o n o f CF^ o b t a i n e d w i t h p r o t e o l y s i s i n h i b i t o r s r e p o r t e d h e r e i s d u e t o i n h i b i t i o n o f p r o t e o l y s i s o r t o some o s m o t i c o r i o n i c e f f e c t o f t h e s e compounds i s u n k n o w n . C h a r a c t e r i z a t i o n o f t h e s u b u n i t s o f w h e a t CF^ by SDS-PAGE g a v e s e v e r a l i n t e r e s t i n g r e s u l t s . M o l e c u l a r w e i g h t s o f t h e y and 6 s u b u n i t s a r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e o f s p i n a c h C F ^ , e s p e c i a l l y t h a t o f t h e 6 s u b u n i t (25 kd v e r s u s 19 kd f o r s p i n a c h ) . In a d d i t i o n , a number o f d i s c r e t e f o r m s o f t h e y s u b u n i t w e r e f o u n d , d e p e n d a n t upon t h e d e g r e e o f r e d u c t i o n p r i o r t o e l e c t r o p h o r e s i s . I t i s n o t s u f f i c i e n t t o d i s s o c i a t e w h e a t C F 1 i n a s t o c k s o l u t i o n o f 2% SDS p l u s 0.2% 8 - m e r c a p t o e t h a n o l 55 or 5 mM DTT. A c r i t i c a l amount of r e d u c i n g agent i s n e c e s s a r y t o i n s u r e c o m p lete r e d u c t i o n o f t h e y s u b u n i t , a t l e a s t 33 nmoles DTT/mg CF^. Care s h o u l d be taken t o c o m p l e t e l y reduce and d i s s o c i a t e a l l p o l y p e p t i d e s when m o l e c u l a r w e i g h t d e t e r m i n a t i o n s a r e made on a b a s i s o f m o b i l i t y i n SDS-PAGE. Because two d i s c r e t e y bands a r e o b t a i n e d when CF^ i s e l e c t r o p h o r e s e d i n low amounts o f r e d u c i n g a g e n t , i t i s t e m p t i n g t o c o n c l u d e t h a t t h e r e i s a t l e a s t one i n t e r n a l d i s u l f i d e b r i d g e i n the y s u b u n i t . F a r r o n and Racker (1970) showed t h a t t h e r e a r e two d i s u l f i d e bonds i n the s p i n a c h CF^ m o l e c u l e . These a r e not broken d u r i n g ATPase a c t i v a t i o n , as t r y p s i n o r heat a c t i v a t i o n o f the l a t e n t ATPase d i d not r e s u l t i n a n . i n c r e a s e i n the number o f t i t r a b l e h a l f c y s t i n e s ( a l s o F a r r o n and R a c k e r , 1970). I t i s proposed here t h a t t h e r e i s a t l e a s t one i n t e r n a l d i s u l f i d e b r i d g e i n the y s u b u n i t of wheat CF^. A s i m i l a r i n t e r n a l c y s t i n e which a l t e r s m o b i l i t y i n e l e c t r o p h o r e s i s has been i d e n t i f i e d i n t h e 33 kd p o l y p e p t i d e o f P hotosystem II p a r t i c l e s i n s p i n a c h (Kuwabara and M u r a t a , 1979). The e f f e c t o f p r o t e o l y s i s i n h i b i t o r s i n p r e v e n t i n g r e l e a s e o f CF^ from t h e t h y l a k o i d i s s t r i k i n g , and s p e c i f i c f o r t h e CF^ complex. In f i g . k ( l a n e s c and d) one can see t h a t p r o t e o l y s i s i n h i b i t o r s do not p r e v e n t the r e l e a s e o f two o t h e r u n i d e n t i f i e d p o l y p e p t i d e s n o r m a l l y e x t r a c t e d by h y p o t o n i c s u c r o s e (arrows) but they c o m p l e t e l y i n h i b i t r e l e a s e o f any o f t h e p o l y p e p t i d e s of CF^. One c o u l d thus argue t h a t t h e i n h i b i t o r s , by t h e i r s p e c i f i c i t y , p r e v e n t CF^ r e l e a s e because p r o t e o l y s i s i s p r e v e n t e d , r a t h e r than because o f some g e n e r a l o s m o t i c e f f e c t . In a d d i t i o n , the d i s c o v e r y t h a t p r o t e o l y s i s i n h i b i t o r s p r e v e n t the l o s s o f the minor s u b u n i t s o f CF^ d u r 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 s u g g e s t s t h a t a p r o t e a s e e x t r a c t s w i t h CF,. 56 A l l a t t e m p t s t o h e a t a c t i v a t e t h e l a t e n t C a A T P a s e o f w h e a t CF^ w e r e u n s u c c e s s f u l . U n d e r i d e n t i c a l c o n d i t i o n s , t h e s p i n a c h CF^ a c t i v a t e d t o a maximum o f 10 u n i t s / m g p r o t e i n . T h i s l e a d s o n e t o c o n c l u d e t h a t t h e r e i s a b a s i c d i f f e r e n c e i n t h e s t r u c t u r e o f t h e s e two e n z y m e s . F a r r o n (1970) and F a r r o n a n d R a c k e r (1970) s t u d i e d t h e p r o c e s s o f h e a t a c t i v a t i o n o f s p i n a c h C F ^ . In b o t h t h e a c t i v a t e d and u n a c t i v a t e d CF^ c o m p l e x e s , e i g h t SH g r o u p s w e r e t i t r a b l e i n u r e a o r S D S . H e a t a c t i v a t i o n d i d n o t r e s u l t i n t h e c l e a v a g e o f e i t h e r o f t h e two d i s u l f i d e b r i d g e s o f s p i n a c h CF . H o w e v e r , m o s t o f t h e e i g h t SH g r o u p s a r e n o t on t h e s u r f a c e o f t h e e n z y m e , s i n c e i n t h e a b s e n c e o f d e n a t u r i n g s o l v e n t s , o n l y two SH g r o u p s p e r m o l e c u l e o f CF^ c o u l d be t i t r a t e d . H e a t a c t i v a t i o n made a c c e s s i b l e f o u r a d d i t i o n a l SH g r o u p s . T h e s e n e w l y - e x p o s e d SH g r o u p s c a n be i n d u c e d t o f o r m i n t r a p e p t i d e d i s u l f i d e b r i d g e s by t h e a d d i t i o n o f t h e s t r o n g o x i d i z i n g a g e n t o - i o d o s o b e n z o a t e d u r i n g h e a t a c t i v a t i o n ( A n d r e o e t a l . , 1979). I t h a s b e e n shown t h a t N - e t h y l m a l e i m i d e o r i o d o a c e t a m i d e i n h i b i t t h e a c t i v a t i o n o f CF^ by h e a t b u t n o t by t r y p s i n ( F a r r o n a n d R a c k e r , 1970), i m p l y i n g t h a t t h e two t y p e s o f a c t i v a t i o n o f t h e l a t e n t C a A T P a s e p r o c e e d by d i f f e r e n t m e c h a n i s m s . T h u s i t i s q u i t e p o s s i b l e t h a t w h e a t CF^ s h o u l d show t r y p s i n a c t i v a t i o n o f t h e l a t e n t C a A T P a s e b u t n o t h e a t a c t i v a t i o n . The o p t i m a l C a r A T P r a t i o f o r A T P a s e a c t i v i t y o f t h e a c t i v a t e d CF^ f r o m s p i n a c h i s 1:1 ( V a m b u t a s and R a c k e r , 1965). S i m i l a r r e s u l t s a r e r e p o r t e d h e r e f o r w h e a t C F ^ . T h i s s u g g e s t s t h a t t h e a c t u a l s u b s t r a t e 2+ f o r t h e A T P a s e r e a c t i o n i s t h e Ca ATP c o m p l e x . The f o r m a t i o n o f t h e c a t i o n - A T P c o m p l e x i s pH d e p e n d e n t a s w e l l a s d e p e n d e n t upon t h e d i s s o c i a t i o n c o n s t a n t f o r t h e c o m p l e x , a c c o r d i n g t o t h e r e l a t i o n s h i p g i v e n i n e q u a t i o n (1). I t i s i m p o r t a n t t o c a l c u l a t e 57 s u b s t r a t e c o n c e n t r a t i o n a s c o m p l e x e d CaATP r a t h e r t h a n f r e e A T P , f o r a t v e r y l o w ATP c o n c e n t r a t i o n s t h e r e i s a l a r g e d i f f e r e n c e b e t w e e n t h e two v a l u e s . T h e c o m p l e x k i n e t i c c u r v e s p u b l i s h e d by C a r r e i r a and Munoz (1975) f o r t h e A T P a s e a c t i v i t y o f E. c o l i w e r e c a l l e d i n t o q u e s t i o n by A h l r e s (1976) f o r t h i s v e r y r e a s o n . In e x p e r i m e n t s w h e r e C a r r e i r a and Munoz u s e d a C a : A T P r a t i o o f 0.5:1 i n d e t e r m i n i n g r e a c t i o n r a t e s w i t h ATP a s t h e s u b s t r a t e , n o n - 1 i n e a r L i n e w e a v e r - B u r k e p l o t s w e r e o b t a i n e d . A h l e r s h a s shown t h a t t h e s e u n u s u a l k i n e t i c c u r v e s a r e d u e t o f a u l t y c h o i c e o f s u b s t r a t e and f a i l u r e t o c a l c u l a t e t h e e f f e c t i v e c o n c e n t r a t i o n o f t h e c a t i o n - A T P c o m p l e x . In t h e s t u d y r e p o r t e d h e r e , by p l o t t i n g i n i t i a l v e l o c i t i e s a g a i n s t t h e c a l c u l a t e d CaATP c o n c e n t r a t i o n , a M i c h a e l i s - M e n t e n r e l a t i o n s h i p i s o b t a i n e d ( f i g s . 13 and 1 4 ) . A number o f K.. v a l u e s f o r t h e C a A T P a s e h a v e b e e n r e p o r t e d i n t h e M l i t e r a t u r e . N e i s o n e t a l . (1972), a s s a y i n g t h e s p i n a c h A T P a s e i n T r i c i n e - m a l e a t e b u f f e r a t pH 8 a n d i n t h e p r e s e n c e o f 8 mM C a C l ^ , f o u n d a o f 0.8 mM A T P . In l o w e r pH medium (pH 6) i n t h e p r e s e n c e o f 2 mM M g C l ^ a MgATPase w i t h a K M o f 0.1 mM ATP was o b s e r v e d , t h o u g h ^ m a x was v e r y l o w a t t h i s p H . In pH 8 Hepes-NaOH b u f f e r , Hochman e t a l . (1976) o b t a i n e d a o f 2.7 mM CaATP f o r t h e l e t t u c e e n z y m e . In t h e r e s u l t s p r e s e n t e d i n t h i s d i s s e r t a t i o n , t h e o b t a i n e d i n t h e p r e s e n c e o f e x c e s s c a l c i u m (0.125 mM CaATP) i s c o m p a r a b l e t o v a l u e s r e p o r t e d i n t h e l i t e r a t u r e , b u t t h e o b t a i n e d i n e x p e r i m e n t s w i t h e q u i m o l a r c a l c i u m and ATP (18 uM CaATP) i s much l o w e r t h a n t h o s e r e p o r t e d f o r s p i n a c h o r l e t t u c e A T P a s e s . T h i s i s p r o b a b l y d u e t o a d i f f e r e n c e i n t h e enzyme f r o m w h e a t . T h e k i n e t i c d a t a o f Hochman e t a l . (1976) i n d i c a t e s t h a t t h e c a t i o n - A T P c o m p l e x i s a l s o t h e s u b s t r a t e o f A T P a s e a c t i v i t y o f l e t t u c e C F ^ . By 2+ 2+ s u b s t i t u t i n g Mn f o r Ca , t h e y m e a s u r e d d i r e c t b i n d i n g o f MnATP by 58 t h e a m p l i t u d e o f t h e M n i T e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s i g n a l . T h e i r r e s u l t s show t w o t i g h t and t h r e e l o o s e c a t i o n b i n d i n g s i t e s on C F ^ . The t i g h t c a t i o n b i n d i n g s i t e s h a v e a i n t h e m i c r o m o l a r r a n g e , s i m i l a r t o t h e K v a l u e s f o r t h e c o m p e t i t i v e i n h i b i t i o n o f f r e e c a t i o n s . T h e s e a u t h o r s p r o p o s e t h a t t h e s e h i g h a f f i n i t y c a t i o n s i t e s a r e t h e c a t a l y t i c s i t e s , w h i l e t h e l o w a f f i n i t y s i t e s a r e r e g u l a t o r y . T h e y c o n c l u d e t h a t t h e r a t e o f c a t a l y s i s i s r e g u l a t e d by t h e l o w a f f i n i t y s i t e , w h i c h h a s a i n t h e m i l l i m o l a r r a n g e . In e x p e r i m e n t s r e p o r t e d h e r e , t h e o f t h e t r y p s i n - a c t i v a t e d A T P a s e was d e t e r m i n e d u s i n g CaATP a s s u b s t r a t e i n t h e p r e s e n c e o f a v a s t e x c e s s o f Ca and w i t h a 1:1 r a t i o o f C a : A T P . W i t h e x c e s s C a , a o f 0.1 t o 0.3 mM was o b t a i n e d , w h i l e w i t h e q u i m o l a r a m o u n t s o f Ca and ATP a o f 18 uM was f o u n d . One may s u g g e s t s e v e r a l r e a s o n s f o r t h e d i f f e r e n t a f f i n i t y c o n s t a n t s i n t h e s e t w o t y p e s o f d e t e r m i n i a t i o n s . I t i s p o s s i b l e t h a t e x c e s s c a l c i u m b i n d s a t o t h e r s i t e s t h a n t h e a c t i v e s i t e s on t h e A T P a s e , d e c r e a s i n g t h e a f f i n i t y o f t h e enzyme f o r s u b s t r a t e . A l t e r n a t i v e l y , t h e i n c r e a s e i n K.. o b s e r v e d w i t h e x c e s s c a l c i u m c o u l d be due t o t h e e x c e s s c a l c i u m b i n d i n g c o m p e t i t i v e l y f o r t h e same h i g h a f f i n i t y s i t e a s C a A T P . The s e c o n d p o s t u l a t e w o u l d c a l l f o r a c o m p e t i t i v e i n h i b i t i o n o f t h e A T P a s e r e a c t i o n by c a l c i u m . Hochman e t a l . ( 1 9 7 6 ) g i v e a K. o f 7 mM f o r C a , w o r k i n g w i t h t h e h e a t - a c t i v a t e d A T P a s e o f l e t t u c e C F ^ . T h e p r e l i m i n a r y n a t u r e o f t h e r e s u l t s p r e s e n t e d h e r e p r e v e n t s m a k i n g a n y c o n c l u s i o n s a b o u t t h e a f f i n i t y o f s u b s t r a t e b i n d i n g i n l o w and h i g h Ca c o n c e n t r a t i o n s . N e g a t i v e - s t a i n e d w h e a t CF^ h a s a p a r t i c l e d i a m e t e r o f 108-110 A when v i e w e d i n t h e e l e c t r o n m i c r o s c o p e u s i n g t w o d i f f e r e n t n e g a t i v e s t a i n s . The m i c r o g r a p h s show a h e x a g o n a l s t r u c t u r e i n p a r t i c l e s o f o n e p a r t i c u l a r o r i e n t a t i o n . T h e e l e c t r o n m i c r o g r a p h s p r e s e n t e d h e r e a r e s t r i k i n g l y s i m i l a r 59 t o p u b l i s h e d m i c r o g r a p h s o f y e a s t m i t o c h o n d r i a l ( S c h a t z e t a l . , 1967) and b e e f h e a r t m i t o c h o n d r i a F^  (Kagawa and R a c k e r , 1966). T h e s e F^  p r e p a r a t i o n s g a v e 80-90 A* s p h e r e s w h i c h c o n s i s t o f a b o u t s i x d i s c r e t e s u b u n i t s . Markham r o t a t i o n o f w h e a t CF^ p a r t i c l e s e n h a n c e s a s i x - f o l d s y m m e t r y . H o w e v e r , i t m u s t be remembered t h a t o n e i n t r o d u c e s b i a s when u s i n g t h i s image e n h a n c e m e n t t e c h n i q u e , by s e l e c t i n g p a r t i c l e s t h a t s u g g e s t a p a r t i c u l a r s y m m e t r y . P a r t i c l e s w i l l f a l l i n many o r i e n t a t i o n s upon t h e e l e c t r o n m i c r o s c o p y g r i d . In a d d i t i o n , o n e e n h a n c e s a r t e f a c t s o f s t a i n i n g a s w e l l a s t h e a c t u a l s t r u c t u r e s . From f i g . 18 o n e c a n c o n c l u d e o n l y t h a t o n e p a r t i c u l a r o r i e n t a t i o n o f w h e a t CF^ shows a s i x - f o l d s y m m e t r y . F o r t h e m o s t p a r t , t h e s e h e x a g o n a l p a r t i c l e s a p p e a r s o l i d , a s i f t h e r e i s a c e n t r a l c o r e o f s u b u n i t o r s u b u n i t s ( f i g . 17). On t h e b a s i s o f my o b s e r v a t i o n s and t h e w o r k o f o t h e r s , I am p r o p o s i n g a model o f w h e a t CF^ ( f i g . 22). The <5 and e s u b u n i t s a r e p l a c e d a t e x p o s e d r e g i o n s o f t h e s o l u b i l i z e d c o m p l e x b e c a u s e o f e v i d e n c e p r e s e n t e d h e r e ( f i g . 5) a n d by o t h e r a u t h o r s ( Y o u n i s e t a l . , 1977; B i n d e r e t a l . , 1978) t h a t t h e s e p o l y p e p t i d e s a r e e a s i l y l o s t d u r i n g enzyme p u r i f i c a t i o n . T h e 6 a n d e s u b u n i t s a r e d r a w n a t o p p o s i t e e n d s o f t h e a^B^Y c o r e o n t h e b a s i s o f t h e c r o s s - 1 i n k i n g s t u d i e s o f B a i r d and Hammes (1976), i n w h i c h i t was f o u n d t h a t t h e s e a r e t h e o n l y s u b u n i t s n o t i n c l o s e e n o u g h p r o x i m i t y t o be c h e m i c a l l y c r o s s - l i n k e d . S t o i c h i o m e t r y and s t r u c t u r e a r e c l o s e l y l i n k e d . As n o t e d i n t h e I n t r o d u c t i o n , t h e q u e s t i o n o f s t o i c h i o m e t r y o f t h e s u b u n i t s o f t h e c o u p l i n g f a c t o r c o m p l e x e s i s s t i l l a c o n t r o v e r s i a l i s s u e . Any a2^2*2 ° r a 2^2 Y s t o i c h i o m e t r y w o u l d n o t be e x p e c t e d t o g i v e a h e x a g o n a l p a r t i c l e b e c a u s e o f t h e i n e q u a l i t y i n s i z e o f t h e s e s u b u n i t s . A c o r e p a r t i c l e o f a^B^Y c a n a c c o u n t f o r a r e g u l a r h e x a g o n a l p a r t i c l e , s i n c e t h e s m a l l s i z e 60 F i g u r e 2 2 : P r o p o s e d model f o r t h e a r r a n g e m e n t o f t h e s u b u n i t s o f w h e a t C F 1 w i t h i n t h e enzyme c o m p l e x . 60a a and j8 S I D E V I E W a and /5 T O P V I E W 61 o f t h e 6 and e s u b u n i t s p r o b a b l y means t h a t t h e y c o n t r i b u t e l i t t l e t o t h e g r o s s s h a p e o f t h e p a r t i c l e . E v i d e n c e f o r a n e a r - p e r f e c t h e x a g o n h a s been r e p o r t e d . Kagawa and h i s c o - w o r k e r s o b t a i n e d o p t i c a l d i f f r a c t i o n p a t t e r n s o f t w o - d i m e n s i o n a l c r y s t a l s o f f r o m t h e t h e r m o p h i l i c b a c t e r i u m PS3 s h o w i n g a h e x a g o n a l s t r u c t u r e ( W a k a b a y a s h i e t a l . , 1977). T h e y f u r t h e r u s e d a F o u r i e r t r a n s f o r m programme t o c o m p u t e r c o n s t r u c t t h e o p t i c a l l y f i l t e r e d image o f t h i s F 1 . T h e t w o - d i m e n s i o n a l c r y s t a l showed a 90 K s p a c i n g w i t h c o n s i d e r a b l e , b u t n o t p e r f e c t , s i x - f o l d and t h r e e - f o l d s y m m e t r y . Much o f t h e p r o b l e m i n d e t e r m i n i n g t h e s u b u n i t s t o i c h i o m e t r y f o r c o u p l i n g f a c t o r s may be d u e t o u n r e l i a b l e m o l e c u l a r w e i g h t d e t e r m i n a t i o n s . Y o s h i d a e t a l . (1979) r e - e v a l u a t e d t h e m o l e c u l a r w e i g h t o f F^  f r o m t h e b a c t e r i u m P S 3 , c h l o r o p l a s t s a n d m i t o c h o n d r i a by e q u i l i b r i u m c e m t r i f u g a t i o n and g e l f i l t r a t i o n , and t h a t o f t h e s u b u n i t s by e q u i l i b r i u m c e n t r i f u g a t i o n and g e l f i l t r a t i o n by h i g h s p e e d l i q u i d c h r o m a t o g r a p h y . T h e y r e p o r t an o v e r - e s t i m a t e o f t h e m o l e c u l a r w e i g h t s o f F^  s u b u n i t s i n a l l c a s e s , and when t h e new m o l e c u l a r w e i g h t s w e r e u s e d t o c a l c u l a t e t h e s t o i c h i o m e t r y o f t h e enzyme c o m p l e x , an a^^y&e, o r " h e x a g o n a l " model c o u l d be a c c o m o d a t e d . In s u p p o r t o f t h i s s t o i c h i o m e t r y , E s c h and A l l i s o n (1979) f o u n d o n e s p e c i f i c s i t e f o r a r r a c h m e n t o f p - f l u o r o s u l f o n y l b e n z o y l - 5 ' - a d e n o s i n e , an i n h i b i t o r o f F ^ - A T P a s e , o n e a c h o f t h e 3 s u b u n i t s o f b e e f h e a r t F^  . A t o t a l o f t h r e e s p e c i f i c s i t e s p e r F^  c o m p l e x w e r e f o u n d , s u g g e s t i n g t h r e e 3 s u b u n i t s p e r c o m p l e x . In a d d i t i o n , 0.5 n o n - s p e c i f i c s i t e s o f a t t a c h m e n t p e r a s u b u n i t w e r e f o u n d , w i t h a t o t a l o f 1.5 p e r F^, g i v i n g t h r e e a p e r c o m p l e x a l s o . 3 C o n f l i c t i n g d a t a a l s o e x i s t . H - N - e t h y l m a l e i m i d e b i n d s t o F^  f r o m b e e f h e a r t w i t h a s t o i c h i o m e t r y o f 2 a : 2 3 : 2 y : ? 6 : 2 e ( S e n i o r , 1975; t h e number o f 6 s u b u n i t s i s unknown b e c a u s e t h i s r e a g e n t d o e s n o t b i n d t o t h i s s u b u n i t ) 62 b u t b i n d i n g o f t h i s m o l e c u l e r e q u i r e s f r e e s u l f h y d r y l g r o u p s , s o a n y s u b u n i t l o w o r l a c k i n g i n SH m o i e t i e s w i l l n o t b i n d i t s t o i c h i o m e t r i c a l l y . E a r l i e r w o r k f r o m t h e same l a b o r a t o r y ( S e n i o r and B r o o k s , 1971) g a v e an a ^ 3 6 E s t o i c h i o m e t r y on t h e b a s i s o f r e l a t i v e s t a i n i n g o f s u b u n i t s i n SDS-PAGE b a n d s . A c c u r a c y i n d e t e r m i n i n g s t o i c h i o m e t r y by t h i s m e t h o d d e p e n d s upon a g o o d v a l u e f o r t h e m o l e c u l a r w e i g h t o f t h e c o m p l e x . As n o t e d e a r l i e r , s e v e r a l a u t h o r s h a v e shown t h a t t h e c o m p l e x i s v e r y f r a g i l e , and t h e l o s s o f t h e s m a l l e r s u b u n i t s w i l l g i v e l o w e r v a l u e s f o r t h e m o l e c u l a r w e i g h t o f F 1 . One s h o u l d n o t p r o p o s e a model s o l e l y on t h e b a s i s o f e l e c t r o n m i c r o s c o p y , b u t t a k e n i n c o n j u n c t i o n w i t h t h e more r e c e n t l i t e r a t u r e , t h e e l e c t r o n m i c r o g r a p h s p r e s e n t e d h e r e t e n d t o s u p p o r t a model o f & ^ f o r t h e m a j o r s u b u n i t s , w i t h t h e 6 and e s u b u n i t s r e l a t i v e l y e x p o s e d f o r e a s y r e m o v a 1. A n t i s e r u m g e n e r a t e d a g a i n s t w h e a t CF^ p r e c i p i t a t e d t h i s enzyme c o m p l e x a s w e l l as i n h i b i t e d t h e l a t e n t C a A T P a s e a c t i v i t y o f C F ^ . R o c k e t I m m u n o e l e c t r o p h o r e s i s h a s shown t h a t t h i s a n t i s e r u m r e a c t s o n l y w i t h t h e two l a r g e s t s u b u n i t s o f w h e a t C F ^ . H o w e v e r , t h i s d o e s n o t c o n c l u s i v e l y p r o v e t h a t t h e enzyme a c t i v e s i t e i s l o c a t e d on t h e a a n d / o r 3 s u b u n i t s o f w h e a t C F ^ . A n t i b o d y b i n d i n g may o c c u r a t a n y s i t e on an a n t i g e n i c p o l y p e p t i d e , and d e p e n d i n g upon w h e t h e r t h i s s i t e i s c r i t i c a l t o enzyme a c t i v i t y , t h e a n t i b o d y may o r may n o t i n t e r f e r e w i t h t h e enzyme f u n c t i o n . T h i s h a s been d e m o n s t r a t e d w i t h a n t i -s e r u m t o t h e y s u b u n i t o f Antirrhinum majus ( s n a p d r a g o n ) C F ^ . In one c a s e , t h e a n t i - y s e r u m i n h i b i t e d t h e MgATPase ( K o e n i g e t a l . , 1978), w h i l e s e r u m f r o m a n o t h e r r a b b i t i m m u n i z e d a g a i n s t t h e y s u b u n i t s t i m u l a t e d t h e same a c t i v i t y ( K o e n i g e t a 1. , 1976). A n t i b o d y b i n d i n g a t one s i t e may 63 c a u s e a c o n f o r m a t i o n a l c h a n g e a t a d i s t a n t s i t e on t h e e n z y m e , r e s u l t i n g i n a c t i v a t i o n o r i n h i b i t i o n , d e p e n d i n g upon t h e l o c a t i o n o f t h e a n t i b o d y . K o e n i g e t a l . (1978) w a r n s t h a t " . . . c o n c l u s i o n s c o n c e r n i n g t h e u n i n v o l v e -ment o f a c o m p o n e n t c a n be d r a w n w i t h c e r t a i n t y o n l y , i f a l a r g e number o f a n t i s e r a t o t h e same c o m p o n e n t h a v e been i n v e s t i g a t e d . " S i n c e s e r u m f r o m o n l y o n e r a b b i t h a s b e e n c h a r a c t e r i z e d no c o n c l u s i o n s c a n y e t be made a b o u t t h e a c t i v e s i t e o f w h e a t C F , . 6k SUMMARY Wheat CF.| e x h i b i t s many c h a r a c t e r i s t i c s i n common w i t h c o u p l i n g f a c t o r c o m p l e x e s f r o m o t h e r p l a n t s o u r c e s , a s w e l l a s m i t o c h o n d r i a l and m i c r o b i a l c o m p l e x e s . T h e s e s i m i l a r i t i e s i n c l u d e t h e number o f s u b u n i t s , e x t r a c t i o n p r o p e r t i e s , s u b s t r a t e s p e c i f i c i t y , and a s i m i l a r s t r u c t u r e when n e g a t i v e s t a i n e d . A number o f p r o p e r t i e s u n i q u e t o t h e w h e a t e n z y m e h a v e b e e n e l u c i d a t e d i n t h i s s t u d y . A r e q u i r e m e n t f o r p r o t e o l y s i s i n h i b i t o r s d u r i n g CF^ e x t r a c t i o n by c h l o r o f o r m and s u b s e q u e n t p u r i f i c a t i o n h a s b e e n d e s c r i b e d h e r e . T h i s may be d u e t o t h e c o n c o m i t a n t e x t r a c t i o n o f a p r o t e a s e w i t h C F ^ . T h e d e m o n s t r a t i o n t h a t e x t r a c t i o n o f CF^ by h y p o t o n i c s u c r o s e w a s h i s i n h i b i t e d by t h e i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s , w h i l e d e s c r i b e d f o r ECF.J , h a s n e v e r b e f o r e b e e n shown w i t h a p l a n t c o u p l i n g f a c t o r . H o w e v e r , u n l i k e ECF^ , t h i s i s n o t d u e t o an i n h i b i t i o n o f p r o t e o l y s i s o f a s u b u n i t o f C F ^ . In a d d i t i o n , I h a v e shown t h a t w h e a t CF^ c o n t a i n s an i n t e r n a l d i s u l f i d e b r i d g e i n i t s y s u b u n i t . U n l i k e s p i n a c h C F ^ , t h e w h e a t enzyme d o e s n o t e x h i b i t a h e a t - a c t i v a t e d C a A T P a s e a c t i v i t y . The t r y p s i n - a c t i v a t e d A T P a s e a c t i v i t y i s b o t h c a l c i u m -d e p e n d e n t and i n h i b i t e d by c a l c i u m . Two d i f f e r e n t v a l u e s f o r t h e C a A T P a s e a c t i v i t y w e r e o b t a i n e d , a l o w when c a l c i u m and ATP a r e i n e q u i m o l a r a m o u n t s , and a h i g h e r when c a l c i u m i s i n e x c e s s . T h e s e v a l u e s d i f f e r by an o r d e r o f m a g n i t u d e . More r e s e a r c h i s n e c e s s a r y t o d e t e r m i n e w h e t h e r t h i s r e f l e c t s a c o m p e t i t i v e i n h i b i t i o n o r an a l l o s t e r i c c o n t r o l by c a l c i u m . E l e c t r o n m i c r o s c o p y o f n e g a t i v e l y s t a i n e d w h e a t CF^ p a r t i c l e s shows a s t r u c t u r e s i m i 1 a r t o o t h e r F ^ ' s v i e w e d i n t h e same m a n n e r . CF^ shows a s i x - f o l d s y m m e t r y i n o n e o r i e n t a t i o n , a s y m m e t r y w h i c h i s b e s t shown w i t h 65 t h e image e n h a n c e m e n t t e c h n i q u e o f Markham r o t a t i o n . A c o m p a r a t i v e a n a l y s i s o f ECF^ by n e g a t i v e s t a i n i n g showed a v e r y s i m i l a r s t r u c t u r e . T h e b e g i n n i n g s o f a s t u d y o f t h e b i o s y n t h e s i s o f w h e a t CF^ a r e r e p o r t e d h e r e . R a b b i t a n t i b o d i e s t o t h e enzyme w e r e c h a r a c t e r i z e d . W i t h a n t i b o d i e s a g a i n s t t h e s m a l l e r s u b u n i t s o f w h e a t C F ^ , o n e w o u l d t h e n be p r e p a r e d t o e x a m i n e t h e s i z e o f CF^ s u b u n i t p o l y p e p t i d e s s y n t h e s i z e d i n an in v i t r o t r a n s l a t i o n o f w h e a t RNA. In v i e w o f t h e r e c e n t w o r k o f N e l s o n e t a l . (1980) s h o w i n g t h e y s u b u n i t made by t h e i n t a c t s p i n a c h c h l o r o p l a s t , a r e s u l t i n d i r e c t c o n f l i c t w i t h r e s u l t s f r o m t h r e e o t h e r g r o u p s ( M e n d i o l a - M o r g e n t h a l e r e t a 1., 1976; E l l i s , 1977; B o u t h y e t t e and J a e g e n d o r f , 1978) an a d d i t i o n a l s t u d y o f t h e s i t e o f s y n t h e s i s o f t h e y and 6 s u b u n i t s o f CF^ w o u l d be m o s t i n f o r m a t i v e . The s i t e o f s y n t h e s i s and m e t h o d o f t r a n s p o r t o f t h o s e p o l y p e p t i d e s made o u t s i d e t h e c h l o r o p l a s t i s an i n t r i g u i n g e x a m p l e o f c o o p e r a t i o n b e t w e e n n u c l e a r and c h l o r o p l a s t g e n o m e s . T h e c o u p l i n g f a c t o r s a r e p r o b a b l y o n e o f t h e m o s t i m p o r t a n t g r o u p s o f p r o t e i n c o m p l e x e s y e t s t u d i e d . T h e i r s t r u c t u r e , mode o f a c t i o n , r e g u l a t i o n and b i o s y n t h e s i s a r e a l l q u e s t i o n s w h i c h f a s c i n a t e s c i e n t i s t s t o d a y . As t e c h n i q u e s and i n s i g h t s a r e g r a d u a l l y i m p r o v e d , t h i s i s o n e enzyme w h i c h w i l l r e c e i v e much a t t e n t i o n . 66 BIBLIOGRAPHY A b r a m s , A . , C . J e n s e n and D. M o r r i s . 1 9 7 6 . R o l e o f M G ^ T i o n s i n t h e s u b -u n i t s t r u c t u r e and membrane b i n d i n g p r o p e r t i e s o f b a c t e r i a l e n e r g y t r a n s -d u c i n g A T P a s e . B i o c h e m . B i o p h y s R e s . Commun. 69: 8 0 4 - 8 1 1. A h l e r s , J . , D. K a b i s c h and T . G u n t h e r . 1975- The f u n c t i o n a l g r o u p s o f t h e M g - C a A T P a s e o f Escherichia c o l i . C a n . J . B i o c h e m . 5 3 : 658-665. A n d r e o , C . S . , R . A . R a v i z z i n i and R . F . V a l l e j o s . 1979- S u l p h y d r y l g r o u p s i n p h o t o s y n t h e t i c e n e r g y c o n s e r v a t i o n . V . L o c a l i z a t i o n o f t h e new d i s u l f i d e b r i d g e s f o r m e d by o-iodosobenzoate i n c o u p l i n g f a c t o r o f s p i n a c h c h l o r o p l a s t s . B i o c h e m . B i o p h y s . A c t a 5 ^ 7 : 3 7 0 - 3 7 9 . A r n o n , D . I . 19^9- C o p p e r e n z y m e s i n i s o l a t e d c h l o r o p l a s t s . P o l y p h e n o l -o x i d a s e i n Beta v u l g a r i s . P I . P h y s i o l . 2 4 : 1-15-A r n o n , D . I . , M. B. A l l e n and F. R. W h a t l e y . 1 9 5 4 . P h o t o s y n t h e s i s by i s o l a t e d c h l o r o p l a s t s . N a t u r e 174: 3 9 4 - 3 9 6 . A v r o n , M. 1 9 6 2 . L i g h t - d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e i n c h l o r o p l a s t s . J . B i o l . Chem. 237: 2011-2017-A v r o n , M. 1963- A c o u p l i n g f a c t o r i n p h o t o p h o s p h o r y l a t i o n . * B i o c h e m . B i o p h y s . A c t a 7 7 : 6 9 9 - 7 0 2 . B a c k u s , R. C. and R . C . W i l l i a m s . 1 9 5 0 . The u s e o f s p r a y i n g m e t h o d s and o f v o l a t i l e s u s p e n d i n g m e d i a i n t h e p r e p a r a t i o n o f s p e c i m e n s f o r e l e c t r o n m i c r o s c o p y . J . A p p l . P h y s . 21: 11-15. B a i r d , B . A . and G . G . Hammes. 1 9 7 6 . C h e m i c a l c r o s s - l i n k i n g s t u d i e s o f c h l o r o p l a s t c o u p l i n g f a c t o r 1 . J . B i o l . Chem. 2 5 1 : 6 9 5 3 - 6 9 . 6 2 . B a k k e r - G r u n w a l d , T . a n d K. Van Dam. 1 9 7 4 . On t h e m e c h a n i s m o f a c t i v a t i o n o f t h e A T P a s e i n c h l o r o p l a s t s . B i o c h e m . B i o p h y s . A c t a 3 4 7 : 290-298. B a l t s c h e f f s k y , H. 1959- A c t a Chem, S c a n d , 13: 393 a s c i t e d i n V a m b u t u s and R a c k e r . 1965- P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y i n g p h o t o p h o s p h o r y l a t i o n . 1. S t i m u l a t i o n o f p h o t o p h o s p h o r y l a t t o n by a p r e p a r a t i o n o f a l a t e n t C a ^ + - d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e f r o m c h l o r o p l a s t s . J . B i o l , Chem, 2 4 0 : 2660-2667, B e e c h e y , B . R . , S . A . H u b b a r d , P . F . L i n n e t t , D. A . M i t c h e l l and E, A , M u n n , 1 9 7 5 . A s i m p l e and r a p i d m e t h o d f o r t h e p r e p a r a t t o n o f a d e n o s i n e t r i p h o s -p h a t a s e f r o m s u b m i t o c h o n d r i a l p a r t i c l e s . B i o c h e m . J . 1 4 8 : 5 3 3 - 5 3 7 , B i n d e r , A . and R. B a c h o f e n . 1979- I s o l a t i o n and c h a r a c t e r i z a t i o n o f a c o u p l i n g f a c t o r 1 A T P a s e o f t h e t h e r m o p l o l i c b l u e - g r e e n a l g a ( C y a n o b a c t e r i u m ) Mastigocladus laminosus. FEBS L e t t . 1 0 4 : 6 6 - 7 0 , . 6 7 B i n d e r , A . , A . J a e g e n d o r f and E. N g o . 1 9 7 8 . I s o l a t i o n and c o m p o s i t i o n o f t h e s u b u n i t s o f s p i n a c h c h l o r o p l a s t c o u p l i n g f a c t o r p r o t e i n . J . B i o l . Chem. 2 5 3 : 3094-3IOO. B o u t h y e t t e , P . - Y . a n d A . T . J a e g e n d o r f . 1 9 7 8 . The s i t e o f s y n t h e s i s o f c h l o r o p l a s t c o u p l i n g f a c t o r 1 . P l a n t C e l l P h y s i o l . 1 9 : 1 1 6 9 - 1 1 7 4 . B o y e r , P . D . , B . C h a n c e , L. E r n s t e r , P . M i t c h e l l , E . R a c k e r and E. C . S l a t e r . 1977. O x i d a t i v e p h o s p h o r y l a t i o n and p h o t o p h o s p h o r y l a t i o n . A n n . R e v . B i o c h e m . 4 6 : 955-1026. B r a d f o r d , M . M . 1 9 7 6 . A r a p i d a n d s e n s i t i v e m e t h o d f o r q u a n t i t a t i o n o f m i c r o g r a m q u a n t i t i e s o f p r o t e i n u t i l i z a t i n g t h e p r i n c i p l e o f p r o t e i n - d y e b i n d i n g . A n a l . B i o c h e m . 7 2 : 2 4 8 - 2 5 4 . B r a g g , P . D . and C ; - ] H o u . - 1 9 7 9 - P r o t e o l y s i s o f t h e d e l t a s u b u n i t i s r e q u i r e d f o r r e l e a s e o f t h e C a + 2 , M g + 2 - a c t i v a t e d A T P a s e f r o m t h e c e l l membrane o f Escherichia c o l i . FEBS L e t t . 1 0 3 : 1 2 - 1 6 . B r o o k s , J . C . and A . E. S e n i o r . 1 9 7 1 - S t u d i e s o f t h e m i t o c h o n d r i a l o l i g o m y c i n - s e n s i t i v e A T P a s e . I I . The r e l a t i o n s h i p o f t h e s p e c i f i c p r o t e i n i n h i b i t o r t o t h e A T P a s e . A r c h . B i o c h e m . B i o p h y s . 1 4 7 : 4 6 7 - 4 7 0 . C a r l i e r , M . - F . , D . A . H o l o w k a a n d G . G . Hammes. 1 9 7 9 - I n t e r a c t i o n o f p h o t o r e a o t i v e and f l u o r e s c e n t n u c l e o t i d e s w i t h c h l o r o p l a s t c o u p l i n g f a c t o r . B i o c h e m 1 8 : 3 4 5 2 - 3 4 5 7 . C a r r e i r a , J . and E. M u n o z . 1975. Membrane bound and s o l u b l e a d e n o s i n e t r i p h o s p h a t a s e o f Escherichia c o l i K12. K i n e t i c p r o p e r t i e s o f t h e b a s a l and t r y p s i n - s t i m u l a t e d a c t i v i t i e s . M o l . C e l l . B i o c h e m . 9 : 8 5 - 9 5 . C a s h m o r e , A . R . , M . K . B r o a d h u r s t and R . E . G r a y . 1 9 7 8 . C e l l - f r e e s y n t h e s i s o f l e a f p r o t e i n : I d e n t i f i c a t i o n o f an a p p a r e n t p r e c u r s o r o f t h e s m a l l s u b u n i t o f r i b u l o s e - 1 , 5 - b i s p h o s p h a t e c a r b o x l y a s e . P r o c . N a t . A c a d . S c i . 7 5 : 6 5 5 - 6 5 9 . C a t t e r a l l , W . A . and P . L . P e d e r s o n . 1 9 7 1 . A d e n o s i n e t r i p h o s p h a t a s e f r o m r a t l i v e r m o t o c h o n d r i a . I . P u r i f i c a t i o n , h o m o g e n i t y , and p h y s i c a l p r o p e r t i e s . J . B i o l . Chem. 246: 4 9 8 7 - 4 9 9 4 . C h e n , P . S . , T . Y . T o r i b a r a and H. W a r n e r . 1 9 5 6 . M i c r o d e t e r m i n a t i o n o f p h o s p h o r u s . A n a l . Chem. 2 8 : 1 7 5 6 - 1 7 5 8 . C h u a , N . - H . and F. B l o m b e r g . 1 9 7 9 . I m m u n o c h e m i c a l s t u d i e s o f t h y l a k o i d membrane p o l y p e p t i d e s f r o m s p i n a c h and C hlamydomonas reinhardtii. J . B i o l . Chem. 2 5 4 : 2 1 5 - 2 2 3 . C h u a , N . - H a n d G. W. S c h m i d t . 1 9 7 8 . P o s t - t r a n s 1 a t i o n a 1 t r a n s p o r t i n t o i n t a c t c h l o r o p l a s t s o f a p r e c u r s o r t o t h e s m a l l s u b u n i t o f r t b u l o s e ' - l , 5 -b i s p h o s p h a t e c a r b o x y l a s e . P r o c . N a t . A c a d . S c i . 7 5 : 6 1 1 0 - 6 1 1 4 . 68 C l a r k e , D . J . , F . M . F u l l e r and J . G . M o r r i s . 1979- The p r o t o n - t r a n s l o c a t i n g a d e n o s i n e t r i p h o s p h a t a s e o f t h e o b l i g a t e l y a n e r o b i c b a c t e r i u m Clostridium pasteurlanum. 1- ATP p h o s p h o h y d r o l a s e a c t i v i t y . E u r J . B i o c h e m 98: 597-612. C l e v e l a n d , D.W. and S . G . F i s c h e r , M. W. K i r s c h n e r and U . K . L a e m m l i . 1977. P e p t i d e m a p p i n g by l i m i t e d p r o t e o l y s i s i n s o d i u m d o d e c y l s u l f a t e a n d a n a l y s i s by g e l e l e c t r o p h o r e s i s . J . B i o l . Chem. 252: 1120-1106. C o x , G . B . , J . A . Downie; , D . R . H . F a y l e , F. G i b s o n and J . R a k i k . 1978. I n h i b i t i o n by a p r o t e a s e i n h i b i t o r , o f a s o l u b i z a t i o n o f t h e F^  p o r t i o n o f t h e M g 2 + - s t i m u l a t e d a d e n o s i n e t r i p h o s p h a t a s e o f Eschericha coli. J . B a c t e r i d . 133: 287-292. E l l e n s o n , J . L . , D. J . P h e a s a n t a n d R. P . L e v i n e . 1978. L i g h t / d a r k l a b e l l i n g p a t t e r n s o f CF^ c o m p o n e n t s . B i o p h y s . J . 21: 81a. E l l i s , R. J . P r o t e i n s y n t h e s i s by i s o l a t e d c h l o r o p l a s t s . 1977. B i o c h e m . B i o p h y s . A c t a 463: 185-215-E s c h , F . S . and W . S . A l l i s o n . 1979- On t h e s u b u n i t s t o i c h i o m e t r y o f t h e F - j - A T P a s e and t h e s i t e s i n i t t h a t r e a c t s p e c i f i c a l l y w i t h p - f l u o r o s u l -f o n y l - 5 ' - a d e n o s i h e . J . B i o l . Chem. 254: 10740-10746. F a r r o n , F. 1 9 7 0 . I s o l a t i o n and p r o p e r t i e s o f a c h l o r o p l a s t c o u p l i n g f a c t o r and h e a t - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e . B i o c h e m . 9: 3823~3828. F a r r o n , F. and E. R a c k e r . 1970. S t u d i e s on t h e m e c h a n s i m o f t h e c o v e r s i o n o f c o u p l i n g f a c t o r 1 f r o m c h l o r o p l a s t s t o an a c t i v e a d e n o s i n e t r i p h o s p h a t a s e . B i o c h e m . 9: 3829-3836. H a r r i s , D . A . 1978. The i n t e r a c t i o n s o f c o u p l i n g A T P a s e s w i t h n u c l e o t i d e s . B i o c h e m . B i o p h y s . A c t a 463: 245-273. H i g h f i e l d , P . E . and R . J . E l l i s . 1978. S y n t h e s i s and t r a n s p o r t o f t h e s m a l l s u b u n i t o f c h l o r o p l a s t r i b u l o u s e b i o s p h o s p h a t e a n d c a r b o x y l a s e . N a t u r e 271: 4 2 0 - 4 2 4 . H o c h m a n , Y . , A . L a n i r a n d C . C a r m e l i . 1976. R e l a t i o n s b e t w e e n d i v a l e n t c a t i o n s b i n d i n g and A T P a s e a c t i v i t y i n c o u p l i n g f a c t o r f r o m c h l o r o p l a s t . FEBS L e t t . 61: 255"259-H o w e l l , S . H . and E. N. M o u d r i a n a k i s . 1967. F u n c t i o n o f t h e " q u a n t a s o m e " i n p h o t o s y n t h e s i s : S t r u c t u r e and p r o p e r t i e s o f m e m b r a n e - b o u n d p a r t i c l e a c t i v e i n t h e d a r k r e a c t i o n s o f p h o t o p h o s p h o r y1 a t i o n . P r o c . N a t . A c a d . S c i . 58: 1261-1268. H u b e r m a n , M. and M . J . R . S a l t o n . 1979- P u r i f i c a t i o n and p r o p e r t i e s o f t h e l a t e n t F . - A T P a s e o f Micrococcus l y s i d e i k t i c u s . B i o c h e m . B i o p h y s . A c t a 547: 230-240. J a e g e n d o r f , A . T . and M. S m i t h . 1961. U n c o u p l i n g p h o s p h o r y l a t i o n i n s p i n a c h c h l o r o p l a s t s i n a b s e n c e o f c a t i o n s . P I . P h y s i o l . 37: 131-141. 69 J a e g e n d o r f , A . T . and E. U r i b e . 1966. ATP f o r m a t i o n c a u s e d by a c i d - b a s e t r a n s i t i o n o f s p i n a c h c h l o r o p l a s t s . P r o c . N a t . A c i d . S c i . 5 5 : 170-177-K a g a w a , Y . and E. R a c k e r . 1 9 6 6 . P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g o x i d a t i v e p h o s p h o r y l a t i o n . X . C o r r e l a t i o n o f m o r p h o l o g y and f u n c t i o n i n s u b m i t o c h o n d r i a l p a r t i c l e s . J . B i o l . Chem. 241: 2475-2482. K e n n e d y , E . P . a n d A . L . L e h n i n g e r . 1949. O x i d a t i o n o f f a t t y a c i d s and t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s by i s o l a t e d r a t l i v e r m i t o c h o n d r i a . J . B i o l . Chem. 1 7 9 : 9 5 7 ~ 9 7 2 . K i r c h a n s k i , S . J . and R . B . P a r k . 1 9 7 6 . C o m p a r a t i v e s t u d i e s o f t h e t h y l a k o i d p r o t e i n s o f m e s o p h y l l and b u n d l e s h e a t h p l a s t i d s o f Zea mays. P h y s i o l . 5 8 : 3 4 5 - 3 4 9 . K o e n i g , F . , G . H . S c h m i d , A . R a d u n z , B . P i n e a u and W. M e n k e . 1 9 7 6 . The i s o l a t i o n o f f u r t h e r p o l y p e p t i d e s f r o m t h e t h y l a k o i d m e m b r a n e , t h e i r l o c a l i z a t i o n and f u n c t i o n . FEBS L e t t . 6 2 : 3 4 3 - 3 4 6 . K o e n i g , F. A . R a d u n z , C G . S c h m i d and W. M e n k e . 1978. A n t i s e r a t o t h e c o u p l i n g f a c t o r p h o t o p h o s p h o r y l a t i o n a n d i t s s u b u n i t s . Z . N a t u r f o r s c h . 3 3 c : 5 2 9 - 5 3 6 . K u w a b a r a , T . and N. M u r a t a . 1979- P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f 33 k d . p r o t e i n o f s p i n a c h c h l o r o p l a s t s . B i o c h e m . B i o p h y s . A c t a 581 : 2 2 8 - 2 3 6 . L i e n , S . , R . J . B e r z e b o r n and E. R a c k e r . 1 9 7 2 . P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . X . S t u d i e s on t h e s u b u n i t s t r u c t u r e o f c o u p l i n g f a c t o r 1 f r o m c h l o r o p l a s t s . J . B i o l . Chem 2 4 7 : 3 5 2 0 - 3 5 2 4 . L i e n , S . and E. R a c k e r . 1 9 7 1 a . P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . V I I I . P r o p e r t i e s o f s i 1 i c o t u n g s t a t e - t r e a t e d s u b c h l o r o p l a s t p a r t i c l e s . J . B i o l . Chem. 2 4 6 : 4 2 9 8 - 4 3 0 7 . L i e n , S . and E. R a c k e r . 1971b. P r e p a r a t i o n and a s s a y o f c h l o r o p l a s t c o u p l i n g f a c t o r CF-j . p p . 5^7-555^.J_n. : A . S a n P i e t r o ( e d . ) M e t h o d s i n E n z y m o l o g y . A c a d e m i c P r e s s , New Y o r k . L i p m a n n , F . 1 9 4 1 . M e t a b o l i c g e n e r a t i o n and u t i l i z a t i o n o f p h o s p h a t e bond e n e r g y . A d v . E n z y m o l . 18: 99-162. M a c c e c c h i n i , M. - L . , Y . R u d i n , G. B l o b e l and G. S c h a t z . 1979- I m p o r t o f p r o t e i n s i n t o m i t o c h o n d r i a : P r e c u r s o r f o r m s o f t h e e x t r a m i t o c h o n d r i a 11y made F - | - A T P a s e s u b u n i t s i n y e a s t . P r o c . N a t . A c a d . S c i . 76: 3 4 3 - 3 4 7 -M a r k h a m , R. S . F r e y and G. J . H i l l s . 1 9 6 3 - M e t h o d f o r t h e e n h a n c e m e n t o f image d e t a i l and a c c e n t u a t i o n o f s t r u c t u r e i n e l e c t r o n m i c r o s c o p y . V i r o l o g y 2 0 : 88-102. 70 M c C a r t y , R. E. and J . F a g a n . 1973- L i g h t - s t i m u l a t e d i n c o r p o r a t i o n o f N - e t h y l m a l e i m i d e i n t o c o u p l i n g f a c t o r 1 i n s p i n a c h c h l o r o p l a s t s . B i o c h e m . 1 2 : 1 5 0 3 - 1 5 0 7 . M e n d i o l a - M o r g e n t h a l e r , L. R . , J . - J M o r g e n t h a l e r and C . A . P r i c e . 1976. S y n t h e s i s o f c o u p l i n g f a c t o r CF-j p r o t e i n s by i s o l a t e d s p i n a c h c h l o r o p l a s t s . FEBS L e t t . 6 2 : 96-100. M i l l e r , K. R. and L. A . S t a e h e l i n . 1976. A n a l y s i s o f t h e t h y l a k o i d o u t e r s u r f a c e . C o u p l i n g f a c t o r i s l i m i t e d t o u n s t a c k e d membrane r e g i o n s . J . C e l 1 B i o l . 68: 30-47. M i r s k y , R. and V . B a r l o s . 1 9 7 3 . M o l e c u l a r w e i g h t , a m i n o a c i d c o m p o s i t i o n and o t h e r p r o p e r t i e s o f m e m b r a n e - b o u n d A T P a s e f r o m Bacillus megaterium KM. B i o c h e m . B i o p h y s . A c t a 2 9 1 : 4 8 0 - 4 8 8 . M i t c h e l l P . 1 9 6 1 . C o u p l i n g o f p h o s p h o r y l a t i o n t o e l e c t r o n t r a n s p o r t and h y d r o g e n t r a n s f e r by a c h e m i - o s m o t i c t y p e o f m e c h a n i s m . N a t u r e 1 9 1 : 1 4 4 - 1 4 8 . M o l l i n e d o , F . , V . L a r r a g a , F . J . C o l l and E. M u n o z . I98O. R o l e o f t h e s u b u n i t s o f t h e e n e r g y - t r a n s d u c i n g a d e n o s i n e t r i p h o s p h a t a s e f r o m Micrococcus lysodeikticus membrane s t u d i e s by p r o t e o l y t i c d i g e s t i o n and i m m u n o l o g i c a l a p p r o a c h e s . B i o c h e m . J . 1 8 6 : 713~723-N a n n i n g a , L . B . 1957- F o r m a t i o n c o n s t a n t s and h y d r o l y s i s a t 1 0 0 ° o f c a l c i u m and m a g n e s i u m c o m p l e x e s o f a d e n o s i n e t r i - , d i - and m o n o p h o s p h a t e . J . P h y s . Chem. 6 1 : 1 1 4 4 - 1 1 4 9 . N e l s o n , N . , D.W. D e t e r s , H . N e l s o n and E. R a c k e r . 1973- P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . X I I I . P r o p e r t i e s o f i s o l a t e d s u b u n i t s o f c o u p l i n g f a c t o r 1 f r o m s p i n a c h c h l o r o p l a s t s . J . B i o l . Chem. 2 4 8 : 2 0 4 9 - 2 0 5 5 . N e l s o n , N . , E. E y t a n , B . - E . N o t s a n i , H. S i g r i s t , K. S i g r i s t - N e l s o n and C . G i t l e r . 1974. I s o l a t i o n o f a c h l o r o p l a s t N , N 1 - d i c y c l o h e x y 1 c a r b o d i i m i d e -b i n d i n g p r o t e o l i p i d , a c t i v e i n p r o t o n t r a n s l o c a t i o n . P r o c . N a t . A c a d . S c i . 74: 2 3 7 5 - 2 3 7 8 , N e l s o n , N . , A . K a m i e n i e t s k y , D.W. D e t e r s and H. N e l s o n . 1975. S u b u n i t s t r u c t u r e a n d f u n c t i o n o f CF-j . p p . 1 4 9 - 1 5 4 . J_n_: E. Q u a g l i a r o , S . P a p a , F. P a l m i e r i a . E . C . S l a t e r and N. S i l i p r a n d i ( e d s . ) E l e c t r o n t r a n s f e r c h a i n s a n d o x i d a t i v e p h o s p h o r y l a t i o n . N o r t h - H o l l a n d P u b l i s h i n g C o . , A m s t e r d a m . N e l s o n , N . , H. N e l s o n , and E. R a c k e r . 1 9 7 2 a . P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . X I . M a g n e s i u m - d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e p r o p e r t i e s o f h e a t - a c t i v a t e d c o u p l i n g f a c t o r 1 f r o m c h l o r o p l a s t s . J . B i o l . Chem. 2 4 7 : 6506-6510. N e l s o n , N . , H. N e l s o n and E. R a c k e r . 1 9 7 2 b . P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . X I I . P u r i f i c a t i o n a n d p r o p e r t i e s o f an i n h i b i t o r i s o l a t e d f r o m c h l o r o p l a s t c o u p l i n g f a c t o r 1 . J . B i o l . Chem. 2 4 7 : 7657-7662. 71 N e l s o n , N . , H. N e l s o n and G. S c h a t z . 1980. B i o s y n t h e s i s and a s s e m b l y o f t h e p r o t o n - t r a n s l o c a t i n g a d e n o s i n e t r i p h o s p h a t a s e c o m p l e x f r o m c h l o r o p l a s t s . P r o c . N a t . A c a d . S c i . ' 77: ' 1361-1 364. O u c h t e r l o n y , 0. 1968. Handbook o f i m m u n o d i f f u s i o n and I m m u n o e l e c t r o p h o r e s i s . Ann A r b o r S c i e n c e P u b l i s h e r s , Ann A r b o r , M i c h . P e n i n , F . , C . G o d i n o t and D. C . G a u t h e r o n . 1979- O p t i m i z a t i o n o f t h e p u r i f i c a t i o n o f m i t o c h o n d r i a l F ^ - a d e n o s i n e t r i p h o s p h a t a s e . B i o c h e m . B i o p h y s . A c t a 5 4 8 : 63-71. P e n e f s k y , H. S . M.F. . P u l l m a n , A . D a t t a and E. R a c k e r . i960. P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g o x i d a t i v e p h o s p h o r y l a t i o n . I I . P a r t i c i p a t i o n o f a s o l u b l e a d e n o s i n e t r i p h o s p h a t a s e i n o x i d a t i v e p h o s p h o r y l a t i o n . J . B i o l . Chem. 235: 3330-3336. P i c k , U . and E. R a c k e r . 1979. P u r i f i c a t i o n and r e c o n s i t i t u t i o n o f t h e N , N 1 - d i c y c l o h e x l y c a r b o d i i m i d e - s e n s i t i v e A T P a s e c o m p l e x f r o m s p i n a c h c h l o r o p l a s t s . J . B i o l . Chem. 254: 2793-2799. P u l l m a n , M . E . H. S . P e n e f s k y , A . D a t t a and E. R a c k e r . i960. P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g o x i d a t i v e p h o s p h o r y l a t i o n . I . P u r i f i c a t i o n and p r o p e r t i e s o f a s o l u b l e , d i n i t r o p h e n o l - s t i m u 1 a t e d a d e n o s i n e t r i p h o s p h a t a s e . J . B i o l . Chem. 235: 3322-3329-R a c k e r . E. G. A . H a u s k a , S . L i e n , R. J . B e r z b o r n and N. N e l s o n . ;" 197-1. R e s o l u t i o n and r e c o n s t i t u t i o n o f t h e s y s t e m o f p h o t o p h o s p h o r y 1 a t i o n p p . 1097-1113. J_n: G. F o r t i , M . A v r o n and A . M e l a n d r i ( e d s . ) The s e c o n d i n t e r n a t i o n a l c o n g r e s s o f p h o t o s y n t h e s i s r e s e a r c h . W. J u n k N. V . P u b l i s h e r s , The H a g u e . R y r i e . I . J . and A . G a l l a g h e r . 1979- The y e a s t m i t o c h o n d r i a l A T P a s e c o m p l e x . S u b u n i t c o m p o s i t i o n a n d e v i d e n c e f o r a l a t e n t p r o t e a s e c o n t a m i n a n t . B i o c h e m . B i o p h y s . A c t a 545: 1-14. S c h a t z , G. H. S . P e n e f s k y and E. R a c k e r . 1967. P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g o x i d a t i v e p h o s p h o r y l a t i o n . X I V . I n t e r a c t i o n o f p u r i f i e d m i t o c h o n d r i a l a d e n o s i n e t r i p h o s p h a t a s e f r o m b a k e r s ' y e a s t w i t h s u b m i t o c h o n d r i a l p a r t i c l e s f r o m b e e f h e a r t . J . B i o l . Chem. 2 4 2 : 2552-2560. S e n i o r , A . E . 1975- M i t o c h o n d r i a l a d e n o s i n e t r i p h o s p h a t a s e . L o c a t i o n o f s u l f h y d r y l g r o u p s and d i s u l f i d e b o n d s i n t h e s o l u b l e enzyme f r o m b e e f h e a r t . B i o c h e m . 14: 660 - 6 6 4 . S e n i o r , A . E. and J . C . B r o o k s . 1971- T h e s u b u n i t c o m p o s i t i o n o f t h e m i t o c h o n d r i a l o l i g o m y c i n - i n s e n s i t i v e A T P a s e . FEBS L e t t 17: 327~329-S e r r a h i m a - Z i e g e r , M. and H. M o n t e i l . 1978. Membrane A T P a s e Bacillus s u b t i t l i s . I . P u r i f i c a t i o n and p r o p e r t i e s . B i o c h e m . B i o p h y s . A c t a 502: 445-457. S i l i e n , L . G . and A . E. M a r t e l l . 1964. S t a b i l i t y C o n s t a n t s o f M e t a l - I o n C o m p l e x e s . T h e C h e m i c a l S o c i e t y . S p e c i a l P u b l i c a t i o n s N o . 17- L o n d o n . 72 S l a t e r , E . C . 1953. M e c h a n i s m o f p h o s p h o r y l a t i o n i n t h e r e s p i r a t o r y c h a i n . N a t u r e 172: 975"978. S p i t s b e r g , V . L . and J . E. B l a i r . 1977- E v i d e n c e s u p p o r t i n g t h e i d e n t i t y o f b e e f h e a r t m i t o c h o n d r i a l c h l o r o f o r m - r e l e a s e d a d e n o s i n e t r i p h o s p h a t a s e ( A T P a s e ) w i t h c o u p l i n g f a c t o r 1. B i o c h e m . B i o p h y s . A c t a 460: 136-141. S t r o t m a n n , H . , H. H e s s e and K. E d e l m a n n . 1973- Q u a n t i t a t i v e d e t e r m i n a t i o n o f c o u p l i n g f a c t o r CF^ o f c h l o r o p l a s t s . B i o c h e m . B i o p h y s . A c t a 314: 202-210. V a m b u t a s , V . K . and E. R a c k e r . 1965- P a r t i a l r e s o l u t i o n o f t h e enzyme c a t a l -y z i n g p h o t o p h o s p h o r y l a t i o n . 1. S t i m u l a t i o n o f p h o t o p h o s p h o r y l a t i o n by a p r e p a r a t i o n o f a l a t e n t C a ^ + - d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e f r o m c h l o r o p l a s t s . J . B i o l . Chem. 2 4 0 : 2660-2667. W a k a b a y a s h i , T . M . K u b o t a , M. Y o s h i d a and Y . K a g a w a . 1977. S t r u c t u r e o f A T P a s e ( c o u p l i n g f a c t o r T F ^ f r o m a t h e r m o p h i l i c b a c t e r i u m . J . M o l . B i o l . 117: 515-519-W e b b e r , K . , and M. O s b o r n e . 1969- The r e l i a b i l i t y o f m o l e c u l a r w e i g h t d e t e r m i n e d by d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . J . B i o l . Chem. 16: 4406-4412. W e s s e l s , J . S . C . a n d H. B a 1 t s c h e f f s k y . A c t a Chem. S c a n d . 14 (i960) 233 a s c i t e d i n V a m b u t u s a n d R a c k e r . 1 9 6 5 - P a r t i a l r e s o l u t i o n o f t h e e n z y m e s c a t a l y z i n g p h o t o p h o s p h o r y l a t i o n . 1. S t i m u l a t i o n o f p h o t o p h o s p h o r y l a t i o n by a p r e p a r a t i o n o f a l a t e n t Ca^" 1"- d e p e n d e n t a d e n o s i n e t r i p h o s p h a t a s e f r o m c h l o r o p l a s t s . J . B i o l . Chem. 2 4 0 : 2600-1667. W i l s o n , D . B . and J . B. S m i t h . 1978. B a c t e r i a l t r a n s p o r t p r o t e i n s , p p . 495" 505. J_n_: B . P . R o s e n ( e d . ) B a c t e r i a l t r a n s p o r t . M. D e c k k e r , I n c . New Y o r k . Y o s h i d a , M . , N. S o n e , H. H i r a t a and Y . K a g a w a . 1975. A h i g h l y s t a b l e a d e n o s i n e t r i p h o s p h a t a s e f r o m a t h e r m o p h i l i c b a c t e r i u m . P u r i f i c a t i o n , p r o p e r t i e s and r e c o n s t i t u t i o n . J . B i o l . Chem. 250: 7910-7916. Y o s h i d a , M . , N. S o n e , H. H i r a t a and Y . K a g a w a . 1977. R e c o n s t i t u t i o n o f a d e n o s i n e t r i p h o s p h a t a s e o f t h e r m o p h l i c b a c t e r i u m f r o m p u r i f i e d i n d i v i d u a l s u b u n i t s . J . B i o l . Chem 252: 3480-3485. Y o s h i d a , M . , N . S o n e , H. H i r a t a , Y . Kagawa a n d N. U r i . 1979- S u b u n i t s t r u c t u r e o f a d e n o s i n e t r i p h o s p h a t a s e . C o m p a r i s o n o f t h e s t r u c t u r e i n t h e r m o p h i l i c b a c t e r i u m PS3 w i t h t h o s e i n m i t o c h o n d r i a , c h l o r o p l a s t s and Escherichia c o l i . J . B i o l . Chem. 254: 9525~9533-Y o u n i s , H . M . and G. D. W i n g e t . 1977- C F ^ - d e p e n d e n t r e s t o r a t i o n o f e n e r g y -l i n k e d r e a c t i o n s r e c o n s t i t u t e d w i t h a h y d r o p h o b i c p r o t e i n f r o m s p i n a c h . B i o c h e m . B i o p h y s . R e s . Commun. 77: 168-174. Y o u n i s , H . M . , G . D . W i n g e t and E. R a c k e r . 1977- R e q u i r e m e n t o f t h e 6 s u b u n i t o f c h l o r o p l a s t c o u p l i n g f a c t o r 1 f o r p h o t o p h o s p h o r y l a t i o n . J . B i o l . Chem. 252: 1814-1818. 

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