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Atpases in plasma membrane enriched fractions from Dictyostelium discoideum MacDonald, James Innes Scott 1986

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ATPASES IN PLASMA MEMBRANE ENRICHED FRACTIONS FROM DICTYOSTELIUM DISCOIDEUM by JAMES INNES SCOTT MACDONALD B.Sc. ( H o n s ) , U n i v e r s i t y o f K i n g s C o l l e g e , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department o f M i c r o b i o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA November, 1986 (c) James Innes S c o t t MacDonald In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten permission. Department of Af tc eotitOLoG-/ The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT E v i d e n c e i s p r e s e n t e d f o r t h e e x i s t e n c e o f an ATPase a c t i v i t y i n D. d i s c o i d e u m p l a s m a membranes. T h i s a c t i v i t y was d i s t i n c t f r o m t h e m i t o c h o n d r i a l ATPase i n t h a t i t was i n s e n s i t i v e t o a z i d e and o l i g o m y c i n . The ATPase was s t i m u l a t e d by M g + 2 and t o a l e s s e r e x t e n t by Ca + 2 and was n o t a f f e c t e d by e q u i m o l a r N a + / K + o r o u a b a i n . V a n a d a t e , DES, t h i m e r o s a l and DCCD a l l p r o v e d t o be p a r t i a l l y i n h i b i t o r y . Enzyme a c t i v i t y was s o l u b i l i z e d w i t h a wide v a r i e t y o f d e t e r g e n t s , w i t h l y s o l e c i t h i n g i v i n g t h e b e s t r e s u l t s . C o n c o m i t a n t w i t h s o l u b i l i z a t i o n was a p a r t i a l l o s s o f DES s e n s i t i v i t y w h i c h was shown t o be due t o t h e p r e s e n c e o f a l a b i l e DES s e n s i t i v e ATPase i n a d d i t i o n t o a s t a b l e DES i n s e n s i t i v e a c t i v i t y . The DES s e n s i t i v e ATPase was s t i m u l a t e d by Mg + 2 b u t o n l y somewhat by C a + 2 whereas t h e DES i n s e n s i t i v e enzyme was s t i m u l a t e d e q u a l l y by e i t h e r . The DES s e n s i t i v e enzyme a l s o d i s p l a y e d M i c h a e l i s - M e n t e n k i n e t i c s when enzyme a c t i v i t y was measured as a f u n c t i o n o f t h e ATP c o n c e n t r a t i o n w h i l e t h e DES i n s e n s i t i v e ATPase d i s p l a y e d k i n e t i c s w h i c h were i n d i c a t i v e o f a s i g m o i d a l r e l a t i o n s h i p between s u b s t r a t e c o n c e n t r a t i o n and enzyme a c t i v i t y . F r a c t i o n a t i o n o f s o l u b i l i z e d p l a s m a membranes by i o n e x c h a n g e c h r o m a t o g r a p h y r e s o l v e d two DES i n s e n s i t i v e ATPase a c t i v i t i e s , d e s i g n a t e d p eaks I and I I . Peak I was i n s e n s i t i v e t o v a n a d a t e and e x p r e s s e d optimum a c t i v i t y w i t h p y r o p h o s p h a t e . O p t i m a l a c t i v i t y was a t a l k a l i n e pH v a l u e s . i i Peak I I was p u r i f i e d by two d i f f e r e n t p r o c e d u r e s . The f i r s t i n v o l v e d an i n i t i a l s e p a r a t i o n on D E A E - S e p h a c e1 f o l l o w e d by c e n t r i f u g a t i o n t h r o u g h a l i n e a r g l y c e r o l g r a d i e n t . The s e c o n d i n v o l v e d an i n i t i a l c h r o m a t o g r a p h i c f r a c t i o n a t i o n by S e p h a c r y l S - 3 0 0 g e l f i l t r a t i o n f o l l o w e d by D E A E - S e p h a c e l i o n e x c h a n g e c h r o m a t o g r a p h y o f t h e ATPase c o n t a i n i n g f r a c t i o n s . B o t h p r o c e d u r e s r e s u l t e d i n p r e p a r a t i o n s t h a t c o n t a i n e d a s i n g l e m a j o r component o f a p p a r e n t m o l e c u l a r w e i g h t 6 4 kDa, as a s s e s s e d by SDS-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 . The p u r i f i e d ATPase was s e n s i t i v e t o v a n a d a t e and f l u o r i d e b u t i n s e n s i t i v e t o DCCD , t h i m e r o s a l and N-e t h y l m a l e i m i d e . The enzyme was a c t i v a t e d e q u a l l y by e i t h e r M g + 2 o r C a + 2 i n m i l l i m o l a r c o n c e n t r a t i o n s . ATP h y d r o l y s i s was a l s o s t i m u l a t e d by m i l l i m o l a r c o n c e n t r a t i o n s o f Mn + 2 , Z n + 2 o r C u + 2 . The ATPase d i s p l a y e d s i g m o i d a l k i n e t i c s when a s s a y e d as a f u n c t i o n o f ATP c o n c e n t r a t i o n i n t h e a b s e n c e o f any d i v a l e n t c a t i o n . A d d i t i o n o f 1 o r 10 mM M g + 2 o r C a + 2 i n c r e a s e d t h e s u b s t r a t e a f f i n i t y o f t h e enzyme, w h i l e 100 mM d i v a l e n t c a t i o n p r o v e d i n h i b i t o r y . The enzyme was n o t s t i m u l a t e d by low c o n c e n t r a t i o n s o f C a + 2 o r by C a + 2 -c a l m o d u l i n , s u g g e s t i n g t h a t i t was p r o b a b l y n o t a C a + 2 pump. i i i TABLE OF CONTENTS A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i i L i s t o f A b b r e v i a t i o n s x i i i A c k n o w l e d g m e n t s x i INTRODUCTION 1 MATERIALS AND METHODS 16 I . M a t e r i a l s 16 I I . Methods A. O r g a n i s m s and C u l t u r e C o n d i t i o n s 16 B. P l a s m a Membrane P r e p a r a t i o n 17 C. S o l u b i l i z a t i o n o f P l a s m a Membranes 19 D. P r o t e i n D e t e r m i n a t i o n 20 E. P r e p a r a t i o n o f P h o s p h a t i d y l c h o l i n e 20 F. Enzyme A s s a y s 20 G. 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 22 H. P u r i f i c a t i o n o f ATPase i . S e p h a c r y l S-300 G e l F i l t r a t i o n 23 i i . I o n E x c h a n g e C h r o m a t o g r a p h y on D E A E - S e p h a c e l 23 i i i . G l y c e r o l G r a d i e n t D e n s i t y C e n t r i f u g a t i o n 2 4 i v . P h e n y l - S e p h a r o s e , R e a c t i v e R e d - A g a r o s e and A T P - A g a r o s e C h r o m a t o g r a p h y 24 I . ^ 2 P L a b e l i n g E x p e r i m e n t s 25 i v RESULTS SECTION I . IDENTIFICATION OF ATPASE ACTIVITY IN PURIFIED D. DISCOIDEUM PLASMA MEMBRANES a. P l a s m a Membrane I s o l a t i o n 26 b . C h a r a c t e r i z a t i o n o f t h e P l a s m a Membrane ATPase 27 c . S o l u b i l i z a t i o n o f t h e P l a s m a Membrane ATPase 39 d . E v i d e n c e f o r a L a b i l e DES S e n s i t i v e P l a s m a Membrane ATPase 43 e. C o m p a r i s o n o f t h e P r o p e r t i e s o f t h e DES S e n s i t i v e and I n s e n s i t i v e A TPase A c t i v i t i e s i n P l a s m a Membrane 50 SECTION I I . PURIFICATION OF THE PLASMA MEMBRANE ATPASE ACTIVITY a. F r a c t i o n a t i o n o f S o l u b i l i z e d ATPase A c t i v i t y by I o n Excha n g e C h r o m a t o g r a p h y 60 b. P u r i f i c a t i o n o f ATPase A c t i v i t y by 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 60 c . A f f i n i t y and H y d r o p h o b i c C h r o m a t o g r a p h y 69 d. P u r i f i c a t i o n by G e l F i l t r a t i o n 69 SECTION I I I . PROPERTIES OF THE FRACTIONATED DES INSENS I T I V E ATPASES a. Peak I f r o m D E A E - S e p h a c e l 78 b. P r o p e r t i e s o f P u r i f i e d Peak I I 78 c . P h o s p h o r y l a t i o n o f t h e ATPase 93 d. E f f e c t o f DIF on ATPase A c t i v i t y 97 DISCUSSION 99 REFERENCES 121 v L I S T OF TABLES D i s t r i b u t i o n o f enzyme m a r k e r s d u r i n g p l a s m a membrane p u r i f i c a t i o n 28 D i s t r i b u t i o n o f h y d r o l y z e d p r o d u c t s r e s u l t i n g f r o m i n c u b a t i o n o f p l a s m a membranes w i t h [ 3 H ] - A T P 30 E f f e c t o f o u a b a i n on p l a s m a membrane ATPase a c t i v i t y 31* U t i l i z a t i o n o f o t h e r n u c l e o t i d e s by t h e p l a s m a membrane M g + 2 - A T P a s e 37 E f f e c t o f 100 mM KC1 on p l a s m a membrane ATPase a c t i v i t y 40 E f f e c t o f e x o g e n o u s p h o s p h o l i p i d on s o l u b i l i z e d DES s e n s i t i v e ATPase a c t i v i t y 53 E f f e c t o f DES on peaks I and I I ATPase a c t i v i t y 62 ATPase a c t i v i t y f r o m p e a k s I and I I f r o m D E A E - S e p h a c e l 63 P u r i f i c a t i o n o f t h e D E S - i n s e n s i t i v e p l a s m a membrane ATPase 68 A f f i n i t y c h r o m a t o g r a p h y o f peak I I ATPase 70 P h e n y l - S e p h a r o s e c h r o m a t o g r a p h y o f peak I I ATPase a c t i v i t y 71 P u r i f i c a t i o n o f t h e p l a s m a membrane ATPase 77 S u b s t r a t e s p e c i f i c i t y o f peak I 80 E f f e c t o f d i v a l e n t c a t i o n s on peak I enzyme a c t i v i t y 81 E f f e c t o f v a r i o u s c a t i o n s on p u r i f i e d A TPase a c t i v i t y 88 E f f e c t o f m o n o v a l e n t c a t i o n s on Mg o r C a + 2 s t i m u l a t e d ATPase a c t i v i t y 89 v i X V I I . 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 p u r i f i e d ATPase 91 X V I I I . E f f e c t o f c a l m o d u l i n on p u r i f i e d A TPase a c t i v i t y 9^ XIX. E f f e c t o f DIF on ATPase a c t i v i t y 98 XX. C o m p a r i s o n o f t h e p u r i f i e d D. d i s c o i d e u m p l a s m a membrane ATPase w i t h s e v e r a l o t h e r membrane bound p h o s p h a t a s e s 110 v i i L I S T OF F I G U R E S F i g u r e 1. F i g u r e 2. F i g u r e 3. F i g u r e 4. F i g u r e 5. F i g u r e 6. F i g u r e 7. F i g u r e 8. F i g u r e 9. F i g u r e 10 F i g u r e 11 F i g u r e 12 F i g u r e 13. F i g u r e 14, L i f e c y c l e o f D i c t y o s t e l i u m d i s c o i d e u m 2 E f f e c t o f a z i d e and o l i g o m y c i n on plasma membrane and m i t o c h o n d r i a l ATPase a c t i v i t y 29 S t i m u l a t i o n o f pl a s m a membrane ATPase a c t i v i t y by Mg + 2 and Ca + 2 32 The e f f e c t o f v a r i o u s c a t i o n s on p l a s m a membrane ATPase a c t i v i t y 33 The e f f e c t s o f v a r i o u s i n h i b i t o r s on p l a s m a membrane ATPase a c t i v i t y 35 I n h i b i t i o n o f pl a s m a membrane ATPase a c t i v i t y by M - e t h y l m a l e i m i d e . . . . 3 6 D e v e l o p m e n t a l c h a nges i n p l a s m a membrane ATPase a c t i v i t y 38 S o l u b i l i z a t i o n o f p l a s m a membrane ATPase a c t i v i t y w i t h l y s o l e c i t h i n 41 D e t e r g e n t s o l u b i l i z a t i o n o f pl a s m a membrane ATPase a c t i v i t y 44 E f f e c t o f exogenous p h o s p h a t i d y l c h o l i n e on d e o x y c h o l a t e and Z-14 e x t r a c t e d ATPase 46 E f f e c t o f DES on t h e p l a s m a membrane bound and s o l u b i l i z e d ATPase a c t i v i t y 47 I n a c t i v a t i o n o f t h e DES s e n s i t i v e ATPase upon s o l u b i l i z a t i o n w i t h s e v e r a l d e t e r g e n t s 48 E f f e c t o f l y s o l e c i t h i n , Z-14 and d e o x y c h o l a t e s o l u b i l i z a t i o n on DES s e n s i t i v e and i n s e n s i t i v e ATPase a c t i v i t y 51 E f f e c t o f C a + 2 and M g + 2 on DES s e n s i t i v e and i n s e n s i t i v e ATPase a c t i v i t y 54 v i i i F i g u r e 15. F i g u r e 16. F i g u r e 17. F i g u r e 18. F i g u r e 19. F i g u r e 20. F i g u r e 21. F i g u r e 22. F i g u r e 23. F i g u r e 24. F i g u r e 25. F i g u r e 26. F i g u r e 27. F i g u r e 28. E f f e c t o f ATP c o n c e n t r a t i o n on DES s e n s i t i v e and i n s e n s i t i v e ATPase a c t i v i t y 56 The e f f e c t o f Mg + 2 a n c j Ca + 2 on ATP h y d r o l y s i s k i n e t i c s o f the DES s e n s i t i v e ATPase 57 The e f f e c t o f Mg + 2 and C a + 2 on ATP h y d r o l y s i s k i n e t i c s o f the DES i n s e n s i t i v e ATPase 58 The e f f e c t o f pH on t h e DES s e n s i t i v e and i n s e n s i t i v e ATPase 59 D E A E - S e p h a c e l c h r o m a t o g r a p h y o f d e t e r g e n t s o l u b i l i z e d p l a s m a membranes 61 S D S - 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 o f v a r i o u s f r a c t i o n s o b t a i n e d d u r i n g the p u r i f i c a t i o n o f t h e DES i n s e n s i t i v e ATPase 64 G l y c e r o l g r a d i e n t c e n t r i f u g a t i o n o f peak I I ATPase 66 C a l i b r a t i o n c u r v e o f p r o t e i n m a r k e r s on g l y c e r o l d e n s i t y g r a d i e n t s 67 Ge l f i l t r a t i o n c h r o m a t o g r a p h y i n t h e p r e s e n c e o f 1$ (w/v) BRIJ-35 o f d e t e r g e n t s o l u b i l i z e d p l a s m a membranes 72 Ge l f i l t r a t i o n c h r o m a t o g r a p h y i n t h e p r e s e n c e o f 0.5$ (w/v) CHAPS o f d e t e r g e n t s o l u b i l i z e d p l a s m a membranes 73 S D S - 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 o f f r a c t i o n s o b t a i n e d d u r i n g the p u r i f i c a t i o n o f the DES i n s e n s i t i v e ATPase 74 D E A E - S e p h a c e l c h r o m a t o g r a p h y o f ATPase f r a c t i o n e l u t e d from t h e g e l f i l t r a t i o n column 75 E f f e c t o f pH on peak I ATPase a c t i v i t y 79 E f f e c t o f pH on the p u r i f i e d ATPase a c t i v i t y .82 i x F i g u r e 29. E f f e c t o f v a r i o u s i n h i b i t o r s on p u r i f i e d ATPase a c t i v i t y 84 F i g u r e 30. E f f e c t o f N - e t h y l m a l e i m i d e on p u r i f i e d ATPase a c t i v i t y 85 F i g u r e 31. I n h i b i t i o n o f p u r i f i e d ATPase a c t i v i t y by h i g h c o n c e n t r a t i o n s o f C a + 2 and Mg + 2 86 F i g u r e 32. E f f e c t o f M g + 2 o r C a + 2 on p u r i f i e d ATPase a c t i v i t y 87 F i g u r e 33. E f f e c t o f d i v a l e n t c a t i o n s on p u r i f i e d ATPase a c t i v i t y 90 F i g u r e 34. E f f e c t o f Mg+ 2 o r C a + 2 on ATPase a c t i v i t y d e t e r m i n e d as a f u n c t i o n o f t h e ATP c o n c e n t r a t i o n 92 F i g u r e 35. A u t o r a d i o g r a p h y o f 3 2 p _ i a o e i e c i d e t e r g e n t s o l u b i l i z e d p l a s m a membranes 95 F i g u r e 36. A u t o r a d i o g r a p h y o f 3 2 p _ l a b e l e d c r u c i e membranes 96 x A B B R E V I A T I O N S ADP AMP AMP-PNP ATP A T P a s e C 1 2 E 9 CHAPS CTP DCCD D C P I P DES D I F EDTA EGTA GTP MES NEM PAGE PC P i pNPP PMSF P P i SDS a d e n o s i n e 5 ' - d i p h o s p h a t e a d e n o s i n e 5 ' - m o n o p h o s p h a t e a d e n y l 5 ' - y l i m i d o d i p h o s p h a t e a d e n o s i n e 5 ' - t r i p h o s p h a t e a d e n o s i n e 5 ' - t r i p h o s p h a t a s e p o l y o x y e t h y l e n e 9 - l a u r y l e t h e r ( 3 - [ 3 - c h o l a m i d o p r o p y l ] d i m e t h y l a m m o n i o ) 1 p r o p a n e s u l f o n a t e c y t i d i n e 5 1 - t r i p h o s p h a t e N , N * - d i c y c l o h e x y l c a r b o d i i m i d e d i c h l o r o p h e n o l i n d o p h e n o l d i e t h y l s t i l b e s t r o l d i f f e r e n t i a t i o n i n d u c i n g f a c t o r e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d e t h y l e n e g l y c o l b i s ( / 2 - a m i n o e t h y l e t h e r ) -N , N , N ' , N * - t e t r a a c e t i c a c i d g u a n o s i n e 5 ' - t r i p h o s p h a t e 2 [ N - M o r p h o l i n e ] e t h a n e s u l f o n i c a c i d N - e t h y l m a l e i m i d 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 p h o s p h a t i d y l c h o l i n e o r t h o p h o s p h a t e p - n i t r o p h e n y l p h o s p h a t e p h e n y l m e t h y l s u l f o n y l f l u o r i d e p y r o p h o s p h a t e s o d i u m d o d e c y l s u l f a t e x i T r i s t r i ( h y d r o x y m e t h y l ) a m i n o m e t h a n e TMB-8 ( 8 - d i e t h y l a m i n o ) o c t y l - 3 , 4 , 5 -t r i m e t h o x y b e n z o a t e UTP u r i d i n e 5 ' - t r i p h o s p h a t e ACKNOWLEDGMENTS I would l i k e t o e x p r e s s my a p p r e c i a t i o n t o Dr. G. Weeks f o r h i s p a t i e n t g u i d a n c e and h e l p f u l s u g g e s t i o n s . I would a l s o l i k e t o thank a l l my f r i e n d s and l a b mates f o r p u t t i n g up w i t h my bad temper and g e n e r a l l y m i s e r a b l e d i s p o s i t i o n . In a d d i t i o n , I thank Dr. P. Bhanot f o r the many s t i m u l a t i n g c o n v e r s a t i o n s o v e r c o f f e e , and t h e U n i v e r s i t y o f B r i t i s h C olumbia f o r f i n a n c i a l a s s i s t a n c e i n t h e form o f a F r a n k Wesbrook F e l l o w s h i p . F i n a l l y , I want t o thank my w i f e K a t h y f o r t y p i n g t h i s t h e s i s and f o r b e i n g a n e v e r e n d i n g s o u r c e o f e n c o u r a g e m e n t . x i i i INTRODUCTION D i c t y o s t e l i u m d i s c o i d e u m i s a e u k a r y o t i c o r g a n i s m t h a t e x h i b i t s a v e r y d i s t i n c t i v e d e v e l o p m e n t a l c y c l e ( L o o m i s , 1975). The myxamoeboid form o f the o r g a n i s m f e e d s on b a c t e r i a and d i v i d e s by b i n a r y f i s s i o n , but when the f o o d s u p p l y i s e x h a u s t e d t h e c e l l s i n i t i a t e a complex s e q u e n c e o f m o r p h o l o g i c a l changes which u l t i m a t e l y r e s u l t s i n the f o r m a t i o n o f a m u l t i c e l l u l a r s t r u c t u r e composed o f two c e l l t y p e s , s t a l k s and s p o r e s ( L o o m i s , 1 9 7 5 ) . These s t a g e s a r e d e t a i l e d i n F i g u r e 1. Upon c e s s a t i o n o f g r o w th the c e l l s a g g r e g a t e i n r e s p o n s e t o p u l s e s o f cAMP ( G e r i s c h e_t a l . , 1975; Darmon e_t ajL. , 1975 ) t o form a m i g r a t o r y s l u g or p s e u doplasmodium. At t h i s s t a g e the two c e l l t y p e s become d i s t i n g u i s h a b l e f o r the f i r s t time as d i s t i n c t p r e - s t a l k and p r e - s p o r e c e l l s . These c e l l t y p e s have a d e f i n i t e s p a t i a l o r i e n t a t i o n , w i t h p r e - s t a l k c e l l s i n t h e a n t e r i o r and p r e -s p o r e c e l l s i n the p o s t e r i o r p o r t i o n o f the p s e u d o p l a s m o d i u m ( L o o m i s , 1975). F o l l o w i n g a p e r i o d o f p s e u d o p l a s m o d i a l m i g r a t i o n , t h e t e r m i n a l s t a g e o f d i f f e r e n t i a t i o n , known as c u l m i n a t i o n , b e g i n s . T h i s p r o c e s s r e s e m b l e s a r e v e r s e f o u n t a i n f l o w ( F a r n s w o r t h , 1973) i n which th e p r e - s t a l k c e l l s move down toward t h e s u b s t r a t u m , m a t u r i n g i n t o s t a l k c e l l s and f o r m i n g a tube w i t h a r i g i d c e l l u l o s i c w a l l . As t h e s t a l k i s formed the p r e - s p o r e c e l l s a r e r a i s e d from t h e s u b s t r a t e to t h e t o p o f t h e column where t h e y mature t o s p o r e c e l l s . ( F a r n s w o r t h , 1 2 1973)- Under l a b o r a t o r y c o n d i t i o n s t h i s p r o c e s s i s c o m p l e t e i n a p p r o x i m a t e l y 24 h o u r s . The r e l a t i v e s i m p l i c i t y o f t h i s d e v e l o p m e n t a l c y c l e makes _D. d i s c o i d e u m a model s y s t e m f o r s t u d i e s on c e l l d i f f e r e n t i a t i o n . T h e r e have been r e p o r t s t h a t a v a r i e t y o f i o n s a r e i n v o l v e d a t v a r i o u s s t a g e s o f t h e d e v e l o p m e n t a l c y c l e . Mason e_t a l . (1971) o b s e r v e d t h a t a g g r e g a t i o n was e f f e c t i v e l y h a l t e d when t h e e x t e r n a l Ca + 2 c o n c e n t r a t i o n was d r o p p e d b e l o w 1 0 ~ 6 M. I n a d d i t i o n , B r a c h e t and K l e i n (1977) r e p o r t e d t h a t a g g r e g a t i o n was g r e a t l y a c c e l e r a t e d when amoebae were e x p o s e d t o t h e Ca + 2 i o n o p h o r e ^ 2 2 1 3 7 * ^ n c o n t r a s t , S a i t o (1979) r e p o r t e d t h a t , w h i l e t h e number o f m ature f r u i t i n g b o d i e s was d e p e n d e n t on t h e Ca + 2 c o n c e n t r a t i o n , amoebae r e s p o n d e d t o e x t r a c e l l u l a r cAMP even when t h e e x t e r n a l C.a * c o n c e n t r a t i o n was b e l o w 10 M. S a i t o c o n c l u d e d t h a t a g g r e g a t i o n was a C a + 2 i n d e p e n d e n t phenomenon, a l t h o u g h t h e c a t i o n was n e c e s s a r y f o r n o r m a l m o r p h o g e n e s i s ( S a i t o , 1 9 7 9 ) . R e c e n t l y , E u r o p e - F i n n e r _e_t a l . (1984) showed t h a t c h e m o t a x i s was s t r o n g l y i n h i b i t e d by EGTA i n d u c e d C a + 2 d e p l e t i o n , i n d i c a t i n g an a c t i v e r o l e f o r t h e c a t i o n d u r i n g a g g r e g a t i o n . C a l c i u m has a l s o been shown t o i n h i b i t a d e n y l a t e c y c l a s e i n v i t r o ( L o o m i s e_t a l . , 1978) and t o i n c r e a s e t h e number o f c e l l s u r f a c e cAMP b i n d i n g s i t e s w i t h o u t a l t e r i n g t h e i r a f f i n i t y c o n s t a n t s ( J u l i a n i and K l e i n , 1 9 7 7 ) . I t has a l s o been p r o p o s e d t h a t C a + 2 r e g u l a t e s t h e p o l y m e r i z a t i o n o f 3 a c t i n and i t s a s s o c i a t i o n w i t h m y o s i n ( M o k r i n and S p u d i c h , 1976; S p u d i c h and S p u d i c h , 1 9 8 2 ) . E v i d e n c e f o r Ca + 2 f l u x d u r i n g a g g r e g a t i o n has a l s o been o b t a i n e d . Wick e t a L l . (1 978) o b s e r v e d a t r a n s i e n t , r a p i d i n f l u x o f ^ C a + 2 i n t o s t a r v e d amoebae upon e x c i t a t i o n w i t h cAMP . T h i s i n i t i a l i n f l u x was f o l l o w e d by a r e a p p e a r a n c e o f 45 +2 t h e s e q u e s t e r e d Ca i n t o t h e medium s e v e r a l m i n u t e s l a t e r (Wick et. a l . , 1 9 7 8 ) . Bumann e t a l . ( 1 984 ) e x t e n d e d t h i s work t o show t h a t C a + 2 u p t a k e o c c u r r e d i n an o s c i l l a t o r y f a s h i o n w i t h a p e r i o d o f r o u g h l y 8 m i n u t e s and an a m p l i t u d e o f 3.5x10-6 i o n s p e r c e l l . T h e r e have been some r e p o r t s r e g a r d i n g t h e mechanism o f Ca + 2 movement a c r o s s t h e p l a s m a membrane i n jD_. d i s c o i d e u m . W o r k i n g w i t h i n t a c t c e l l s , P a r i s h and W e i b e l (1980) p r e s e n t e d e v i d e n c e f o r an e c t o - A T P a s e i n t h a t e x t r a c e l l u l a r r l 4 n 4 5 + 2 L C]-ATP was h y d r o l y z e d and Ca u p t a k e was r e d u c e d by t h e u n c o u p l e r s KCN, CCCP and d i n i t r o p h e n o l and by non-h y d r o l y s a b l e ATP a n a l o g u e s . I n most i n s t a n c e s t h i s r e d u c t i o n was l e s s t h a n 50$ and C a + 2 u p t a k e was g e n e r a l l y v e r y s l o w , r e a c h i n g s a t u r a t i o n a f t e r 20 m i n u t e s . E u r o p e -F i n n e r and N e w e l l ( 1 9 8 5 a ) , on t h e o t h e r hand, r e p o r t e d C a + 2 u p t a k e t o be e x t r e m e l y r a p i d and u n a f f e c t e d by p r e - t r e a t m e n t o f c e l l s w i t h a z i d e , s u g g e s t i n g t h a t Ca + 2 moved a c r o s s t h e membrane v i a p a s s i v e f a c i l i t a t e d d i f f u s i o n . R e c e n t l y , C a + 2 has been i m p l i c a t e d i n t h e cAMP o r f o l a t e i n d u c e d f o r m a t i o n o f i n t r a c e l l u l a r cGMP ( S m a l l e t a l . . 1 9 8 6 ) . The a c c u m u l a t e d e v i d e n c e s u g g e s t e d t h a t t h i s 4 phenomenon was due t o m o b i l i z a t i o n o f i n t r a c e l l u l a r C a + 2 r a t h e r t h a n t r a n s p o r t a c r o s s t h e membrane, s i n c e t h e e f f e c t o f cAMP c o u l d be m i m i c k e d by i n o s i t o l 1 ,4 , 5 - t r i p h o s p h a t e ( E u r o p e - F i n n e r and N e w e l l , 1 9 8 5 b ) , w h i c h has s u b s e q u e n t l y been shown t o c a u s e Ca + 2 r e l e a s e f r o m n o n - m i t o c h o n d r i a l p o o l s i n D i c t y o s t e l i u m amoebae ( E u r o p e - F i n n e r and N e w e l l , 1 9 8 6 ) . F u r t h e r m o r e , cGMP f o r m a t i o n was i n h i b i t e d by t h e i n t r a c e l l u l a r C a + 2 a n t a g o n i s t ( 8 - d i e t h y l a m i n o ) o c t y l - 3 , 4 , 5 -t r i m e t h o x y b e n z o a t e (TMB-8) ( E u r o p e - F i n n e r and N e w e l l , 1984; E u r o p e - F i n n e r ^ t a l . , 1 9 8 5 ) . P r o t o n f l u x has a l s o been i m p l i c a t e d i n J3. d i s c o i d e u m d e v e l o p m e n t . I t was p r o p o s e d t h a t c h a n g e s i n i n t r a c e l l u l a r pH p l a y an i m p o r t a n t r o l e i n d i f f e r e n t i a t i o n w i t h a c i d pH f a v o r i n g s t a l k c e l l f o r m a t i o n and a l k a l i n e pH f a v o r i n g s p o r e c e l l f o r m a t i o n ( G r o s s e_t a_l. , 1 983 ) . F u r t h e r m o r e , t h e s t a l k c e l l d i f f e r e n t i a t i o n i n d u c i n g f a c t o r ( D I F ) was h y p o t h e s i z e d to i n h i b i t a p u t a t i v e c e l l s u r f a c e p r o t o n pump, t h u s e f f e c t i n g a d e c r e a s e i n t h e i n t r a c e l l u l a r pH and d i v e r t i n g c e l l s f r o m t h e s p o r e t o t h e s t a l k pathway ( G r o s s e t a l . , 1 983) . S u b s e q u e n t work has f a i l e d t o d e t e c t any s u s t a i n e d change i n t h e i n t r a c e l l u l a r pH d u r i n g d i f f e r e n t i a t i o n . J e n t o f t and Town (1985) d e t e c t e d two pH p e a k s u s i n g ^ 1P-NMR s p e c t r o s c o p y , t h e m i t o c h o n d r i a l peak a t pH 7.16 + 0.03 and t h e c y t o s o l i c peak a t pH 6.48 + 0.02. No change i n t h e pH o c c u r r e d up t o 13.5 h o u r s o f d e v e l o p m e n t ( J e n t o f t and Town, 1 9 8 5 ) . A s i m i l a r r e s u l t was o b t a i n e d by Kay e_t a l _ . (1986) 5 a l t h o u g h t h e y o b t a i n e d pH measurements w h i c h were somewhat h i g h e r t h a n t h o s e s e e n by J e n t o f t and Town ( 1 9 8 5 ) . I n a n o t h e r s t u d y , R a t n e r (1986) f o u n d t h e i n t r a c e l l u l a r pH v a l u e s o f p r e - s p o r e and p r e - s t a l k c e l l s t o be i n d i s t i n g u i s h a b l e . The o n l y r e p o r t o f a d e v e l o p m e n t a l c h a n g e i n i n t r a c e l l u l a r pH i s t h a t o f J a m e i s o n e_t a l . ( 1 9 8 4 ) , who o b s e r v e d a s h i f t f r o m pH 6.3 t o pH 7.13 a f t e r a b o u t two h o u r s o f d e v e l o p m e n t . T h i s s h i f t , h owever, was e x t r e m e l y s h o r t l i v e d ( a p p r o x i m a t e l y 15 m i n u t e s ) and t h e i n t r a c e l l u l a r pH q u i c k l y r e t u r n e d t o t h e o r i g i n a l v a l u e . P r o t o n f l u x has been o b s e r v e d d u r i n g t h e e a r l y s t a g e s o f I), d i s c o i d e u m d e v e l o p m e n t . Malchow e_t_ a l . ( 1978a) r e p o r t e d t r a n s i e n t d e c r e a s e s i n e x t r a c e l l u l a r pH when u n b u f f e r e d c e l l s u s p e n s i o n s were e x p o s e d t o cAMP . S u b s e q u e n t work r e v e a l e d t h e e x t r a c e l l u l a r pH t o o s c i l l a t e i n c o n j u n c t i o n w i t h t h e p u l s a t i l e r e l e a s e o f cAMP (Malchow e t a l . . 1978b) s u g g e s t i n g t h a t t h e two phenomena were l i n k e d . R e c e n t l y Gottmann and W e i j e r (19 86) have shown t h e s e p r o t o n o s c i l l a t i o n s t o be i n h i b i t e d by c a f f e i n e , i n d i c a t i n g t h a t t h e y a r e c o u p l e d t o cAMP o s c i l l a t i o n s . C a f f e i n e has been shown t o i n h i b i t a g g r e g a t i o n by i n t e r f e r i n g w i t h a d e n l y a t e c y c l a s e a c t i v a t i o n ( B r e n n e r and Thorns, 1 9 8 4 ) . In a d d i t i o n t o t h e s e o b s e r v a t i o n s i t has been r e p o r t e d t h a t t h e N a + / H + a n t i p o r t i n h i b i t o r a m i l o r i d e d i s r u p t s n o r m a l d e v e l o p m e n t ( J a m i e s o n e_t _a_l. , 1 9 8 4 ) . These d a t a s u g g e s t t h a t p r o t o n f l u x e s may p l a y an a c t i v e r o l e i n d i f f e r e n t i a t i o n , b u t t h e r e i s as y e t no d i r e c t e v i d e n c e t o 6 i n d i c a t e t h a t c h a n g e s i n i n t e r n a l pH a r e i n v o l v e d i n t h e r e g u l a t i o n o f s t a l k and s p o r e c e l l d e v e l o p m e n t . O t h e r i o n s have a l s o been i m p l i c a t e d i n t h e d i f f e r e n t i a t i o n p r o c e s s . Town ( 1 9 8 4 ) o b s e r v e d t h a t b o t h t h e N a + ,K + -ATPase i n h i b i t o r s c i l l a r e n and t h e K + i o n o p h o r e v a l i n o m y c i n i n h i b i t e d s p o r e c e l l d e v e l o p m e n t w i t h o u t a f f e c t i n g s t a l k s when c e l l s were d i f f e r e n t i a t i n g i n submerged m o n o l a y e r s . T h e s e d a t a s u g g e s t e d a r o l e f o r K + i n s p o r e c e l l d e v e l o p m e n t s i n c e , u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s u s e d , t h e p r i m a r y e f f e c t o f b o t h o f t h e s e compounds would be t o r e d u c e t h e i n t r a c e l l u l a r K + c o n c e n t r a t i o n . O t h e r i o n o p h o r e s s u c h as t h e N a + - s p e c i f i c i o n o p h o r e m o n e n s i n and t h e K /H e x c h a n g e r n i g e r i c i n i n h i b i t e d t h e d i f f e r e n t i a t i o n o f b o t h c e l l t y p e s (Town, 1984 ) . I n v i e w o f t h e a p p a r e n t i m p o r t a n c e o f i o n s and i o n f l u x i n D_. d i s c o i d e u m d e v e l o p m e n t , i t would be a d v a n t a g e o u s t o u n d e r s t a n d t h e u n d e r l y i n g mechanism o f i o n t r a n s p o r t i n more d e t a i l . I n many c a s e s t h e t r a n s p o r t o f i o n s a c r o s s t h e membrane i s f a c i l i t a t e d by ATP d r i v e n pumps . The b e s t c h a r a c t e r i z e d o f t h e s e enzymes a r e t h e N a + , K + - A T P a s e s r e s p o n s i b l e f o r N a + / K + t r a n s p o r t , t h e C a + 2 - A T P a s e s w h i c h t r a n s l o c a t e C a + 2 i o n s and t h e p r o t o n pumping M g + 2 - A T P a s e s . The N a + , K + - A T P a s e has been p u r i f i e d f r o m a number o f s o u r c e s i n c l u d i n g s h a r k r e c t a l g l a n d ( H o k i n ^ t a ^ l . , 1 9 7 3 ; D i x o n and H o k i n , 1 9 7 8 ; H a s t i n g s and R e y n o l d s , 1 9 7 9 ) , e l e c t r o p l a x t i s s u e f r o m e l e c t r i c e e l s ( D i x o n and H o k i n , 7 1 9 7 1 * ; D i x o n and H o k i n , 1 9 7 8 ) and k i d n e y m e d u l l a from dogs ( K y t e , 1 9 7 1 ) and p i g s ( J o r g e n s e n , 1 9 7 4 ) . T h i s enzyme c a t a l y z e s the exchange o f t h r e e Na + f o r two K + ( S c a r b o r o u g h , 1 9 8 2 ) and i s g e n e r a l l y c h a r a c t e r i z e d by i t s s e n s i t i v i t y t o o u a b a i n (Lane e_t a l . , 1 973 ; K y t e , 1 9 7 4 ; W a l l i c k e t a l . , 1 9 7 8 ; Schoner e t a l . , 1 9 7 9 ) . The p u r i f i e d N a + , K + - A T P a s e c o n t a i n s two p o l y p e p t i d e s , a l a r g e , r e l a t i v e l y h y d r o p h o b i c o( s u b u n i t and a s m a l l g l y c o s y l a t e d ^ s u b u n i t (Lane e t a l . , 1 9 7 3 ; D i x o n and H o k i n , 1 9 7 4 ; K y t e , 1 974 ; P e r r o n e e t a l . , 1 9 7 5 ). The s u b u n i t has been shown to be p h o s p h o r y l a t e d ( K y t e , 1 9 7 1 ; Lane e t a l . , 1 9 7 3 ; W a l l i c k e t a l . , 1 9 7 8 ; B o n t i n g e t a l . , 1 9 7 9 ) and t o c o n t a i n the o u a b a i n b i n d i n g s i t e (Ruoho and K y t e , 1 9 7 4 ; Rogers e t a l . , 1 9 7 9 ; Charlemagne e t a l . , 1 9 8 6 ). The f u n c t i o n o f the s u b u n i t i s not known. R e p o r t e d v a l u e s f o r the m o l e c u l a r w e i g h t o f the e n c h a i n v a r y between 85 t o 140 kDa ( H a s t i n g s and R e y n o l d s , 1 9 7 9 ; Esmann e t a l . , 1 9 7 9 ; W i n t e r and Moss, 1 9 7 9 ; Lane e t a_l . , 1 9 7 9 ; C r a i g and K y t e , 1 9 8 0 ) w h i l e t h o s e f o r the ^ c h a i n v a r y between 35 t o 70 kDa (Lane e t a l . , 1 9 7 3 ; H a s t i n g s and R e y n o l d s , 1 9 7 9 ; Esmann e_fc a l . , 1 9 7 9 ; W i n t e r and Moss, 1 9 7 9 ; Lane e t a l . , 1 9 7 9 ; C r a i g and K y t e , 1980 ) . Much work has f o c u s s e d on the s t o i c h i o m e t r y o f the two s u b u n i t s . P e r r o n e e_t a l . (1 9 7 5 ) p r o p o s e d a s t o i c h i o m e t r y o f 2 /3 based on a minimum m o l e c u l a r w e i g h t o f a c t i v e ATPase o f 2 5 0 kDa, as d e t e r m i n e d by r a d i a t i o n i n a c t i v a t i o n . H a s t i n g s and R e y n o l d s ( 1 9 7 9 ) used e q u i l i b r i u m a n a l y t i c a l 8 c e n t r i f u g a t i o n t o d e t e r m i n e a minimum m o l e c u l a r w e i g h t o f 3 7 9 . 9 kDa, w i t h t h e p r o t e i n composed o f two c< and f o u r ^ 4 s u b u n i t s . Esmann e_t a l . ( 1 9 7 9 ) e s t i m a t e d a 1:1 s t o i c h i o m e t r y and d e t e r m i n e d the enzyme s t r u c t u r e to be 0 ^ 2 / ^ 2 C r a i g and K y t e ( 1 9 8 0 ) , i n a c o m p r e h e n s i v e c r o s s l i n k i n g s t u d y , a r r i v e d a t a m o l e c u l a r w e i g h t o f 1 7 7 kDa and the s u b u n i t c o m p o s i t i o n oy<^  . The f i n d i n g s o f C r a i g and Kyte (1980) have r e c e n t l y been c o n f i r m e d by J o r g e n s e n and A n d e r s e n (1986) who used s e d i m e n t a t i o n 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 t o show a c t i v e ATPase w i t h a q u a t e r n a r y s t r u c t u r e o f The p r o t o n t r a n s l o c a t i n g M g + 2 - A T P a s e s t h a t have been b e s t c h a r a c t e r i z e d a r e t h o s e from t h e f i l a m e n t o u s fungus N e u r o s p o r a c r a s s a ( S c a r b o r o u g h , 1 9 80 ; Bowman e t a l . , 1981a; A d d i s o n and S c a r b o r o u g h , 1981) and t h e f i s s i o n y e a s t S c h i z o s a c c h a r o m y c e s pombe ( D u f o u r and G o f f e a u , 1978; V i l l a l o b o e t a l . , 1 9 8 1 ) . These p r o t e i n s a r e major membrane c o n s t i t u e n t s and r e l a t i v e l y l i t t l e p u r i f i c a t i o n has been r e q u i r e d t o p r o d u c e homogeneous enzyme, which m i g r a t e s d u r i n g SDS-PAGE as a s i n g l e p o l y p e p t i d e o f 104 kDa (D u f o u r and G o f f e a u , 1 978 ; A d d i s o n and S c a r b o r o u g h , 1981 ; Bowman e£ a l . , 1981a; S c a r b o r o u g h and Dame, 1984). ATP d r i v e n p r o t o n t r a n s l o c a t i o n by t h e s e enzymes has been shown t o be e l e c t r o g e n i c i n t h a t i t o c c u r s c o n c u r r e n t l y w i t h t h e f o r m a t i o n o f a.membrane p o t e n t i a l ( S c a r b o r o u g h , 1980 ; V i l l a l o b o e t a l . , 1981; D u f o u r e t a l . , 1 982 ; P e r l i n e t a l . , 1984 ). I t i s g e n e r a l l y b e l i e v e d t h a t t h e s e enzymes 9 c a t a l y z e o n l y the movement o f p r o t o n s a c r o s s the membrane, a l t h o u g h V i l l a l o b o (1982, 1984) has p r e s e n t e d e v i d e n c e f o r an e n e r g y d e p e n d e n t , v a n a d a t e s e n s i t i v e H +-K + exchange. I t was p r o p o s e d t h a t the K + g a t e opened o n l y upon the i m p o s i t i o n o f a membrane p o t e n t i a l ( V i l l a l o b o , 1984). The s t o i c h i o m e t r y o f the H +,K + exchange was n o t d e t e r m i n e d . P r o t o n t r a n s l o c a t i o n and ATPase a c t i v i t y a r e s e n s i t i v e to v a n a d a t e , d i e t h y l s t i l b e s t r o l ( DES), 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) and m e r c u r i a l s such as t h i m e r o s a l and p - c h l o r o - m e r c u r i p h e n y l s u l f o n a t e (Bowman and Slayman, 1 9 7 9 ; S c a r b o r o u g h , 1980. Bowman e t a l . ; 1981; Amory and G o f f e a u , 1982 ; D u f o u r e t a l . , 1 982; P e r l i n e t a l . . 1984). The N e u r o s p o r a enzyme i s a l s o s t r o n g l y i n h i b i t e d by N - e t h y l m a l e i m i d e ( B r o o k e r and Slayman, 1982, 1983a). L i k e the N a + , K + - A T P a s e the p r o t o n ATPases o f y e a s t and N e u r o s p o r a form p h o s p h o r y l a t e d i n t e r m e d i a t e s as p a r t o f t h e r e a c t i o n c y c l e (Dame and S c a r b o r o u g h , 1980; Amory e t a l . , 1980). T h i s i n t e r m e d i a t e was c h a r a c t e r i z e d as b e i n g an a c i d - s t a b l e ^ - a s p a r t y l p h o s p h a t e (Dame and S c a r b o r o u g h , 1981; Amory and G o f f e a u , 1982). S i m i l a r l y , l i k e t h e Na +-K +-ATPase, the N e u r o s p o r a enzyme has been shown to undergo a s e r i e s o f c o n f o r m a t i o n a l changes d u r i n g the c a t a l y t i c c y c l e ( A d d i s o n and S c a r b o r o u g h , 1982). Plasma membrane p r o t o n t r a n s l o c a t i n g ATPases have a l s o been c h a r a c t e r i z e d from a v a r i e t y o f p l a n t s p e c i e s ( V a r a and S e r r a n o , 1982; S z e , 1 9 8 3 ; O ' N e i l l and Sp a n s w i c k , 1 9 8 4 a , 1 9 8 4 b , V a r a and S e r a n o , 1982). These enzymes a r e v e r y 10 s i m i l a r t o t h o s e o f N e u r o s p o r a and y e a s t i n t h a t t h e y a r e i n h i b i t e d by v a n a d a t e , DCCD , DES and m e r c u r i a l s . However, t h e p l a n t A T P a s e s a r e s t i m u l a t e d c o n s i d e r a b l y by m o n o v a l e n t c a t i o n s , p a r t i c u l a r l y K + ( V a r a and S e r r a n o , 1982; O ' N e i l l and S p a n s w i c k , 1984a, 1984b, B r i s k e n and P o o l e , 1 984; I m b r i e and Murphy, 1 9 8 4 ) , so i n t h i s r e s p e c t t h e y d i f f e r f r o m t h e N e u r o s p o r a and y e a s t enzymes w h i c h a r e o n l y m a r g i n a l l y ' s t i m u l a t e d by t h e s e c a t i o n s (Bowman e t a l . , 1 9 8 1 ) . The t h i r d m a j o r c l a s s o f c e l l s u r f a c e A T P a s e s a r e t h e C a + 2 - A T P a s e s . I n i t i a l work on r a t k i d n e y ( P a r k i n s o n and Radde , 1 9 7 1 ) , g u i n e a p i g p l a c e n t a (Shami and Radde, 1971) and mouse l i v e r ( G a r n e t t and Kemp, 19 7 5 ) , r e v e a l e d an ATPase w h i c h was s t i m u l a t e d by m i l l i m o l a r c o n c e n t r a t i o n s o f e i t h e r Ca + 2 o r M g + 2 . S u b s e q u e n t l y , i n a d d i t i o n t o t h i s low a f f i n i t y ATPase a c t i v i t y , a h i g h a f f i n i t y a c t i v i t y , s t i m u l a t e d by s u b m i c r o m o l a r c o n c e n t r a t i o n s o f C a + 2 i n t h e p r e s e n c e o f low (5juM) c o n c e n t r a t i o n s o f Mg + 2 , has a l s o been i d e n t i f i e d ( L y n c h and Cheung, 1979; P e n n i s t o n , 1 983; C a r a f o l i e_t a l _ . , 1 9 8 4 ) . T h a t t h e low and h i g h a f f i n i t y a c t i v i t i e s a r e a t t r i b u t a b l e t o d i f f e r e n t enzymes has been shown by t h e f a c t t h a t t h e y c a n be s e p a r a t e d by g e l f i l t r a t i o n and i o n exch a n g e c h r o m a t o g r a p h y ( L o t e r s z t a j n e t a l . , 1981; N i g g l i e t a l . , 1 9 8 1 ) . The h i g h a f f i n i t y enzyme i s g e n e r a l l y r e f e r r e d t o as t h e ( C a + 2 : M g + 2 ) - A T P a s e w h i l e t h e low a f f i n i t y enzyme i s t h e C a + 2 o r M g + 2 - A T P a s e . Many h i g h a f f i n i t y ( C a + 2 : M g + 2 ) - A T P a s e s a r e s t i m u l a t e d by c a l m o d u l i n ( L y n c h and Cheung, 1979; N i g g l i e t a l . , 1981; 1 1 K o t a g a l e t a l . , 1 983; C a r o n i e t a l . , 1 983 ; O l i v a e_t aj.. , 1983; Ansah e t a l . , 1984) w h i c h a c t s by i n c r e a s i n g t h e v e l o c i t y o f ATP h y d r o l y s i s and d e c r e a s i n g t h e K m Q f t h e enzyme f o r Ca + 2 . I n some c a s e s , a d d i t i o n o f ex o g e n o u s c a l m o d u l i n t o p l a s m a membranes had no e f f e c t on ATPase a c t i v i t y (Verma and P e n n i s t o n , 1981; Ochs and Reed, 1984; D e b e t t o and C a n t l e y , 1984; Tsukamoto e t a l . . , 1 9 8 6 ) , a l t h o u g h t h i s may have been due t o t h e d i f f i c u l t y e n c o u n t e r e d i n r e m o v i n g endogenous c a l m o d u l i n f r o m t h e membranes (Verma and P e n n i s t o n , 1981; Ochs and Reed, 1984; D e b e t t o and C a n t l e y , 1 9 8 4 ) . L o t e r s z t a j n e t a_l. (1981) have r e p o r t e d t h e p u r i f i c a t i o n o f a ( C a + 2 : M g + 2 ) - A T P a s e f r o m r a t l i v e r p l a s m a membranes w h i c h was a c t i v a t e d by an endogenous p r o t e i n d i s t i n c t f r o m c a l m o d u l i n and a c i d i c p h o s p h o l i p i d s have been o b s e r v e d t o s u b s t a n t i a l l y a c t i v a t e t h e ( C a + 2 : M g + 2 ) - A T P a s e s f r o m e r y t h r o c y t i c and p a n c r e a t i c a c i n a r p l a s m a membranes ( N i g g l i e t a l - , 1981; Ansah e t a l . , 1 9 8 4 ) . The p l a s m a membrane ( C a + 2 : M g + 2 ) - A T P a s e s a r e r e s i s t a n t t o t h e m i t o c h o n d r i a l i n h i b i t o r s a z i d e , o l i g o m y c i n and DCCD and t o t o o u a b a i n (Verma and P e n n i s t o n , 1981; Ochs and Reed, 1984; D e b e t t o and C a n t l e y , 1 9 8 4 ) . Many o f t h e s e enzymes a r e , however, s e n s i t i v e t o v a n a d a t e ( N i g g l i e_t a l _ . , 1981 ; Gmaj e t a l . , 1982; D e b e t t o and C a n t l e y , 1 9 8 4 ) , a l t h o u g h Tsukamoto e t a l . (1986) have r e p o r t e d t h e e x i s t e n c e o f a v a n a d a t e r e s i s t a n t , M g + 2 - i n d e p e n d e n t C a + 2 - A T P a s e f r o m r e n a l b a s o l a t e r a l p l a s m a membranes. D e b e t t o and C a n t l e y (1984) 1 2 have a l s o r e p o r t e d t h a t the ( C a + :Mg )-ATPase from murine e r y t h r o l e u k e m i a membranes i s s e n s i t i v e to l a n t h a n u m i o n s . The ( C a + 2 : M g + 2 ) - A T P a s e s a r e b e l i e v e d to p l a y a r o l e i n the m a i n t e n a n c e o f i n t r a c e l l u l a r C a + 2 by means o f e n e r g y dependent e x t r u s i o n . Many su c h enzymes have been shown t o t r a n s p o r t C a + 2 a c r o s s the membrane ( N i g g l i e t a l . , 1 9 8 1 ; C a r o n i e t a_l. , 1 9 8 3 ; K o t a g a l e t a l . , 1 9 8 3 ; B r e i t b a r t e t a l . . 1 9 8 3 , Ochs and Reed, 1 9 8 4 ) however the transmembrane o r i e n t a t i o n o f the enzyme i n i n t a c t c e l l s was not d e t e r m i n e d . E v i d e n c e has been p r e s e n t e d w h i c h s u g g e s t s t h a t the h i g h a f f i n i t y C a + 2 - A T P a s e s from p a n c r e a t i c a c i n a r and r e n a l b a s o l a t e r a l plasma membranes a r e e c t o - A T P a s e s (Ansah et a l . , 1 9 8 4 ; Tsukamoto e t a l . , 1986). However, n e i t h e r o f t h e s e r e p o r t s c o n t a i n e d any d a t a on C a + 2 pumping. The ( C a + 2 : M g + 2 ) - A T P a s e s f o r m p h o s p h o r y l a t e d i n t e r m e d i a t e s o f m o l e c u l a r w e i g h t 1 3 0 t o 1 4 0 kDa d u r i n g the c a t a l y t i c c y c l e (De Smedt e t a l . , 1 9 8 3 ; D e b e t t o and C a n t l e y , 1 9 8 4 ; Ansah e t a l . , 1 9 8 4 ) . T h i s i n t e r m e d i a t e i s s i m i l a r t o t h a t seen f o r the H + - A T P a s e s i n t h a t i t i s s t a b l e a t a c i d pH but i s r a p i d l y h y d r o l y z e d a t m i l d l y a l k a l i n e pH ( D e b e t t o and C a n t l e y , 1 9 8 4 ; De Smedt e t a l . , 1 9 8 3 ) . There a p p e a r s to be a c e r t a i n amount o f s t r u c t u r a l homology between the v a r i o u s t y p e s o f membrane t r a n s p o r t A T P a s e s . Walderhaug e t a l . ( 1 9 8 5 ) have d e m o n s t r a t e d a c t i v e s i t e sequence homology between t h e N a + , K + - A T P a s e from dog k i d n e y , Na +,H +-ATPase from g a s t r i c mucosa and the H +-ATPase from c o r n r o o t s . The common s e q u e n c e a r o u n d the a c t i v e s i t e 13 was f o u n d t o be c y s - ( s e r / t h r ) - a s p - l y s and i n a l l c a s e s asp was t h e t a r g e t o f p h o s p h o r y l a t i o n . S e r r a n o e t a l . (1986) have d e t e r m i n e d t h e DNA s e q u e n c e f o r t h e gene e n c o d i n g t h e y e a s t H + - A T P a s e . C o m p a r i s o n between t h i s s e q u e n c e and t h o s e o f t h e Na +,K + -ATPase f r o m k i d n e y ( S h u l l et_ a l . , 1 985 ) and t h e s a r c o p l a s m i c r e t i c u l u m C a + 2 - A T P a s e (McLennan e t a l . , 1985) r e v e a l e d n i n e a r e a s o f e x t e n s i v e homology ( S e r r a n o e t a l . , 1 9 8 6 ) . I n t e r e s t i n g l y , v e r y l i t t l e s e q u e n c e homology was s e e n when t h e s e enzymes were compared w i t h F Q _ p ^ A T P a s e s o f b a c t e r i a , 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 ( S e r r a n o e t a l . , 1986) . At t h e o u t s e t o f t h e work p r e s e n t e d i n t h i s t h e s i s o n l y two r e p o r t s e x i s t e d c o n c e r n i n g p l a s m a membrane A T P a s e s i n D_., d i s c o i d e u m . P a r i s h and W e i b e l (1980) had p r e s e n t e d e v i d e n c e s u g g e s t i n g t h e e x i s t e n c e o f a c e l l s u r f a c e e c t o ATPase and B l a n c o (1 982) had r e p o r t e d a N a + , K + s t i m u l a t e d , a z i d e s e n s i t i v e A T P a s e . I n a d d i t i o n , P a r i s h and W e i b e l (1980) a l s o s u g g e s t e d a l i n k between e c t o p i c ATPase a c t i v i t y and + 2 4 5 +2 Ca t r a n s p o r t i n t h a t Ca t r a n s p o r t was d e p r e s s e d by a g e n t s w h i c h s e r v e d t o d e c r e a s e ATPase a c t i v i t y . In l i g h t o f t h e s e r e s u l t s , and i n c o n s i d e r a t i o n o f t h e a p p a r e n t r o l e o f i o n s and i o n t r a n s p o r t i n p_. d i s c o i d e u m i t would seem a d v a n t a g e o u s t o u n d e r s t a n d t h e u n d e r l y i n g mechanism w h i c h f a c i l i t a t e s t h e s e phenomena. P l a s m a membrane A T P a s e s have been w e l l c h a r a c t e r i z e d i n many o t h e r s y s t e m s and have been shown t o p e r f o r m an i m p o r t a n t r o l e i n t h e r e g u l a t i o n o f i n t r a c e l l u l a r i o n i c h o m e o s t a s i s . I t i s 14 p o s s i b l e t h a t s u c h enzymes p l a y an e q u a l l y i m p o r t a n t r o l e i n D. d i s c o i d e u m d e v e l o p m e n t by r e g u l a t i n g t h e t r a nsmembranous movement o f i o n s d e s c r i b e d e a r l i e r i n t h i s i n t r o d u c t i o n . The p r i m a r y g o a l o f t h i s s t u d y was t o s y s t e m a t i c a l l y i d e n t i f y and c h a r a c t e r i z e p l a s m a membrane ATPase a c t i v i t y i n D. d i s c o i d e u m and t o a t t e m p t t o d e f i n e a r o l e f o r t h e p u t a t i v e ATPase i n d e v e l o p m e n t a l r e g u l a t i o n . E v i d e n c e i s p r e s e n t e d f o r t h e e x i s t e n c e o f t h r e e , s e p a r a t e p l a s m a membrane A T P a s e s . I n a d d i t i o n , one o f t h e s e enzymes was p u r i f i e d and some o f i t s p r o p e r t i e s were e x p l o r e d . A p r e l i m i n a r y d e s c r i p t i o n o f t h i s work has been p u b l i s h e d ( MacDonald and Weeks, 1 9 8 4 ) . W h i l e t h i s work was i n p r o g r e s s , Pogge-von Strandmann e t a l . ( 1984) and S e r r a n o e t a l . ( 1985) p u b l i s h e d work on t h e e x i s t e n c e o f a v a n a d a t e and DES s e n s i t i v e ' A T P a s e i n p u r i f i e d p l a s m a membranes. Pogge-von Strandmann ejt a l . (1984) a l s o d e m o n s t r a t e d ATP d e p e n d e n t p r o t o n t r a n s l o c a t i o n i n r e c o n s t i t u t e d p h o s p h a t i d y l c h o l i n e v e s i c l e s . 15 MATERIALS AND METHODS I . MATERIALS M a t e r i a l s . B a c t e r i o l o g i c a l p e p t o n e and y e a s t e x t r a c t were f r o m O x o i d and B a c t o - A g a r was f r o m D i f c o . T r i s b a s e , s o d i u m o r t h o v a n a d a t e and s o d i u m a z i d e were f r o m F i s h e r S c i e n t i f i c . S u c r o s e was o f s p e c i a l enzyme g r a d e and was o b t a i n e d f r o m Schwarz-Mann. T r i t o n X-100 was f r o m Amersham. L y s o l e c i t h i n and Z-14 were f r o m C a l b i o c h e m and s o d i u m d o d e c y l s u l f a t e ( s p e c i a l l y p u r e g r a d e ) was f r o m BDH c h e m i c a l s . A c r y l a m i d e and b i s - a c r y l a m i d e , ammonium p e r s u l f a t e , g l y c i n e , TEMED and t h e s i l v e r s t a i n i n g k i t were f r o m B i o - R a d . [ 2 , 8 - 3 H ] - A T P was f r o m ICN and [ Y 3 2 P ] - A T P and L i q u i f l u o r were f r o m New E n g l a n d N u c l e a r . S e p h a c r y l S-300, D E A E - S e p h a c e l and P h e n y l - S e p h a r o s e were p u r c h a s e d as p r e -s w o l l e n b e a d s f r o m P h a r m a c i a . R e a c t i v e R e d - A g a r o s e and A T P -A g a r o s e were f r o m S i g m a . 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 f r o m F i s h e r S c i e n t i f i c o r Sigma C h e m i c a l Co. I I . METHODS A ) O r g a n i s m s and C u l t u r e C o n d i t i o n s D. d i s c o i d e u m w i l d t y p e s t r a i n V 1 2 M 2 and t h e a x e n i c mutant Ax-2, o b t a i n e d o r i g i n a l l y f r o m D r s . G. G e r i s c h and J.M. A s h w o r t h , were u s e d t h r o u g h o u t . S t r a i n V ^ M g w a s grown i n a s s o c i a t i o n w i t h E n t e r o b a c t e r  a e r o g e n e s on n u t r i e n t a g a r (Sussman, 1966) p l a t e s u n t i l c l e a r i n g o f t h e b a c t e r i a l lawn was d i s c e r n i b l e . The c e l l s 16 were h a r v e s t e d f r o m g r o w t h p l a t e s u s i n g B o n n e r ' s s a l t s ( B o n n e r , 1947) and washed f r e e o f r e s i d u a l b a c t e r i a by r e p e a t e d d i f f e r e n t i a l c e n t r i f u g a t i o n a t 700 x g . R o u t i n e l y f o u r c e n t r i f u g a t i o n s were u s e d . The a x e n i c mutant Ax-2 was grown i n HL-5 m edia (Weeks and Weeks, 1975) a t 22°C on a g y r a t o r y s h a k e r , t o a d e n s i t y 6 7 o f 5 x 10° t o 10' c e l l s / m l . The c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 700 x g and washed t w i c e w i t h B o n n e r ' s s a l t s ( B o n n e r , 1 9 4 7 ) . B) P l a s m a Membrane P r e p a r a t i o n P l a s m a membranes were p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e o f G i l k e s and Weeks ( 1 9 7 7 b ) , w i t h s e v e r a l m o d i f i c a t i o n s . H a r v e s t e d c e l l s w h i c h had been washed w i t h B o n n e r ' s s a l t s were r e s u s p e n d e d i n b u f f e r c o n t a i n i n g 10 mM T r i s - C l pH 7.5, 8.6$ (w/v) s u c r o s e , 0.1 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF) and 1 mM p - a m i n o - b e n z a m i d i n e , c e n t r i f u g e d a t 700 x g and r e s u s p e n d e d i n t h e same b u f f e r a t a d e n s i t y o f 10^ c e l l s / m l . The c e l l s were b r o k e n by m e c h a n i c a l a g i t a t i o n w i t h g l a s s beads (0.45 Q t o 0.5 mm d i a m e t e r , 3.3 g/10 c e l l s ) . U nbroken c e l l s , n u c l e i and i n t a c t m i t o c h o n d r i a were removed by c e n t r i f u g a t i o n a t 1 0,000 RPM f o r 15 m i n u t e s (10K p e l l e t ) i n an SS34 r o t o r . The s u p e r n a t a n t was c o l l e c t e d and c e n t r i f u g e d a t 100,000 x g f o r 1 h r . The r e s u l t i n g p e l l e t (40K p e l l e t ) was r e s u s p e n d e d i n b u f f e r c o n t a i n i n g 10 mM T r i s - C l pH 7.5, 20% (w/v) s u c r o s e , 0.1 mM PMSF, 1 mM p-a m i n o b e n z a m i d i n e and 0.1 mM EDTA and was c e n t r i f u g e d a t 17 100,000 x g . The p e l l e t was t h e n r e s u s p e n d e d i n 12 ml o f t h e same b u f f e r to g i v e a p r o t e i n c o n c e n t r a t i o n o f a p p r o x i m a t e l y 15 mg/ml and 2 ml was l a y e r e d o n t o e a c h o f 6 d i s c o n t i n u o u s s u c r o s e g r a d i e n t s (15 ml 44$; 10 ml 41.6$ and 10 ml 39.3$ (w/v) s u c r o s e i n 10 mM T r i s - C l pH 7.5 b u f f e r c o n t a i n i n g 0.1 mM PMSF , 0.1 mM EDTA and 1 mM p-a m i n o b e n z a m i d i n e ) . The g r a d i e n t s were c e n t r i f u g e d a t 23,000 RPM i n a Beckman SW27 r o t o r f o r 16 t o 18 h o u r s and t h e bands e n r i c h e d f o r p l a s m a membranes were c o l l e c t e d . The p l a s m a membranes were d i l u t e d w i t h 10 t o 15 volumes o f 10 mM T r i s - C l pH 7.5 and c e n t r i f u g e d a t 100,000 x g f o r 1 h o u r . The r e s u l t i n g p e l l e t was r e s u s p e n d e d i n 10 mM T r i s - C l pH 7.5,"100 mM KC1, 2 mM EDTA, c e n t r i f u g e d and washed once i n 10 mM T r i s - C l pH 7.5. F o l l o w i n g a f i n a l c e n t r i f u g a t i o n a t 100,000 x g t h e p e l l e t was r e s u s p e n d e d i n b u f f e r c o n t a i n i n g 10 mM T r i s - C l pH 7.5, 20$ ( v / v ) g l y c e r o l , 40 ^ig/ml l e u p e p t i n and s t o r e d a t -70°C . Crude membranes were p r e p a r e d i n a s i m i l a r manner. C e l l s were d i s r u p t e d w i t h g l a s s beads and t h e homogenate c e n t r i f u g e d a t 10,000 RPM i n an SS34 r o t o r . The r e s u l t i n g s u p e r n a t a n t was t h e n c e n t r i f u g e d a t 100,000 x g f o r 1 h o u r . The p e l l e t was r e s u s p e n d e d i n 100 mM KCl/2mM EDTA i n 10 mM T r i s - C l pH 7.5. F o l l o w i n g an a d d i t i o n a l c e n t r i f u g a t i o n a t 100,000 x g t h e p e l l e t was s u s p e n d e d i n 10 mM T r i s - C l pH 7.5, 20$ ( v / v ) g l y c e r o l and 40 jxg/ml l e u p e p t i n and s t o r e d a t -70°C . 1 8 F o r some e x p e r i m e n t s c r u d e membranes were o b t a i n e d a c c o r d i n g t o the p r o c e d u r e o f Das and Henderson (1983) w i t h minor m o d i f i c a t i o n s . C e l l s were h a r v e s t e d and washed as Q d e s c r i b e d above, r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 2 x 10° c e l l s / m l i n b u f f e r c o n t a i n i n g 5 mM T r i s - C l pH 8.5, 1 mM p-a m i n o b e n z a m i d i n e and 0.1 mM PMSF and l y s e d by f o r c e d p a s s a g e t h r o u g h a N u c l e o p o r e p o l y c a r b o n a t e f i l t e r (25 mm d i a m e t e r , 5 urn p ore s i z e ) p l a c e d i n a Swin-Lok H o l d e r ( N u c l e o p o r e C o r p o r a t i o n ) . The homogenate was t h e n c e n t r i f u g e d a t 5,000 x g f o r 30 m i n u t e s and the p e l l e t was r e s u s p e n d e d i n 10 mM T r i s - C l pH 7.5 and s t o r e d a t - 7 0 ° C . C) S o l u b i l i z a t i o n o f Plasma Membranes The p l a s m a membranes were r e s u s p e n d e d a t a p r o t e i n c o n c e n t r a t i o n o f 5 mg/ml i n 10 mM T r i s - C l pH 7.5, 20$ ( v / v ) g l y c e r o l . D e t e r g e n t was added from a 10$ (w/v) s t o c k s o l u t i o n t o g i v e t h e d e s i r e d f i n a l c o n c e n t r a t i o n and the s o l u t i o n was i n c u b a t e d , w i t h s t i r r i n g , a t 0°C f o r 15 m i n u t e s . The s u p e r n a t a n t from c e n t r i f u g a t i o n a t 100,000 x g was t h e s o l u b i l i z e d p r o t e i n f r a c t i o n . The r e m a i n i n g p e l l e t was r e s u s p e n d e d i n an e q u a l volume o f 10 mM T r i s - C l pH 7.5, 20$ ( v / v ) g l y c e r o l . 19 D) P r o t e i n D e t e r m i n a t i o n The F o l i n p r o c e d u r e , as m o d i f i e d by Sandermann and S t r o m i n g e r ( 1 9 7 2 ) , was u s e d t h r o u g h o u t . E) P r e p a r a t i o n o f P h o s p h a t i d y l c h o l i n e Egg y o l k (Type I I ) p h o s p h a t i d y l c h o l i n e was washed a c c o r d i n g t o t h e p r o c e d u r e o f Kogawa and R a c k e r ( 1 9 7 1 ) , w i t h s e v e r a l m o d i f i c a t i o n s . A p p r o x i m a t e l y 700 mg o f t h e p h o s p h o l i p i d was s t i r r e d a t 4°C f o r 48 h o u r s i n 100 ml a c e t o n e c o n t a i n i n g 1 mg/ml b u t y l a t e d h y d r o x y t o l u e n e . The a c e t o n e was t h e n d e c a n t e d and t h e p h o s p h o l i p i d t r a n s f e r r e d t o a 35 ml s i l i c o n i z e d g l a s s s c r e w cap t e s t t u b e and washed t w i c e w i t h 30 ml a c e t o n e . The p h o s p h o l i p i d was d i s s o l v e d i n a n h y d r o u s e t h e r and was t r a n s f e r r e d t o a t a r e d g l a s s b e a k e r . Most o f t h e e t h e r was t h e n e v a p o r a t e d o f f u n d e r a s t r e a m o f n i t r o g e n and r e s i d u a l s o l v e n t removed by s t o r i n g t h e l i p i d i n v a c u o f o r 24 h o u r s . The p h o s p h o l i p i d was r e c o n s t i t u t e d t o a c o n c e n t r a t i o n o f 50 mg/ml i n 10 mM M E S / T r i s pH 6,8 and s o n i c a t e d on i c e u n t i l c l e a r . T h i s p r e p a r a t i o n was t h e n u s e d f o r s t u d i e s on ATPase a c t i v i t y . F ) Enzyme A s s a y s The ATPase a c t i v i t y was a s s a y e d a t 30°C by t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e . U n l e s s i n d i c a t e d o t h e r w i s e i n t h e f i g u r e l e g e n d s t h e r e a c t i o n m i x t u r e s c o n t a i n e d , i n a f i n a l volume o f 1.0 m l , 3 mM ATP, 10 mM M g C l 2 a n d enzyme p r o t e i n i n 10 mM M E S / T r i s pH 6.8. A f t e r a 10 m i n u t e p r e - i n c u b a t i o n 20 r e a c t i o n s were i n i t i a t e d by t h e a d d i t i o n o f ATP and were t e r m i n a t e d a f t e r 15 t o 60 m i n u t e s by t h e a d d i t i o n o f 0.1 ml 10$ (w/v) TCA and 1$ (w/v) SDS. I n o r g a n i c p h o s p h a t e was measured as d e s c r i b e d by Ames (1966) by t h e a d d i t i o n o f 0.1 ml o f t h e r e a c t i o n m i x t u r e t o 0.2 ml 1$ (w/v) SDS and 0.7 ml o f t h e ammonium m o l y b d a t e r e a g e n t ( s i x p a r t s 0.42$ (w/v) ammonium m o l y b d a t e , 0.5$ (w/v) SDS, i n 1N H^so^ t o one p a r t 1$ (w/v) SDS, 1$ (w/v) a s c o r b i c a c i d ) . S o l u t i o n s were i n c u b a t e d a t 45°C f o r 15 m i n u t e s and t h e a b s o r b a n c e was r e a d a t 660 nm. I n some c a s e s [ 2 , 8 - 3 H ] - A T P ( s p e c i f i c a c t i v i t y 200,000 cpm/umol) was u s e d as t h e s u b s t r a t e . F o l l o w i n g t h e t e r m i n a t i o n o f t h e r e a c t i o n 0.025 ml a l i q u o t s were s p o t t e d o n t o P E I - c e l l u l o s e TLC p l a t e s ( P o l y g r a m CEL 300; M a c k e r y -N a g e l , West Germany). The p l a s t i c p l a t e s were d e v e l o p e d i n i t i a l l y w i t h a m o b i l e p h a s e o f 0.1 M L i C l t o 4 cm above t h e o r i g i n , f o l l o w e d d i r e c t l y by 1 M L i C l t o 13 cm above t h e o r i g i n . N u c l e o t i d e s p o t s were v i s u a l i z e d u n d e r UV l i g h t , e x c i s e d w i t h s c i s s o r s and t h e r a d i o a c t i v i t y d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A l k a l i n e p h o s p h a t a s e was d e t e r m i n e d by t h e h y d r o l y s i s o f p - n i t r o p h e n y l p h o s p h a t e . The i n c u b a t i o n m i x t u r e s c o n t a i n e d 1 mM p - n i t r o p h e n y l p h o s p h a t e (pNPP), 20 mM M g C l 2 f 30 mM NaF , 20 mM T r i s - C l pH 8.5 and enzyme i n a f i n a l volume o f 1 m l . R e a c t i o n s were c a r r i e d o u t a t 30°C and were t e r m i n a t e d by t h e a d d i t i o n o f 1.0 ml o f 1 M N a ^ O ^ . Enzyme a c t i v i t y was measured s p e c t r o p h o t o m e t r i c a l l y a t 410 nm and 21 an e x t i n c t i o n c o e f f i c i e n t o f E ^ = 1.62 x 10 was used t o c a l c u l a t e the q u a n t i t y o f p - n i t r o p h e n o l f o r m e d . S u c c i n a t e d e h y d r o g e n a s e was a s s a y e d as d e s c r i b e d by G i l k e s and Weeks (1977a) i n a m i x t u r e c o n t a i n i n g 2 mM KCN, 10 mM EDTA,0.1 mM d i c h l o r o p h e n o l - i n d o p h e n o l ( D C P I P ) , 0.2$ ( v / v ) T r i t o n X-100, 1 mg/ml p h e n a z i n e m e t h o s u l f a t e , 12.5 mM p h o s p h a t e b u f f e r pH 7.6, 20 mM sodium s u c c i n a t e and membrane p r o t e i n (0.1 t o 0.2 mg) i n a f i n a l volume o f 3.0 ml. Enzyme a c t i v i t y was measured by the d e c r e a s e i n a b s o r b a n c e a t 600 nm and the amount o f DCPIP r e d u c e d was c a l c u l a t e d u s i n g t h e e x t i n c t i o n c o e f f i c i e n t = 2.1 x 10^. G) 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 Sodium 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 was p e r f o r m e d by the p r o c e d u r e o f Laemmli ( 1 9 7 0 ) . The s e p a r a t i o n g e l c o n t a i n e d 7$ a c r y l a m i d e , 0.1$ SDS i n 0.15 M T r i s - C l pH 8.8 and t h e s t a c k i n g g e l c o n t a i n e d 1$ a c r y l a m i d e , 0.1$ SDS i n 0.083 M T r i s - C l pH 6.8. 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 0.2 M g l y c i n e , 0.083 M T r i s - C l and 0.1$ SDS and t h e sample b u f f e r c o n t a i n e d g l y c e r o l , 0.083 M T r i s - C l pH 6.8 and 2$ (w/v) SDS. The g e l s were s u b j e c t e d t o 5 mA f o r 1 hour and t h e n 30 mA u n t i l t h e b r o m o p h e n o l b l u e t r a c k i n g dye had r e a c h e d the bottom o f the g e l . The g e l s were f i x e d o v e r n i g h t i n m e t h a n o l : g l a c i a l a c e t i c a c i d r d e -i o n i z e d w a ter (0.8:0.2:1.0, v / v / v ) and s i l v e r s t a i n e d the f o l l o w i n g day w i t h t h e BioRad s i l v e r s t a i n k i t ( B i o R a d T e c h n i c a l B u l l e t i n # 1089), e x c e p t t h a t the o x i d i z i n g s t e p 22 was r e p l a c e d by t r e a t m e n t w i t h 3 mg/ml d i t h i o t h r e i t o l as d e s c r i b e d by M o r r i s s e y ( 1 9 8 1 ) . H) P u r i f i c a t i o n o f ATPase A c t i v i t y A l l p r o c e d u r e s were p e r f o r m e d a t 4°C. i ) S e p h a c r y l S-300 g e l f i l t r a t i o n . S e p h a c r y l S-300 was packed i n t o a 2 x 80 cm column and e q u i l i b r a t e d w i t h 25 mM T r i s - C l pH 7.5 c o n t a i n i n g 1$ BRIJ-35 or 0.5$ CHAPS and 0.3 M N a C l . No more t h a n 4 ml o f the s o l u b i l i z e d membrane p r e p a r a t i o n ( a p p r o x i m a t e l y 20 mg p r o t e i n ) was a p p l i e d t o t h e column and f r a c t i o n s o f 2.5 ml were c o l l e c t e d and m o n i t o r e d f o r p r o t e i n by a b s o r b a n c e a t 280 nm and ATPase a c t i v i t y . F r a c t i o n s e x p r e s s i n g enzyme a c t i v i t y were p o o l e d and d i a l y z e d f o r 12 h o u r s a g a i n s t a t l e a s t 100 volumes o f 10 mM T r i s - C l pH 7.5 c o n t a i n i n g 10$ ( v / v ) g l y c e r o l and 0.1$ CHAPS. The b u f f e r was g e n e r a l l y changed once d u r i n g t h e d i a l y s i s p r o c e d u r e . i i ) Ion exchange c h r o m a t o g r a p h y on D E A E - S e p h a c e l . A p p r o x i m a t e l y 5 ml o f D E A E - S e p h a c e l was p o u r e d i n t o a 1 x 10 cm column and e q u i l i b r a t e d w i t h 100 mis o f 10 mM T r i s - C l pH 7.5 c o n t a i n i n g 0.5$ BRIJ o r CHAPS. Enzyme p r e p a r a t i o n s were l o a d e d o n t o the column and, a f t e r t h e unbound p r o t e i n was removed by w ashing w i t h 75 ml o f e q u i l i b r a t i o n b u f f e r , bound p r o t e i n was e l u t e d by s u c c e s s i v e l y w a s h i n g the column w i t h 0.1, 0.3 and 0.5 M N a C l i n e q u i l i b r a t i o n b u f f e r . F r a c t i o n s o f 1.5 ml were c o l l e c t e d and m o n i t o r e d f o r p r o t e i n and ATPase a c t i v i t y as d e s c r i b e d b e f o r e . ATPase c o n t a i n i n g f r a c t i o n s were p o o l e d and 23 d i a l y z e d a g a i n s t 100 volumes o f T r i s - C l pH 7.5 c o n t a i n i n g 10$ ( v / v ) g l y c e r o l and 0.1$ CHAPS. i i i ) G l y c e r o l 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 . A p p r o x i m a t e l y 0.5 ml o f t h e ATPase p r e p a r a t i o n was l a y e r e d o n t o each o f s i x 9 ml l i n e a r g l y c e r o l g r a d i e n t s (15 t o 35$ ( v / v ) g l y c e r o l , 0.25$ (w/v) CHAPS i n 10 mM T r i s - C l pH 7.5) and c e n t r i f u g e d a t 120,000 x g f o r 24 t o 36 h o u r s i n an SW41 s w i n g i n g b u c k e t r o t o r . F o l l o w i n g t h i s , the g r a d i e n t s were d r i p p e d from th e bottom and 20 d r o p f r a c t i o n s were c o l l e c t e d and a s s a y e d f o r enzyme a c t i v i t y . P r o t e i n was e s t i m a t e d by i t s a b s o r b a n c e a t 280 nm, f o l l o w i n g the r e m o v a l o f g l y c e r o l by o v e r n i g h t d i a l y s i s a g a i n s t 10 mM T r i s - C l pH 7.5. i v ) P h e n y l - S e p h a r o s e , R e a c t i v e Red-Agarose and ATP-A g a r o s e c h r o m a t o g r a p h y . P h e n y l - S e p h a r o s e (5 ml) was p o u r e d i n t o a 1 x 10 cm column and e q u i l i b r a t e d w i t h 100 mis o f 25 mM T r i s - C l pH 7.5 c o n t a i n i n g 0.5 M N a C l . Enzyme p r e p a r a t i o n s were l o a d e d o n t o the column and e l u t e d f i r s t w i t h e q u i l i b r a t i o n b u f f e r f o l l o w e d by 10 mM T r i s - C l pH 7.5 c o n t a i n i n g 10$ ( v / v ) g l y c e r o l and f i n a l l y by a l i n e a r g r a d i e n t o f 0 t o 1$ BRIJ-35 i n 10 mM T r i s - C l pH 7.5. R e a c t i v e Red-Agarose was s w o l l e n a t room t e m p e r a t u r e (1 g d r y beads/5 ml g e l ) i n 10 mM T r i s - C l pH 7.5 f o r 4 h o u r s . The g e l was t h e n p o u r e d i n t o a 0.5 x 5 cm column and washed w i t h 0.5 M NaCl as d e s c r i b e d by C o l l and Murphy (1984) b e f o r e b e i n g e q u i l i b r a t e d w i t h 10 mM T r i s - C l pH 7.5 c o n t a i n i n g 10$ ( v / v ) g l y c e r o l and 0.5$ (w/v) CHAPS. The ATPase p r e p a r a t i o n s were a p p l i e d t o t h e column and e l u t e d 24 f i r s t w i t h e q u i l i b r a t i o n b u f f e r f o l l o w e d by e q u i l i b r a t i o n b u f f e r c o n t a i n i n g 0.4 mM a d e n y l 5 ' - y l i m i d o d i p h o s p h a t e (AMP-PNP ) . P r e - s w o l l e n A T P - A g a r o s e was p a c k e d i n t o a 0.5 x 5 cm colu m n and e q u i l i b r a t e d w i t h t h e same b u f f e r as t h e r e a c t i v e r e d c olumn e x c e p t t h a t i t c o n t a i n e d 1 mM M g C l 2 . P r o t e i n was e l u t e d w i t h t h e e q u i l i b r a t i o n b u f f e r f o l l o w e d by e q u i l i b r a t i o n b u f f e r c o n t a i n i n g 1 mM ATP. I ) 3 2 P l a b e l i n g e x p e r i m e n t s Enzyme p r e p a r a t i o n s were i n c u b a t e d i n a r e a c t i o n m i x t u r e c o n t a i n i n g 0.01 t o 0.02 ml p l a s m a membrane p r o t e i n , 10 mM M g C l 2 > 1 0 mM M E S / T r i s pH 6.8 and v a r i o u s c o n c e n t r a t i o n s o f [y - 3 2 P ] - A T P ( s e e a p p r o p r i a t e f i g u r e l e g e n d s f o r d e t a i l s ) i n a f i n a l volume o f 0.05 m l . The r e a c t i o n s were i n i t i a t e d by t h e a d d i t i o n o f ATP and were t e r m i n a t e d by t h e a d d i t i o n o f 0.05 ml o f e l e c t r o p h o r e s i s s ample b u f f e r . The s a m p l e s were t h e n s u b j e c t e d t o e l e c t r o p h o r e s i s a c c o r d i n g t o t h e p r o c e d u r e o f Laemmli (1970) a t 4°C . The g e l s were t h e n s o a k e d o v e r n i g h t i n 0.4 1 o f m e t h a n o l : g l a c i a l a c e t i c a c i d : d e - i o n i z e d w a t e r ( 0 . 8 : 0 . 2 : 1 ) , s i l v e r s t a i n e d , d r i e d and p l a c e d a g a i n s t r a d i o g r a p h i c X - r a y f i l m f o r 24 t o 172 h o u r s b e f o r e b e i n g d e v e l o p e d . 25 RESULTS SECTION I . IDENTIFICATION OF ATPASE ACTIVITY IN PURIFIED D. DISCOIDEUM PLASMA MEMBRANES a) Plasma Membrane I s o l a t i o n The i n i t i a l s t e p i n t h i s work was t o s e l e c t s p e c i f i c marker enzymes f o r the m i t o c h o n d r i a l and p l a s m a membranes. A l k a l i n e p h o s p h a t a s e was s e l e c t e d as t h e c e l l s u r f a c e marker s i n c e i t had been used e x t e n s i v e l y i n p r e v i o u s work on D. d i s c o i d e u m plasma membranes (Green and N e w e l l , 1974; Rossomando and C u t l e r , 1975; G i l k e s and Weeks, 1977a, 1977b). Recent c y t o c h e m i c a l work has c o n f i r m e d t h e p l a s m a membrane l o c a l i z a t i o n o f t h i s enzyme (Glomp e t a l . , 1 9 85). S u c c i n a t e d e h y d r o g e n a s e was s e l e c t e d as t h e m i t o c h o n d r i a l membrane marker. In o r d e r t o e s t a b l i s h t h e e x i s t e n c e o f a p l a s m a membrane ATPase a c t i v i t y i t was o f c r u c i a l i m p o r t a n c e f o r p lasma membrane p r e p a r a t i o n s t o be f r e e o f m i t o c h o n d r i a l ATPase c o n t a m i n a t i o n . I n i t i a l l y a number o f s i m p l e and r a p i d t e c h n i q u e s , s u c h as d i f f e r e n t i a l c e n t r i f u g a t i o n and t h e two phase polymer p a r t i t i o n i n g s y s t e m o f B r u n e t t e and T i l l ( 1 9 7 1 ) , were u s e d , but t h e s e t e c h n i q u e s d i d n o t g i v e s a t i s f a c t o r y r e s u l t s and were abandoned ( d a t a n o t shown). A somewhat time consuming method, d e v i s e d e a r l i e r i n t h i s l a b o r a t o r y ( G i l k e s and Weeks, 1977a. 1977b) was u s e d and 26 y i e l d e d h i g h l y e n r i c h e d p l a s m a membranes as r e v e a l e d by t h e d i s t r i b u t i o n o f the marker enzymes ( T a b l e I ) . M o r e o v e r , t h e s e membranes e x p r e s s e d a h i g h l e v e l o f ATPase a c t i v i t y ( T a b l e I ) , s u g g e s t i n g t h a t t h e r e was a s p e c i f i c p lasma membrane ATPase. b) C h a r a c t e r i z a t i o n o f the Plasma Membrane ATPase The plasma membrane ATPase a c t i v i t y was f o u n d t o be r e s i s t a n t t o t h e m i t o c h o n d r i a l ATPase i n h i b i t o r s a z i d e and o l i g o m y c i n ( F i g . 2 ) , whereas the ATPase i n t h e m i t o c h o n d r i a l f r a c t i o n t h a t p e l l e t e d a t 10,000 RPM was s e n s i t i v e . These d a t a p r o v i d e d f u r t h e r e v i d e n c e t h a t t h e p l a s m a membrane a c t i v i t y was d i s t i n c t from th e m i t o c h o n d r i a l enzyme. P r e v i o u s r e p o r t s had s u g g e s t e d t h e p r e s e n c e o f an A T P : p y r o p h o s p h o h y d r o l a s e i n D. d i s c o i d e u m p l a s m a membranes (Rossomando and Sussman, 1973; Rossomando and Hodge-Jahngen, 1983), n e c e s s i t a t i n g an a n a l y s i s o f t h e r e a c t i o n p r o d u c t s p r o d u c e d under the p r e s e n t ATPase a s s a y c o n d i t i o n s . When plasma membranes were i n c u b a t e d w i t h [ 3H]-ATP and t h e p r o d u c t s s e p a r a t e d by P E I - c e l l u l o s e TLC i t was f o u n d t h a t ADP c o n s t i t u t e d t h e m a j o r i t y o f t h e h y d r o l y z e d r a d i o a c t i v i t y ( T a b l e I I ) . Even a f t e r a 60 m i n u t e i n c u b a t i o n , AMP a c c o u n t e d f o r o n l y s l i g h t l y more t h a n 10$ o f t h e t o t a l p r o d u c t . T h i s r e s u l t i n d i c a t e d t h a t t h e a c t i v i t y was a t t r i b u t a b l e t o a c l a s s i c a l ATPase i n t h a t ATP was h y d r o l y z e d t o ADP and P.. 27 T A B L E I DISTRIBUTION OF ENZYME MARKERS DURING PLASMA MEMBRANE PURIFICATION Activity (nmol min - 1 mg protein l) Succinate Alkaline Fraction dehydrogenase phosphatase Mg+2-ATPase Homogenate 22.5 6.4 75.2 10K pellet 34.0 5.0 92.0 40K pellet 4.7 17.2 41.6 Plasma membranes 1.1 65.0 160.0 2 8 F i g u r e 2. E f f e c t o f A z i d e and O l i g o m y c i n on P lasma Membrane and M i t o c h o n d r i a l ATPase A c t i v i t y . Plasma membranes ( • ) o r 10K p e l l e t f r a c t i o n s ( O ) were p r e i n c u b a t e d a t 30°C f o r 10 min. i n a m i x t u r e c o n t a i n i n g 30 mM i m i d a z o l e - C l pH 7.5, 3 mM MgCl and the i n d i c a t e d amounts o f o l i g o m y c i n (A) and a z i a e ( B ) . R e a c t i o n s were i n i t i a t e d by the a d d i t i o n o f 3 mM ATP. The f i n a l volume o f the r e a c t i o n m i x t u r e was 1.0 ml. The d a t a r e p r e s e n t s s i n g l e e x p e r i m e n t s , a l t h o u g h s i m i l a r r e s u l t s were o b t a i n e d upon r e p e t i t i o n . The a d d i t i o n a l e x p e r i m e n t s a r e n o t i n c l u d e d as t h e s p e c i f i c a c t i v i t i e s v a r y between p r e p a r a t i o n s . One u n i t o f ATPase a c t i v i t y r e p r e s e n t s 1 nmol Pi/min/mg p r o t e i n . 29 TABLE II D I S T R I B U T I O N O F H Y D R O L Y Z E D P R O D U C T S R E S U L T I N G F R O M I N C U B A T I O N O F P L A S M A M E M B R A N E S W I T H [ 3H]ATP Time of Percentage total radioactivity incubation (min) ADP AMP Adenosine 5 2.2 0 0 10 6 0.1 0 15 8.7 0.3 0 30 13.5 1.0 0 45 14.6 1.5 0 60 18.2 2.2 0 R e a c t i o n s o l u t i o n s c o n t a i n e d , i n a d d i t i o n t o membrane p r o t e i n , 3 mM M g C l 2 , 30 mM i m i d a z o l e - C l pH 7.5 and 3 mM (2,8- 3H)Na2ATP (200,000 cpm/^imol). The f i n a l volume was 0.2 ml. 30 H y d r o l y s i s o f ATP was s t i m u l a t e d by m i l l i m o l a r c o n c e n t r a t i o n s o f M g + 2 and t o a l e s s e r e x t e n t by C a + 2 ( F i g . 3 ) . However, when b o t h o f t h e s e c a t i o n s were p r e s e n t a t h i g h c o n c e n t r a t i o n s , enzyme a c t i v i t y was s u b s t a n t i a l l y i n h i b i t e d ( F i g . 4 ) . N e i t h e r o u a b a i n ( T a b l e I I I ) n o r e q u i m o l a r c o n c e n t r a t i o n s o f N a + and K + ( F i g . 4) had any e f f e c t on a c t i v i t y i n d i c a t i n g t h a t t h e enzyme was n o t a N a + -K + - A T P a s e . F i v e mM N a + and K + w i l l r e s u l t i n f u l l a c t i v a t i o n o f t h e N a + , K + - A T P a s e ( K y t e , 1971,1974; D i x o n and H o k i n , 1974; J o r g e n s e n , 1 9 7 4 ) . The p l a s m a membrane ATPase a c t i v i t y was p a r t i a l l y i n h i b i t e d by DES, DCCD , t h i m e r o s a l and v a n a d a t e ( F i g . 5) and a l s o by N - e t h y l m a l e i m i d e (NEM) ( F i g . 6 ) . The p l a s m a membrane ATPase was a s s a y e d f o r i t s a b i l i t y t o h y d r o l y z e o t h e r n u c l e o t i d e s . T a b l e I V shows t h a t a l l t h e n u c l e o s i d e t r i p h o s p h a t e s e x c e p t UTP were u t i l i z e d t o a c e r t a i n e x t e n t a l t h o u g h ATP was h y d r o l y z e d most r a p i d l y . N e i t h e r AMP n o r ADP were d e g r a d e d t o any s i g n i f i c a n t e x t e n t ( T a b l e I V ) . Be c a u s e t h e p l a s m a membrane bound enzyme was r e s i s t a n t t o a z i d e and o l i g o m y c i n i t was p o s s i b l e t o a s s a y s e l e c t i v e l y f o r i t s a c t i v i t y i n c r u d e membrane p r e p a r a t i o n s . The r e s u l t s o f an e x p e r i m e n t where t h e o l i g o m y c i n r e s i s t a n t a c t i v i t y o f c r u d e t o t a l membranes o r a 100,000 x g p e l l e t o f a 10,000 RPM s u p e r n a t a n t was a s s a y e d a t v a r i o u s s t a g e s o f d e v e l o p m e n t i s i l l u s t r a t e d i n F i g . 1. The s p e c i f i c a c t i v i t y d e c r e a s e d as t h e c e l l s p r o c e e d e d t h r o u g h a g g r e g a t i o n and 31 c '5 o a O) c I o £ c >» *> o < o o 0) Q. to Cat ions (-log M ) F i g u r e 3. S t i m u l a t i o n o f Plasma Membrane ATPase A c t i v i t y by M g + 2 and C a + 2 . Plasma membranes were i n c u b a t e d . i n a m i x t u r e c o n t a i n i n g 30 mM i m i d a z o l e - C l pH 7 . 5 and the i n d i c a t e d c o n c e n t r a t i o n s o f M g C l 2 ( • ) o r C a C l 2 (0). R e a c t i o n s were i n i t i a t e d by the a d d i t i o n o f 3 mM Na_ATP and were a l l o w e d t o p r o c e e d f o r 15 m i n u t e s . I n c u b a t i o n volumes were 1 . 0 ml. The e x p e r i m e n t s was r e p e a t e d s e v e r a l t i m e s w i t h s i m i l a r r e s u l t s . S i n c e t h e i n i t i a l s p e c i f i c a c t i v i t y was v a r i a b l e from p r e p a r a t i o n t o p r e p a r a t i o n the a d d i t i o n a l e x p e r i m e n t s a r e n o t shown. 32 C A T I O N S (-log M) F i g u r e 4. The E f f e c t o f V a r i o u s C a t i o n s on Plasma Membrane ATPase A c t i v i t y . Plasma membranes were i n c u b a t e d a t 30°C i n 30 mM i m i d a z o l e - C l pH 7.5 w i t h t h e i n d i c a t e d c o n c e n t r a t i o n s o f c h o l i n e - C l ( A ) , C a C l ( O ) o r an e q u i m o l a r m i x t u r e o f N a C l and KC1 (• ). R e a c t i o n s were i n i t i a t e d by t h e a d d i t i o n o f 3 mM Mg-ATP. The f i n a l r e a c t i o n volume was 1.0 m l . The d a t a r e p r e s e n t s s i n g l e e x p e r i m e n t s , b ut s i m i l a r r e s u l t s were o b t a i n e d upon r e p e t i t i o n . F o r t h e sake o f c l a r i t y t h e a d d i t i o n a l e x p e r i m e n t s a r e n o t shown. One u n i t o f ATPase a c t i v i t y e q u a l s 1 nmol Pi/min/mg p r o t e i n . 3 3 TABLE I I I EFFECT OF OUABAIN ON PLASMA MEMBRANE ATPASE ACTIVITY Ouabain (mM) 1 .0 0 .1 0.01 0.001 no a d d i t i o n S p e c i f i c A c t i v i t y 3 (nmol POjj/min/mg p r o t e i n ) 56.4 + 2.7 55.0 + 1.8 55.0 + 2.6 57.5 + 1.1 59.2 + 2.1 The numbers a r e t h e means o f 3 e x p e r i m e n t s + the s t a n d a r d d e v i a t i o n . A s s a y m i x t u r e s c o n t a i n e d , i n a d d i t i o n t o enzyme p r o t e i n , 3 mM M g C l 2 > 30 mM i m i d a z o l e - C l pH 7.5, 3 mM Na pATP and t h e i n d i c a t e d amounts o f o u a b a i n . The f i n a l volume was 1 ml. 34 Hf . r 6 5 Inhibitor (-log M ) F i g u r e 5. The E f f e c t s o f V a r i o u s I n h i b i t o r s on Plasma Membrane ATPase A c t i v i t y . Plasma membrane ATPase a c t i v i t y was d e t e r m i n e d i n a m i x t u r e c o n t a i n i n g 30 mM i m i d a z o l e - C l pH 7.5, 3 mM MgCl and 3mM Na-ATP and t h e i n d i c a t e d amounts o f the i n h i b i t o r s DES ( C T ) , DCCD ( A ) , t h i m e r o s a l ( # ) o r sodium o r t h o v a n a d a t e ( A ) . The f i n a l r e a c t i o n volume was 1.0 m l . R e a c t i o n s were t e r m i n a t e d a f t e r 15 m i n u t e s and 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 as d e s c r i b e d i n Methods. Each c u r v e r e p r e s e n t s the a v e r a g e o f f o u r s e p a r a t e e x p e r i m e n t s +_ the s t a n d a r d d e v i a t i o n . 35 10 20 3 0 4 0 Time (minsj F i g u r e 6. I n h i b i t i o n o f Plasma Membrane ATPase A c t i v i t y by N - e t h y l m a l e i m i d e . Plasma membranes were i n c u b a t e d a t 0°C i n 0.1 ml 30 mM i m i d a z o l e - C l pH 7.5 and 5 mM N - e t h y l m a l e i m i d e . At the i n d i c a t e d t i m e s 0.9 ml o f the ATPase a s s a y m i x t u r e was added t o g i v e f i n a l c o n c e n t r a t i o n s o f 3 mM ATP, 10 mM M g C l 2 and 30 mM i m i d a z o l e - C l pH 7.0 and i n c u b a t e d f o r a f u r t h e r 15 m i n u t e s a t 30 C. T h i s e x p e r i m e n t was p e r f o r m e d o n l y o n c e . 36 T A B L E I V U T I L I Z A T I O N O F O T H E R N U C L E O T I D E S B Y T H E P L A S M A M E M B R A N E O F Mg+ 2-ATPase Specific activity Substrate (nmol P0 4 min - 1 mg protein-1) AMP 1 ADP 1 UTP 1 CTP 6.1 GTP 7.0 ATP 21.2 R e a c t i o n s o l u t i o n s c o n t a i n e d , i n a d d i t i o n t o membrane p r o t e i n , 3 mM MgCl2, 30 mM i m i d a z o l e - C l pH 7.5 and 3 mM n u c l e o t i d e . The f i n a l v o l -ume was 1 ml. 37 _1 1 — J L_ 4 8 12 16 TIME (hrs) F i g u r e 7. D e v e l o p m e n t a l Changes i n Plasma Membrane ATPase A c t i v i t y . C e l l s were h a r v e s t e d and washed as d e s c r i b e d i n Methods and 2 x 10° c e l l s were p l a t e d o n t o e a c h o f 12 n o n - n u t r i e n t a g a r p l a t e s (2% a g a r i n Bonners S a l t s ) . The c e l l s were h a r v e s t e d a t the i n d i c a t e d t i m e s and d i s r u p t e d w i t h g l a s s beads as d e s c r i b e d i n methods. Homogenates were t h e n d i v i d e d i n h a l f and a t o t a l membrane f r a c t i o n ( • ) was o b t a i n e d from one h a l f by c e n t r i f u g a t i o n a t 100,000 x g. The o t h e r h a l f was c e n t r i f u g e d a t 10,000 RPM and t h e s u p e r n a t a n t was d e c a n t e d and r e c e n t r i f u g e d a t 100,000 x g t o g i v e a 40 K p e l l e t f r a c t i o n ( O ) . ATPase a c t i v i t y was measured i n a r e a c t i o n m i x t u r e c o n t a i n i n g 30 mM i m i d a z o l e - C l (pH 7 . 5 ) , 3 mM M g C l 2 , 10 jig/ml o l i g o m y c i n and 3 mM Na 2ATP. The f i n a l volume was 1.0 ml. 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 as d e s c r i b e d i n methods. Both the t o t a l membrane and 40 K p e l l e t s were r e s u s p e n d e d i n 5 mM T r i s - C l pH 7.5 f o l l o w i n g c e n t r i f u g a t i o n . T h i s e x p e r i m e n t was p e r f o r m e d o n l y o n c e . 38 t h e n r e m a i n e d c o n s t a n t t h r o u g h p s e u d o p l a s m o d i u m f o r m a t i o n and m i g r a t i o n ( F i g . 7 ) . c ) S o l u b i l i z a t i o n o f t h e P l a s m a Membrane ATPase A c t i v i t y A l l o f t h e p r e v i o u s l y d e s c r i b e d r e s u l t s on t h e p l a s m a membrane ATPase o f D. d i s c o i d e u m were o b t a i n e d u s i n g t h e w i l d t y p e s t r a i n V<\2M2. However, i n i t i a l r e s u l t s i n d i c a t e d t h a t t h e ATPase i n ^^2H2 a n d t h e a x e n i c mutant Ax-2 had s i m i l a r p r o p e r t i e s ( d a t a n o t shown, Pogge von Strandmann e_t a l . , 1983) and i n v i e w o f t h e e a s e w i t h w h i c h l a r g e amounts o f Ax-2 c o u l d be o b t a i n e d t h i s s t r a i n was u s e d f o r s u b s e q u e n t s t u d i e s . I t was f o u n d t h a t t h e s p e c i f i c a c t i v i t y o f t h e p l a s m a membrane bound ATPase a c t i v i t y i n c r e a s e d upon w a s h i n g t h e membranes w i t h a m i x t u r e o f 100 mM KC1 and 2 mM EDTA ( T a b l e V ) . F o r t h i s r e a s o n t h e membranes were r o u t i n e l y t r e a t e d w i t h KC1 and EDTA, p r i o r t o s o l u b i l i z a t i o n . I n o r d e r t o e s t a b l i s h t h e optimum c o n d i t i o n s f o r t h e s o l u b i l i z a t i o n o f t h e p l a s m a membrane ATPase a v a r i e t y o f d e t e r g e n t s were t e s t e d . The b e s t d e t e r g e n t was l y s o l e c i t h i n ( F i g . 8 ) . T h i s d e t e r g e n t s t i m u l a t e d t o t a l enzyme a c t i v i t y a t a l l c o n c e n t r a t i o n s t e s t e d ( F i g . 8a) and a t t h e h i g h e s t c o n c e n t r a t i o n , s o l u b i l i z e d most o f t h e a c t i v i t y ( F i g . 8 b ) . The h i g h e s t s p e c i f i c a c t i v i t y o f t h e s o l u b i l i z e d ATPase was, however, o b t a i n e d a t low d e t e r g e n t c o n c e n t r a t i o n , c o n c o m i t a n t w i t h low t o t a l s o l u b i l i z a t i o n ( F i g . 8 c ) . 39 TABLE V EFFECT OF 100 mM KCL/2 mM EDTA ON PLASMA MEMBRANE ATPASE ACTIVITY F r a c t i o n ATPase A c t i v i t y (nmol Pi/min/mg p r o t e i n ) P l a s ma membranes 18.5 Washed Plasma 33.5 membranes 40 F i g u r e 8. S o l u b i l i z a t i o n o f Plasma Membrane ATPase A c t i v i t y w i t h L y s o l e c i t h i n . Plasma membranes were t r e a t e d w i t h t h e i n d i c a t e d c o n c e n t r a t i o n s o f l y s o l e c i t h i n and c e n t r i f u g e d a t 1 0 0 , 0 0 0 x g as d e s c r i b e d i n Methods. F i g u r e A r e p r e s e n t s the t o t a l a d d i t i v e a c t i v i t y i n p e l l e t s and s u p e r n a t a n t s , F i g . B the s o l u b i l i z e d a c t i v i t y as a p e r c e n t o f the t o t a l a c t i v i t y and F i g . C the s p e c i f i c a c t i v i t y o f t h e s o l u b i l i z e d ATPase, a t e a c h d e t e r g e n t c o n c e n t r a t i o n . 4i 4 2 The d e t e r g e n t s CHAPS, o c t y l g l u c o s i d e , c 1 2 E g and T r i t o n X-100 were o n l y s t i m u l a t o r y a t low c o n c e n t r a t i o n s , h i g h e r c o n c e n t r a t i o n s b e i n g i n h i b i t o r y ( F i g . 9 a ) , r e s u l t i n g i n t h e maximum s o l u b i l i z a t i o n o f o n l y 5 0 " t o 65$ o f t h e i n i t i a l a c t i v i t y ( F i g . 9 b ) . The h i g h e s t s p e c i f i c a c t i v i t i e s o f t h e s o l u b i l i z e d m a t e r i a l was a g a i n o b t a i n e d a t low d e t e r g e n t c o n c e n t r a t i o n s ( F i g . 9 c ) . D e o x y c h o l a t e and Z-14 gave p o o r e r e x t r a c t i o n o f ATPase a c t i v i t y , o s t e n s i b l y due t o t h e even g r e a t e r i n h i b i t o r y e f f e c t s o f t h e s e d e t e r g e n t s , r a t h e r t h a n t o i n s u f f i c i e n t e x t r a c t i o n o f membrane p r o t e i n ( F i g . 9 a and b ) . I n t e r e s t i n g l y , t h e a d d i t i o n o f exogenous p h o s p h o l i p i d r e v e r s e d t h e i n h i b i t o r y e f f e c t o f d e o x y c h o l a t e but no s u c h phenomenon was o b s e r v e d f o r Z-14 ( F i g . 1 0 ) . d) E v i d e n c e f o r a L a b i l e DES S e n s i t i v e Plasma Membrane ATPase A c o m p a r i s o n o f the membrane bound and o c t y l g l u c o s i d e s o l u b i l i z e d a c t i v i t i e s r e v e a l e d a l o s s o f DES s e n s i t i v i t y upon e x t r a c t i o n ( F i g . 1 1 ) . T h i s f i n d i n g , i n c o n j u n c t i o n w i t h t h e f a c t t h a t a c e r t a i n amount o f ATPase a c t i v i t y was r o u t i n e l y l o s t upon d e t e r g e n t t r e a t m e n t ( F i g . 9a) s u g g e s t e d e i t h e r t h e e x i s t e n c e o f a s p e c i f i c l a b i l e DES s e n s i t i v e plasma membrane ATPase o r t h e l o s s , d u r i n g s o l u b i l i z a t i o n , o f a c o f a c t o r w h i c h c o n f e r r e d DES s e n s i t i v i t y on t h e enzyme. E v i d e n c e s u p p o r t i n g the f o r m e r h y p o t h e s i s i s shown i n F i g u r e 12. Plasma membranes were s o l u b i l i z e d w i t h v a r i o u s 43 F i g u r e 9. D e t e r g e n t S o l u b i l i z a t i o n o f Plasma Membrane ATPase A c t i v i t y . Plasma membranes were s o l u b i l i z e d by i n c u b a t i o n w i t h the i n d i c a t e d c o n c e n t r a t i o n s o f o c t y l g l u c o s i d e ( A ) . CHAPS ( • ) , C E ( A ) , Z-14 ( • ) , d e o x y c h o l a t e ( O ) and T r i t o n X-100 T L T ) . ATPase a c t i v i t y was d e t e r m i n e d i n b o t h p e l l e t s and s u p e r n a t a n t s f o l l o w i n g c e n t r i f u g a t i o n a t 100,000 x g as d e s c r i b e d i n Methods. The f i g u r e d e s i g n a t i o n s a r e i d e n t i c a l t o t h o s e i n F i g . 8. 44 0-1 0-5 1 0 2 0 0-1 0-5 10 2 0 Detergent •/. (w/v) Detergent •/. (w/v) o - r - 1 — i 1— 0-1 0-5 10 2 0 DetergentV.(w/v) 45 1 5 10 15 20 25 Phosphatidylcholine (/*g/ml) F i g u r e 1 0 . E f f e c t o f E x o g e n o u s P h o s p h a t i d y l c h o l i n e o n D e o x y c h o l a t e a n d Z - 1 4 E x t r a c t e d A T P a s e . P l a s m a m e m b r a n e A T P a s e a c t i v i t y w a s s o l u b i l i z e d w i t h 1$ d e o x y c h o l a t e ( • ) a n d Z - 1 4 ( A ) a n d t h e 1 0 0 , 0 0 0 x g s u p e r n a t a n t s w e r e a s s a y e d f o r 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 t h e i n d i c a t e d c o n c e n t r a t i o n s o f p h o s p h a t i d y l c h o l i n e a s d e s c r i b e d i n M e t h o d s . T h e r e s u l t s a r e r e p r e s e n t a t i v e o f a s i n g l e e x p e r i m e n t w h i c h w a s p e r f o r m e d t w i c e w i t h s i m i l a r r e s u l t s . 46 20 H-l 1 . 1 . 1 1 7 6 5 4 3 DES (-log M) F i g u r e 1 1 . E f f e c t o f DES on t h e Plasma Membrane Bound and S o l u b i l i z e d ATPase A c t i v i t y . ATPase a c t i v i t y was d e t e r m i n e d i n p l a s m a membranes ( • ) and the 1 0 0 , 0 0 0 x g s u p e r n a t a n t f o l l o w i n g s o l u b i l i z a t i o n w i t h \% o c t y l g l u c o s i d e ( • ) i n t h e p r e s e n c e o f the i n d i c a t e d c o n c e n t r a t i o n s o f DES. The r e s u l t s r e p r e s e n t the a v e r a g e o f f o u r s e p a r a t e e x p e r i m e n t s + the s t a n d a r d d e v i a t i o n o f the mean. 47 F i g u r e 12. I n a c t i v a t i o n o f t h e DES S e n s i t i v e A T P a s e U p o n S o l u b i l i z a t i o n w i t h S e v e r a l D e t e r g e n t s . P l a s m a m e m b r a n e s w e r e s o l u b i l i z e d w i t h v a r i o u s d e t e r g e n t s a s d e s c r i b e d i n M e t h o d s a n d t h e A T P a s e a c t i v i t y w a s d e t e r m i n e d i n t h e p r e s e n c e a n d a b s e n c e o f 0.2 mM D E S . T h e DES i n s e n s i t i v e a c t i v i t y ( • ) i s g i v e n a s t h e f o r m e r v a l u e , w h i l e t h e DES s e n s i t i v e a c t i v i t y ( A ) i s t h e d i f f e r e n c e b e t w e e n t h e a c t i v i t i e s i n t h e p r e s e n c e a n d a b s e n c e o f D E S . T h e d e t e r g e n t s w e r e T r i t o n X-100 ( A ) , o c t y l g l u c o s i d e ( B ) , C^Eg ( C ) a n d CHAPS ( D ) . 48 Detergent '/. (w/v) Detergent •/. w/v 49 d e t e r g e n t s and t h e t o t a l DES s e n s i t i v e and i n s e n s i t i v e ATPase a c t i v i t i e s were d e t e r m i n e d . The DES s e n s i t i v e ATPase was p a r t i a l l y i n a c t i v a t e d by a l l t h e d e t e r g e n t s t e s t e d w i t h C ^ E g b e i n g t h e l e a s t and Z-14 t h e most i n h i b i t o r y ( F i g s . 12 and 1 3 ) . I n d e e d t h e e n h a n c e d i n h i b i t o r y e f f e c t o f Z-14 d e s c r i b e d e a r l i e r ( F i g . 9) was due a l m o s t s o l e l y t o i t s e x t r e m e l y d e l e t e r i o u s e f f e c t on t h i s enzyme ( F i g . 1 3 b ) . To t e s t w h e t h e r t h e DES s e n s i t i v e ATPase c o u l d be r e a c t i v a t e d by t h e a d d i t i o n o f p h o s p h o l i p i d s s o l u b i l i z e d p l a s m a membrane ATPase a c t i v i t y was a s s a y e d i n t h e p r e s e n c e o f DES w i t h o r w i t h o u t 2 0 m g / m l e x o g e n o u s p h o s p h a t i d y l c h o l i n e ( P C ) . A d d i t i o n o f 0.2 mM DES r e s u l t e d i n a 57 and 22% i n h i b i t i o n o f p l a s m a membrane bound and d e t e r g e n t s o l u b i l i z e d ATPase a c t i v i t y r e s p e c t i v e l y ( T a b l e V I ) . P h o s p h a t i d y l c h o l i n e f a i l e d t o r e c o n s t i t u t e t h e DES s e n s i t i v e component t o any s i g n i f i c a n t e x t e n t ( T a b l e V I ) . The DES i n s e n s i t i v e enzyme was s t i m u l a t e d by l y s o l e c i t h i n ( F i g . 13a) and i n h i b i t e d by d e o x y c h o l a t e ( F i g . 1 3 c ) , b u t w i t h t h e o t h e r d e t e r g e n t s t h e a c t i v i t y r e m a i n e d r e l a t i v e l y c o n s t a n t ( F i g . 1 2 ) . e) C o m p a r i s o n o f t h e P r o p e r t i e s o f t h e DES S e n s i t i v e and I n s e n s i t i v e ATPase A c t i v i t i e s i n P l a s m a Membranes The DES s e n s i t i v e ATPase was s t i m u l a t e d t o a much l a r g e r e x t e n t by M g + 2 as compared t o C a + 2 ( F i g . 14) whereas t h e DES i n s e n s i t i v e a c t i v i t y was s t i m u l a t e d e q u a l l y w e l l by b o t h c a t i o n s , f u r t h e r s u g g e s t i n g two d i s t i n c t enzymes. T h i s 50 F i g u r e 13. E f f e c t o f Z-14, D e o x y c h o l a t e and L y s o l e c i t h i n S o l u b i l i z a t i o n on DES S e n s i t i v e and I n s e n s i t i v e ATPase A c t i v i t y . P l a s ma membranes were s o l u b i l i z e d and DES s e n s i t i v e and i n s e n s i t i v e ATPase a c t i v i t i e s were d e t e r m i n e d e x a c t l y as d e s c r i b e d i n F i g . 12, e x c e p t t h a t the d e t e r g e n t s were l y s o l e c i t h i n ( A ) , Z-14 (B) and d e o x y c h o l a t e ( C ) . T r i a n g l e s d e n o t e DES s e n s i t i v e and s q u a r e s DES i n s e n s i t i v e ATPase a c t i v i t y . 51 o i l TABLE VI THE EFFECT OF EXOGENOUS PHOSPHOLIPID ON SOLUBILIZED DES SENSITIVE ATPASE ACTIVITY. Intact Plasma S o l u b i l i z e d DES Membrane ATPase Plasma Membrane ( mM ) • A c t i v i t y ATPase A c t i v i t y 5 + PC b - PC ( nmol Pi/min/mg p r o t e i n ) no DES 31.4 43.2 38.8 0.002 31.0 46.0 38.4 0.020 28.2 40.4 39.6 0.200 13.7 31.0 30.4 a Washed plasma membranes were s o l u b i l i z e d with 1% C i 2 E 9 a s ^es-cribed i n the methods. k The phosphatidylcholine concentration was 20 mg/ml. 53 F i g u r e 14. E f f e c t o f Ca+* and Mg c on DES S e n s i t i v e and I n s e n s i t i v e ATPase A c t i v i t y . Plasma membranes were i n c u b a t e d i n t h e p r e s e n c e and a b s e n c e o f 0.2 mM DES i n a m i x t u r e c o n t a i n i n g 3 mM Na 2ATP and t h e i n d i c a t e d c o n c e n t r a t i o n s o f M g C l 2 ( A ) or C a C l - ( B ) i n 10 mM M E S / T r i s pH 6.8. DES s e n s i t i v e (M) and i n s e n s i t i v e ( • ) ATPase a c t i v i t i e s were d e t e r m i n e d as d e s c r i b e d i n F i g . 12. The d a t a r e p r e s e n t s a s i n g l e e x p e r i m e n t but i t was r e p e a t e d s e v e r a l t i m e s w i t h s i m i l a r r e s u l t s . 54 c o n c l u s i o n was c o r r o b o r a t e d when enzyme a c t i v i t y was measured as a f u n c t i o n o f ATP c o n c e n t r a t i o n i n t h e p r e s e n c e o f e i t h e r M g + 2 o r C a + 2 ( F i g . 1 5 ) . I n t h e p r e s e n c e o f M g + 2 DES s e n s i t i v e ATPase a c t i v i t y i n c r e a s e d w i t h i n c r e a s i n g l e v e l s o f ATP t o a s a t u r a t i o n p o i n t a t 1.0 mM ( F i g . 1 5 a ) . When M g + 2 was r e p l a c e d w i t h C a + 2 a s i m i l a r s a t u r a t i o n o c c u r r e d , a l t h o u g h t h e a c t i v i t y was c o n s i d e r a b l y l o w e r ( F i g . 1 5 b ) . I n c o n t r a s t , DES i n s e n s i t i v e ATPase a c t i v i t y d i d n o t d i s p l a y ATP s a t u r a t i o n i n t h i s c o n c e n t r a t i o n r a n g e w i t h e i t h e r c a t i o n ( F i g . 15a and b ) . D o u b l e r e c i p r o c a l p l o t s o f t h e d a t a i n F i g . 15 r e v e a l e d t h a t , w h i l e t h e DES s e n s i t i v e ATPase was c o n s i d e r a b l y l e s s a c t i v e i n t h e p r e s e n c e o f C a + 2 r a t h e r t h a n M g + 2 , t h e a p p a r e n t Km o f t h e enzyme was t h e same (Km = 0.42 mM) r e g a r d l e s s as t o w h i c h c a t i o n was p r e s e n t ( F i g . 1 6 ) . I n c o n t r a s t , t h e DES i n s e n s i t i v e ATPase d i d n o t d i s p l a y M i c h a e l i s - M e n t e n k i n e t i c s w i t h e i t h e r Mg + 2 o r C a + 2 , as d o u b l e r e c i p r o c a l p l o t s showed a d i s t i n c t n o n - l i n e a r p a t t e r n ( F i g . 1 7 ) . N o n - l i n e a r k i n e t i c s a r e c h a r a c t e r i s t i c o f a l l o s t e r i c enzymes. The two a c t i v i t i e s were a s s a y e d a t d i f f e r e n t pH v a l u e s t h e DES s e n s i t i v e ATPase e x h i b i t e d a b e l l - s h a p e d c u r v e w i t h a pH optimum between pH 7.0 t o 7.5 ( F i g . 1 8 ) , w h i l e t h e DES i n s e n s i t i v e enzyme d i s p l a y e d a b r o a d pH optimum between pH 5.5 and 7.5 ( F i g . 16) . 55 8 2 0-2-o E f > • < . 0-1-cn / a ' i 1 r i I 1 ^ 1 1 1 1 1 1 0-2 0< 0-6 0-8 1-0 0-2 0-4 0-6 0-8 1-0 [ATP] mM ATP m M F i g u r e 15. E f f e c t o f ATP C o n c e n t r a t i o n on DES S e n s i t i v e and I n s e n s i t i v e ATPase A c t i v i t y i n the + 2 +2 P r e s e n c e o f Mg or Ca . Plasma membrane ATPasee a c t i v i t y was measured as a f u n c t i o n o f t h e ATP c o n c e n t r a t i o n i n t h e p r e s e n c e and a b s e n c e o f 0.2 mM DES. The r e a c t i o n m i x t u r e s c o n t a i n e d , i n a d d i t o n t o the i n d i c a t e d c o n c e n t r a t i o n s o f ATP, 10 mM MgCl„ ( A ) o r C a C l ? (B) i n 10 mM M E S / T r i s pH 6.8. DES s e n s i t i v e ( • ) and i n s e n s i t i v e ( O ) a c t i v i t y was d e t e r m i n e d as d e s c r i b e d i n F i g . 12. The d a t a r e p r e s e n t s a s i n g l e e x p e r i m e n t but i t was r e p e a t e d s e v e r a l t i m e s w i t h s i m i l a r r e s u l t s . 56 - 1 0 - 8 - 6 - 4 - 2 0 2 4 6 8 10 1 / A T P (mM) F i g u r e 16. The E f f e c t o f M g + 2 and C a + 2 on ATP H y d r o l y s i s K i n e t i c s o f the DES S e n s i t i v e A TPase. The ATP s a t u r a t i o n c u r v e s i l l u s t r a t e d i n F i g . 15 f o r t h e DES s e n s i t i v e enzyme were a n a l y z e d as d o u b l e r e c i p r o c a l p l o t s o f ATP c o n c e n t r a t i o n a g a i n s t v e l o c i t y . F i g u r e s r e p r e s e n t enzyme a c t i v i t y i n t h e p r e s e n c e o f ( O ) 10 mM M g C l 2 or (•) 10 mM C a C l p . The l i n e o f b e s t f i t was d e t e r m i n e d by l i n e a r r e g r e s s i o n . 57 —r-4 —T" 6 -r-8 -10 - 8 - 6 - 4 - 2 10 VftTP (mM) F i g u r e 1 7 . The E f f e c t o f M g + 2 and C a + 2 on ATP H y d r o l y s i s K i n e t i c s o f t h e DES I n s e n s i t i v e ATPase. The ATP s a t u r a t i o n c u r v e s i l l u s t r a t e d i n F i g . 15 f o r t h e DES i n s e n s i t i v e enzyme were a n a l y z e d as d o u b l e r e c i p r o c a l p l o t s o f ATP c o n c e n t r a t i o n a g a i n s t v e l o c i t y . F i g u r e s r e p r e s e n t enzyme a c t i v i t y i n the p r e s e n c e o f ( O ) 10 mM M g C l 2 or ( • ) 10 mM C a C l 2 . 58 F i g u r e 18. The E f f e c t o f pH on the DES S e n s i t i v e and I n s e n s i t i v e ATPase. Plasma membranes were i n c u b a t e d i n the p r e s e n c e and absence o f 0.2 mM DES i n a m i x t u r e c o n t a i n i n g 2 mM Na_ATP, 10 mM M g C l 2 and 10 mM M E S / T r i s a t the i n d i c a t e d pH v a l u e s . DES s e n s i t i v e ( • ) and i n s e n s i t i v e ( • ) a c t i v i t i e s were d e t e r m i n e d as d e s c r i b e d i n F i g . 12. T h i s e x p e r i m e n t was p e r f o r m e d o n c e . 59 S E C T I O N II . P U R I F I C A T I O N OF THE PLASMA MEMBRANE ATPASE A C T I V I T Y a ) F r a c t i o n a t i o n o f S o l u b i l i z e d A T P a s e A c t i v i t y b y I o n E x c h a n g e C h r o m a t o g r a p h y P l a s m a m e m b r a n e s w e r e s o l u b i l i z e d b y 1$ ^12^9» c o n d i t i o n s t h a t r e s u l t e d i n o p t i m u m s o l u b i l i z a t i o n o f A T P a s e a c t i v i t y w i t h t h i s d e t e r g e n t . When t h e s o l u b i l i z e d m e m b r a n e s w e r e a p p l i e d t o a D E A E - S e p h a c e l c o l u m n a n d e l u t e d w i t h a s t e p g r a d i e n t o f N a C l , t w o p e a k s o f A T P a s e a c t i v i t y w e r e r e s o l v e d , o n e e l u t i n g w i t h 0.1 M N a C l ( p e a k I ) a n d t h e s e c o n d ( p e a k I I ) e l u t i n g w i t h 0.3 M N a C l ( F i g . 1 9 ) . N e i t h e r o f t h e A T P a s e a c t i v i t i e s w a s i n h i b i t e d b y DES ( T a b l e V I I ) , i n d i c a t i n g a c o m p l e t e l o s s o f t h e DES s e n s i t i v e a c t i v i t y d u r i n g c h r o m a t o g r a p h y . T h e s p e c i f i c a c t i v i t y o f p e a k I I w a s c o n s i d e r a b l y h i g h e r t h a n t h a t f o r p e a k I ( T a b l e V I I I ) , b u t b o t h f r a c t i o n s c o n t a i n e d a l a r g e n u m b e r o f p r o t e i n b a n d s a s r e v e a l e d b y S D S - 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 ( S D S - P A G E ; F i g . 2 0 ) , i n d i c a t i n g t h a t n e i t h e r a c t i v i t y was h o m o g e n e o u s . b ) P u r i f i c a t i o n o f A T P a s e A c t i v i t y b y 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 t h a d b e e n s h o w n t h a t t h e p l a s m a m e m b r a n e A T P a s e s f r o m N e u r o s p o r a c r a s s a a n d S c h i z o s a c c h a r o m y c e s p o m b e c o u l d b e p u r i f i e d i n o n e s t e p b y c e n t r i f u g a t i o n o f s o l u b i l i z e d m e m b r a n e p r o t e i n s t h r o u g h g l y c e r o l o r s u c r o s e g r a d i e n t s 60 F i g u r e 19- D E A E - S e p h a e e l C h r o m a t o g r a p h y o f D e t e r g e n t S o l u b i l i z e d P l a s m a Membranes. Plasma membranes were t r e a t e d w i t h 1* C , 2 E g and t h e 100,000 x g s u p e r n a t a n t (6 t o 7 ml, 50 mg p r o t e i n ) was l o a d e d o n t o a DEAE-S e p h a c e l column (1.0 x 10 cm) w h i c h had been e q u i l i b r a t e d as d e s c r i b e d u n d e r M e t h o d s . Unbound p r o t e i n was e l u t e d w i t h e q u i l i b r a t i o n b u f f e r and was f o u n d t o c o n t a i n no ATPase a c t i v i t y . Bound p r o t e i n ( # ) and ATPase a c t i v i t y ( O ) were e l u t e d s t e p w i s e w i t h b u f f e r c o n t a i n i n g 0.1, 0.3 and 0.5 M N a C l a t t h e p o i n t s i n d i c a t e d by t h e a r r o w s . P r o t e i n was m o n i t o r e d by a b s o r b a n c e a t 280 nm. The ATPase a c t i v i t y r e p r e s e n t s t h e a b s o r b a n c e a t 660 nm t h a t r e s u l t e d when 0.2 ml a l i q u o t s o f t h e f r a c t i o n s were a s s a y e d f o r enzyme a c t i v i t y as d e s c r i b e d i n M e t h o d s , e x c e p t t h a t the i n c u b a t i o n t i m e was 1 h o u r . 6 1 TABLE V I I EFFECT OF DES ON PEAK I AND I I ATPASE ACTIVITY DES S p e c i f i c A c t i v i t y (mM) (nmol Pi/min/mg p r o t e i n ) Peak I Peak I I a d d i t i o n 11.9 + 1.1 76.0 + 3.5 0.02 13.1 ± 0.9 72.0 + 4.0 0.20 - 12.8 + 0.4 68.0 + 4.5 2.00 1 3 . 4 + 0 . 7 68.0 + 2 . 5 62 TABLE V I I I ATPASE ACTIVITY IN PEAKS I AND I I FROM DEAE-SEPHACEL P r o t e i n T o t a l A c t i v i t y S p e c i f i c A c t i v i t y (mg) (nmol P i / m i n ) (nmol Pi/min/mg p r o t e i n ) Peak I 2.15 29.0 13.5 Peak I I 4.30 400 .0 91 .0 63 F i g u r e 20. S D S - 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 o f V a r i o u s F r a c t i o n s o b t a i n e d D u r i n g the P u r i f i c a t i o n o f the DES I n s e n s i t i v e ATPase. Samples were e l e c t r o p h o r e s e d t h r o u g h S D S - p o l y a c r y l a m i d e g e l s and p r o t e i n s were v i s u a l i z e d u s i n g t h e s i l v e r s t a i n i n g p r o c e d u r e d e s c r i b e d i n Methods. Lane A, 1$ o c t y l g l u c o s i d e s o l u b i l i z e d p lasma membranes (20 jag p r o t e i n ) . Lane B, g l y c e r o l g r a d i e n t c e n t r i f u g a t i o n o f the s o l u b i l i z e d ATPase (10 ^ug p r o t e i n ) . Lane C, peak I from the D E A E - S e p h a c e l c h r o m a t o g r a p h y (20 ,ug p r o t e i n ) . Lane D, peak I I from D E A E - S e p h a c e l (10 ^ug p r o t e i n ) . Lane E, ATPase f r a c t i o n a f t e r g l y c e r o l g r a d i e n t c e n t r i f u g a t i o n o f peak I I (3 ,ug p r o t e i n ) . The m o l e c u l a r w e i g h t markers were myosi n (207 k D a ) , /S -g a l a c t o s i d a s e (116 k D a ) , p h o s p h o r y l a s e b (97.1 k D a ) , b o v i n e serum a l b u m i n (66 kDa) and o v a l b u m i n (45 k D a ) . 6 4 ( D u f o u r and G o f f e a u , 1978; Bowman e t a l . , 1981a; A d d i s o n and S c a r b o r o u g h , 1 9 81). However, when C 1 2 E g s o l u b i l i z e d £ . d i s c o i d e u m p l a s m a membranes were c e n t r i f u g e d t h r o u g h a 15 t o 35$ ( v / v ) g l y c e r o l g r a d i e n t , the ATPase f r a c t i o n s t i l l c o n t a i n e d c o n s i d e r a b l e amounts o f p r o t e i n c o n t a m i n a t i o n ( F i g . 20) i n d i c a t i n g t h a t 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 a l o n e was i n s u f f i c i e n t f o r p u r i f i c a t i o n t o h o m o g e n e i t y . In view o f the h i g h e r s p e c i f i c and t o t a l ATPase a c t i v i t y i n peak I I from D E A E - S e p h a c e l f u r t h e r p u r i f i c a t i o n , e f f o r t s were d i r e c t e d toward t h i s f r a c t i o n . C e n t r i f u g a t i o n o f peak I I a c t i v i t y t h r o u g h a 15 t o 35$ ( v / v ) g l y c e r o l g r a d i e n t r e s u l t e d i n t h e s e p a r a t i o n o f ATPase a c t i v i t y from t h e b u l k o f t h e p r o t e i n ( F i g . 2 1 ) . P o l y a c r y l a m i d e g e l s r e v e a l e d a s i n g l e major p r o t e i n band o f a p p a r e n t m o l e c u l a r w e i g h t 64 kDa and r e l a t i v e l y few o t h e r bands ( F i g . 2 0 ) . That the 64 kDa band r e p r e s e n t e d t h e ATPase was i n d i c a t e d by c o m p a r i s o n o f the s e d i m e n t a t i o n o f ATPase a c t i v i t y i n g l y c e r o l g r a d i e n t s w i t h t h e s e d i m e n t a t i o n o f o t h e r p r o t e i n s o f known m o l e c u l a r w e i g h t . A m o l e c u l a r w e i g h t o f 65 t o 77 kDa was c a l c u l a t e d f o r t h e a c t i v e ATPase ( F i g . 2 2 ) . D e s p i t e the r e l a t i v e s u c c e s s o f t h i s p u r i f i c a t i o n p r o c e d u r e the f i n a l y i e l d o f t h e ATPase was e x t r e m e l y low, i n d i c a t i n g a s u b s t a n t i a l l o s s o f a c t i v i t y ( T a b l e I X ) . A l t e r n a t i v e s t o t h e g l y c e r o l g r a d i e n t were t h e r e f o r e e x p l o r e d . 65 1 5 1 0 1 5 2 0 Fract ion Number F i g u r e 2 1 . G l y c e r o l G r a d i e n t C e n t r i f u g a t i o n o f Peak I I ATPase. A l i q u o t s ( 0 . 5 ml, 0 . 3 5 mg p r o t e i n ) o f peak I I ATPase were a p p l i e d to each o f s i x , 9 ml l i n e a r g l y c e r o l g r a d i e n t s ( 1 5 to 3 5 $ v/v) and c e n t r i f u g e d a t 1 2 0 , 0 0 0 x g f o r 36 to 48 h o u r s . F o l l o w i n g t h i s , f r a c t i o n s were c o l l e c t e d from t h e bottom o f t h e t u b e s and p r o t e i n c o n c e n t r a t i o n ( © ) and ATPase a c t i v i t y ( O ) were d e t e r m i n e d . P r o t e i n i s e x p r e s s e d as the 0 D a t 2 8 0 nm. Enzyme a c t i v i t y was d e t e r m i n e d f o r 0 . 2 ml a l i q u o t s as d e s c r i b e d i n Methods, e x c e p t t h a t the r e a c t i o n time was 2 h o u r s , and i s e x p r e s s e d as t h e a b s o r b a n c e a t 6 6 0 nm. F i g u r e 22. C a l i b r a t i o n C urve o f P r o t e i n M a r k e r s on G l y c e r o l D e n s i t y G r a d i e n t s . S e v e r a l p r o t e i n s o f known m o l e c u l a r w e i g h t were c e n t r i f u g e d t h r o u g h 15 t o 35$ (v / v ) g l y c e r o l g r a d i e n t s as d e s c r i b e d i n Methods and i n F i g . 19. S t a n d a r d p r o t e i n s were g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (102 k D a ) , b o v i n e k i d n e y a l k a l i n e p h o s p h a t a s e (96 k D a ) , E. c o l i a l k a l i n e p h o s p h a t a s e (86 kDa) and c y t o c h r o m e c (12.4 k D a ) . A l k a l i n e p h o s p h a t a s e was d e t e r m i n e d as d e s c r i b e d i n Methods. G l u c o s e - 6 -p h o s p h a t e d e h y d r o g e n a s e a c t i v i t y was d e t e r m i n e d by m e a s u r i n g the r a t e o f NAD + r e d u c t i o n a t 340 nm. The r e a c t i o n m i x t u r e s c o n t a i n e d , i n a f i n a l volume o f 3 ml, 55 mM T r i s - C l pH 7.8, 3-3 mM M g C l 2 and 2 mM NAD + and 0.3 ml o f g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e . The arrow i n d i c a t e s t h e R_ and t h e b r o k e n l i n e i n d i c a t e s t h e m o l e c u l a r w e i g h t r a n g e o f the D. d i s c o i d e u m ATPase. 67 TABLE IX PURIFICATION OF A DES-INSENSITIVE PLASMA MEMBRANE ATPASE P r o t e i n ATPase % R e c o v e r y S p e c i f i c (mg) (nmol/min) o f A c t i v i t y ATPase (nmol/min/mg p r o t e i n ) Washed membranes 1$ o c y t l g l u c o s i d e e x t r a c t 73.2 60.0 1200 .0 1 100.0 100.0 91 .7 16.4 18.3 DEAE-Sephacel column G l y c e r o l g r a d i e n t 4.3 0.2 400.0 51 .0 33 .3 4.2 92.0 243 .0 c ) A f f i n i t y and H y d r o p h o b i c C h r o m a t o g r a p h y A f f i n i t y c h r o m a t o g r a p h y o f peak I I on e i t h e r R e a c t i v e R e d - A g a r o s e ( C o l l and Murphy, 1984) o r A T P - A g a r o s e were u n s u c c e s s f u l i n t h a t t h e enzyme f a i l e d t o b i n d and no a d d i t i o n a l p u r i f i c a t i o n was o b t a i n e d ( T a b l e X ) . H y d r o p h o b i c c h r o m a t o g r a p h y o f peak I I on P h e n y l - S e p h a r o s e was a l s o u n s u c c e s s f u l ( T a b l e X I ) as t h e a c t i v i t y d i d n o t b i n d t o t h e c o l u m n . d) P u r i f i c a t i o n by G e l F i l t r a t i o n I n i t i a l a t t e m p t s a t f r a c t i o n a t i o n o f s o l u b i l i z e d membranes by g e l f i l t r a t i o n on S e p h a c r y l S-300 i n t h e p r e s e n c e o f 1$ BRIJ-35 (w/v) were u n s u c c e s s f u l i n t h a t t h e enzyme a c t i v i t y and most o f t h e p r o t e i n e l u t e d i n t h e c o l u m n v o i d v o lume, s u g g e s t i n g t h a t u n d e r t h e s e c o n d i t i o n s t h e enzyme was h i g h l y a g g r e g a t e d ( F i g 2 3 ) . However, when a p p l i e d t o t h e c o l u m n i n t h e p r e s e n c e o f 0.5% CHAPS (w/v), t h e e l u t i o n o f t h e enzyme was r e t a r d e d and was w e l l s e p a r a t e d f r o m t h e m a j o r p r o t e i n peak ( F i g . 2 4 ) . The g e l f i l t r a t i o n p u r i f i e d ATPase s t i l l c o n t a i n e d a c o n s i d e r a b l e number o f p r o t e i n bands as d e t e r m i n e d by SDS-PAGE ( F i g . 25) and was t h e r e f o r e f u r t h e r f r a c t i o n a t e d by DEAE-Sephace1 c h r o m a t o g r a p h y ( F i g . 2 6 ) . A l m o s t a l l o f t h e a c t i v i t y e l u t e d w i t h 0.3 M N a C l , c o r r e s p o n d i n g t o t h e peak I I a c t i v i t y p r e v i o u s l y d e s c r i b e d ( F i g . 1 9 ) . T h i s m a t e r i a l e x h i b i t e d a s i n g l e m a j o r band o f 64 kDa when s u b j e c t e d t o SDS-PAGE ( F i g . 2 5 ) . M o r e o v e r , as shown i n T a b l e X I I , t h e 69 TABLE X AFFINITY CHROMATOGRAPHY OF PEAK I I ATPASE ACTIVITY ATPase A c t i v i t y G e l u s e d (nmol Pi/min/mg p r o t e i n ) A p p l i e d R e c o v e r e d unbound E l u t e d A T P - a g a r o s e 91.2 80.7 0 R e a c t i v e 86.5 91.8 0 Re d - A g a r o s e 70 TABLE XI PHENYL-SEPHAROSE CHROMATOGRAPHY OF PEAK I I ATPASE ACTIVITY ATPase A c t i v i t y (nmol Pi/min/mg p r o t e i n ) A c t i v i t y a p p l i e d 103.2 E l u t i o n w i t h 10 mM T r i s - C l pH 7.5, 90.1 300 mM NaCl E l u t i o n w i t h 10 mM T r i s - C l pH 7.5, 0.0 10$ ( v / v ) g l y c e r o l E l u t i o n w i t h 0-15$ (w/v) BRIJ-35 0.0 g r a d i e n t E l u t i o n w i t h 1$ BRIJ-35 0.0 71 Fraction Number F i g u r e 23. G e l F i l t r a t i o n i n t h e P r e s e n c e o f 1% (w/v) BRIJ-35 o f D e t e r g e n t S o l u b i l i z e d P l a s ma Membranes. Plasma membranes were s o l u b i l i z e d w i t h 1$ C12^Q a n c * ^ ml (16 mg p r o t e i n ) o f the 100,000 x g s u p e r n a t a n t was a p p l i e d t o a S e p h a c r y l S-300 column (3 x 80 cm) w h i c h had been e q u i l i b r a t e d i n 25 mM T r i s - C l pH 7.5 c o n t a i n i n g 1$ (w/v) BRIJ-35 and 0.3 M N a C l . F r a c t i o n s o f 2.5 ml were c o l l e c t e d and p r o t e i n ( # ) was m o n i t o r e d by a b s o r b a n c e a t 280 nm. ATPase a c t i v i t y ( O ) was d e t e r m i n e d f o r 0.1 ml o f each f r a c t i o n as d e s c r i b e d i n Methods, e x c e p t t h a t a 1 hr i n c u b a t i o n t i m e was u s e d , and i 3 e x p r e s s e d as t h e a b s o r b a n c e a t 660 nm. The arrow i n d i c a t e s t h e volume a t w h i c h t h e maximum amount o f B l u e D e x t r a n e l u t e d and i 3 a measure o f t h e v o i d volume o f t h e c o l u m n . 72 Fract ion N u m b e r F i g u r e 24. G e l F i l t r a t i o n i n t h e P r e s e n c e o f 0.5$ (w/v) CHAPS o f D e t e r g e n t S o l u b i l i z e d P l a s m a Membranes. E x p e r i m e n t a l p r o c e d u r e s and c o n d i t i o n s were i d e n t i c a l to t h o s e o f F i g . 21, e x c e p t t h a t 1$ BRIJ-35 was r e p l a c e d by 0.5$ (w/v) CHAPS. C l o s e d c i r c l e s d e n o t e p r o t e i n as m o n i t o r e d a t 280 nm and t h e open c i r c l e s i n d i c a t e ATPase a c t i v i t y . 7 3 A B C F i g u r e 25. S D S - 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 o f F r a c t i o n s O b t a i n e d D u r i n g t h e P u r i f i c a t i o n o f t h e DES I n s e n s i t i v e ATPAse. Samples o f s o l u b i l i z e d p l a s m a membranes (Lane A, 20 ^ g p r o t e i n ) , ATPase f r a c t i o n f r o m S e p h a c r y l S-300 (Lane B, 10 jig p r o t e i n ) and p u r i f i e d enzyme from D E A E - S e p h a c e l (Lane C, 2.5 Jig p r o t e i n ) were s u b j e c t e d t o S D S - 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 as d e s c r i b e d i n Methods. The m o l e c u l a r w e i g h t m a r k e r s were the same as f o r F i g . 18. P r o t e i n s were v i s u a l i z e d u s i n g the s i l v e r s t a i n i n g p r o c e d u r e d e s c r i b e d i n Methods. 74 F i g u r e 26. D E A E - S e p h a c e l C h r o m a t o g r a p h y o f ATPase F r a c t i o n E l u t e d f r o m t h e G e l F i l t r a t i o n Column. The ATPase c o n t a i n i n g f r a c t i o n s e l u t e d . f r o m t h e S e p h a c r y l S-300 column (0.8 mg p r o t e i n ) i n t h e p r e s e n c e o f CHAPS were p o o l e d and d i a l y z e d a g a i n s t 200 volume s o f 10 mM T r i s - C l pH 7.5 c o n t a i n i n g 0.1$ CHAPS and 10$ v/v) g l y c e r o l f o r 12 h o u r s w i t h one change o f b u f f e r . The d i a l y z e d m a t e r i a l was t h e n a p p l i e d t o a D E A E - S e p h a c e l column w h i c h had been e q u i l i b r a t e d as d e s c r i b e d i n Methods. Bound p r o t e i n was e l u t e d as d e s c r i b e d i n F i g . 1 7 . P r o t e i n was m o n i t o r e d by a b s o r b a n c e a t 280 nm ( # ) and ATPase a c t i v i t y ( O ) was d e t e r m i n e d f o r 0.1 ml a l i q u o t s as d e s c r i b e d i n M e t h o d s , e x c e p t t h a t i n c u b a t i o n s were f o r 2 hr3. Enzyme a c t i v i t y i s e x p r e s s e d as t h e a b s o r b a n c e a t 660 nm. 75 shown i n T a b l e X I I , the r e c o v e r y o f a c t i v i t y a f t e r the c o m b i n a t i o n o f g e l f i l t r a t i o n and D E A E - S e p h a c e l c h r o m a t o g r a p h y was a p p r o x i m a t e l y t w o - f o l d g r e a t e r than t h a t o b s e r v e d f o r t h e i o n e x c h a n g e / g l y c e r o l g r a d i e n t p r e p a r a t i o n , a l t h o u g h the r e l a t i v e e n r i c h m e n t was a p p r o x i m a t e l y the same. I n t e r e s t i n g l y , the a c t i v i t y e l u t e d by 0.1 M Na C l (peak I , F i g . 19) was v e r y much d i m i n i s h e d ( F i g . 24,) and i n o t h e r e x p e r i m e n t s t o t a l l y e l i m i n a t e d , when t h e s o l u b i l i z e d enzyme was f i r s t s u b j e c t e d t o g e l f i l t r a t i o n . 76 TABLE XII PURIFICATION OF THE PLASMA MEMBRANE ATPASE F r a c t i o n P r o t e i n T o t a l (mg) A c t i v i t y (nmol P i / m i n ) % R e c o v e r y S p e c i f i c A c t i v i t y (nmol Pi/min/mg p r o t e i n ) Membranes \% o c t y l -g l u c o s i d e e x t r a c t S e p h a c r y l S-300 DEAE-S e p h a c e l 22 .0 16.0 0 .83 0.11 680 .0 690 .0 290 .0 56.7 100 101 43 8.3 31.0 46.1 343 .6 515.2 SECTION I I I PROPERTIES OF THE FRACTIONATED DES I N S E N S I T I V E A T P a s e s a) Peak I f r o m D E A E - S e p h a c e l The m a t e r i a l e l u t e d by 0.1 M N a C l f r o m D E A E - S e p h a c e l c h r o m a t o g r a p h y e x h i b i t e d maximum ATPase a c t i v i t y a t a l k a l i n e pH ( F i g . 2 7 ) . I n a d d i t i o n t o b e i n g i n s e n s i t i v e t o DES, t h i s A TPase a c t i v i t y was a l s o i n s e n s i t i v e t o v a n a d a t e ( T a b l e X I I I ) . The r e l a t i v e l y low ATPase a c t i v i t y o f peak I s u g g e s t e d t h e p o s s i b i l i t y t h a t ATP m i g h t n o t be t h e t r u e s u b s t r a t e f o r t h e enzyme. When a c t i v i t y was a s s a y e d w i t h a v a r i e t y o f p h o s p h o r y l a t e d s u b s t r a t e s , t h e g r e a t e s t a c t i v i t y was o b s e r v e d f o r i n o r g a n i c p y r o p h o s p h a t e ( T a b l e X I I I ) . UTP was h y d r o l y z e d p r e f e r e n t i a l l y among t h e n u c l e o t i d e s t e s t e d and AMP and p - n i t r o p h e n y l p h o s p h a t e were n o t h y d r o l y z e d a t a l l ( T a b l e X I I I ) . I n t e r e s t i n g l y , a l t h o u g h no marked p r e f e r e n c e t o w a r d C a + 2 o r M g + 2 was o b s e r v e d when t h e n u c l e o s i d e t r i p h o s p h a t e s were h y d r o l y z e d , t h e h y d r o l y s i s o f p y r o p h o s p h a t e was c o n s i d e r a b l y h i g h e r i n t h e p r e s e n c e o f M g + 2 ( T a b l e X I V ) . b) P r o p e r t i e s o f P u r i f i e d Peak I I The enzyme p u r i f i e d by a c o m b i n a t i o n o f g e l f i l t r a t i o n and D E A E - S e p h a c e l c h r o m a t o g r a p h y t h a t e l u t e d w i t h 0.3 M N a C l (peak I I ) e x h i b i t e d a b r o a d pH optimum between pH 6.0 and 8.0 ( F i g . 2 8 ) , a r e s u l t s i m i l a r t o t h a t o b s e r v e d e a r l i e r f o r t h e membrane bound DES i n s e n s i t i v e enzyme ( F i g . 1 8 ) . The 78 P H F i g u r e 27. E f f e c t o f pH on Peak I A c t i v i t y . Peak I was e l u t e d from D E A E - S e p h a c e l and a s s a y e d as d e s c r i b e d i n Methods, e x c e p t t h a t t h e pH was v a r i e d by t i t r a t i o n o f MES w i t h T r i s b a s e . S i m i l a r r e s u l t s were o b t a i n e d w i t h o t h e r n u c l e o s i d e t r i p h o s p h a t e s . 79 TABLE X I I I SUBSTRATE SPECIFICITY OF PEAK I S u b s t r a t e a S p e c i f i c A c t i v i t y (nmol Pi/min/mg p r o t e i n ) ATP 12.8 +_ 0.6 GTP 9.3 + 1 .1 CTP 10.9 +_ 0.3 UTP 16.0 +_ 1 .9 AMP 0 ADP 7.4 + 1 .2 pNPP 0 P P i 82 .1 +_ 7.8 ATP + 1 mM VOjj 12.8 +_ 0.5 R e a c t i o n s were c a r r i e d o ut i n t h e p r e s e n c e o f 10 mM MgCl The f i n a l s u b s t r a t e c o n c e n t r a t i o n s were 3 mM f o r t h e n u c l e o t i d e s and 1 mM f o r pNPP and P P i . S i n c e one mole o f P P i y i e l d s two moles o f P i the a c t i v i t y w i t h P P i was c a l c u l a t e d as h a l f t h e r a t e o f P i l i b e r a t i o n . The d a t a shown a r e the means o f f o u r i n d e p e n d e n t e x p e r i m e n t s + t h e s t a n d a r d d e v i a t i o n . 80 TABLE XIV EFFECT OF DIVALENT CATIONS ON PEAK I ENZYME ACTIVITY S u b s t r a t e 3 A c t i v i t y (nmol Pi/min/mg p r o t e i n ) C a + 2 M g + 2 ATP 13.6 + 1 .2 12.8 0.6 GTP 12.0 +_ 2.3 9.3 + 1 .1 CTP 10.6 +_ 0.5 10.9 + 0.3 UTP 17.8 + 0.7 16.0 + 1 .9 P P i 10.0 + 1 .1 82 .1 + 7.8 R e a c t i o n c o n d i t i o n s were t h e same as f o r T a b l e XV e x c e p t i n some c a s e s M g + 2 was r e p l a c e d w i t h C a + 2 . The d a t a shown a r e th e means f o r f o u r i n d e p e n d e n t e x p e r i m e n t s + t h e s t a n d a r d d e v i a t i o n . 81 _l I I 1 1 1 1 1— 5 5 6-0 6-5 7 0 7-5 8 0 8-5 9 0 pH F i g u r e 28. E f f e c t o f pH on the P u r i f i e d ATPase A c t i v i t y . P u r i f i e d ATPase was a s s a y e d as d e s c r i b e d i n Methods, e x c e p t t h a t the pH was v a r i e d by t i t r a t i o n o f MES w i t h T r i s b a s e . T h i s e x p e r i m e n t was p e r f o r m e d once. 8 2 ATPase was s e n s i t i v e t o v a n a d a t e and f l u o r i d e b u t was o n l y m a r g i n a l l y i n h i b i t e d by t h i m e r o s a l and was c o m p l e t e l y i n s e n s i t i v e t o DCCD ( F i g . 29) and N - e t h y l m a l e i m i d e ( F i g . 3 0 ) . The ATPase was i n h i b i t e d by m i l l i m o l a r c o n c e n t r a t i o n s o f b o t h C a + 2 and M g + 2 ( F i g . 3 1 ) , a l t h o u g h i t was s t i m u l a t e d e q u a l l y w e l l by e i t h e r c a t i o n a l o n e ( F i g . 32, T a b l e X V I ) . I t s h o u l d be n o t e d t h a t t h e 20 t o 30% o f maximum s t i m u l a t e d a c t i v i t y o b s e r v e d a t z e r o o r low M g + 2 o r Ca + 2 c o n c e n t r a t i o n s ( F i g . 32) was n o t a b o l i s h e d by e i t h e r EDTA o r EGTA ( d a t a n o t shown) . M o n o v a l e n t c a t i o n s f a i l e d t o enha n c e t h e M g + 2 o r C a + 2 - A T P a s e a c t i v i t y ( T a b l e XVI) o r s t i m u l a t e t h e enzyme i n t h e a b s e n c e o f t h e d i v a l e n t c a t i o n s ( T a b l e XV ) . Enzyme a c t i v i t y was a l s o g r e a t l y s t i m u l a t e d by Zn + 2 , Cu + 2 and Mn + 2 b u t l e s s e f f i c i e n t l y by B a + 2 ( T a b l e XV ) . As w i t h C a + 2 and M g + 2 t h e s t i m u l a t i o n n e c e s s i t a t e d m i l l i m o l a r d i v a l e n t c a t i o n c o n c e n t r a t i o n s ( F i g . 33) f o r maximum s t i m u l a t i o n , a l t h o u g h s l i g h t s t i m u l a t i o n was o b t a i n e d w i t h 10 ^ iM Zn + 2 . ATP was t h e p r e f e r r e d s u b s t r a t e f o r t h e enzyme, a l t h o u g h a l l t h e n u c l e o s i d e t r i p h o s p h a t e s t e s t e d and ADP were h y d r o l y z e d t o a s i g n i f i c a n t e x t e n t , b u t AMP and pNPP were n o t u t i l i z e d a t a l l ( T a b l e X V I I ) . When enzyme a c t i v i t y was measured as a f u n c t i o n o f t h e s u b s t r a t e c o n c e n t r a t i o n i t was f o u n d t h a t t h e ATP c o n c e n t r a t i o n c u r v e was s i g m o i d a l i n t h e a b s e n c e o f d i v a l e n t c a t i o n s ( F i g . 3 2 ) . A d d i t i o n o f 1.0 and 10.0 mM M g + 2 o r C a + 2 p r o d u c e d a p o s i t i v e a l l o s t e r i c e f f e c t r e s u l t i n g i n an a p p a r e n t i n c r e a s e i n t h e a f f i n i t y o f t h e enzyme f o r ATP 83 I n h i b i t o r (- log M) F i g u r e 29. E f f e c t o f V a r i o u s I n h i b i t o r s on P u r i f i e d ATPase A c t i v i t y . P u r i f i e d ATPase was a s s a y e d as d e s c r i b e d i n Methods, w i t h the i n d i c a t e d c o n c e n t r a t i o n s o f v a n a d a t e ( • ) , t h i m e r o s a l ( O ) , DCCD ( • ) and NaF ( 0 ) . The d a t a r e p r e s e n t s the mean f o r t h r e e s e p a r a t e e x p e r i m e n t s ± the s t a n d a r d d e v i a t i o n . 84 F i g u r e 30. E f f e c t o f N - e t h y l m a l e i m i d e on P u r i f i e d ATPase A c t i v i t y . P u r i f i e d ATPase (100 u l ) was p r e i n c u b a t e d a t 0°C i n 0.2 ml 25 mM T r i s - C l pH 8.0 and 10 mM N - e t h y l m a l e i m i d e . At the i n d i c a t e d t i m e s 0.8 ml o f the ATPase a s s a y m i x t u r e was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 4 mM ATP, 10 mM M g C l 2 a n d 10 mM M E S / T r i s pH 6.8 and f u r t h e r i n c u b a t e d f o r 2 h o u r s . The r e a c t i o n s were t e r m i n a t e d and 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 as d e s c r i b e d i n Methods. The d a t a r e p r e s e n t s the mean o f t h r e e s e p a r a t e e x p e r i m e n t s + the s t a n d a r d d e v i a t i o n . 85 2 0 -I / 1 1 1 r-6 5 4 3 p C a C l 2 F i g u r e 3 1 . I n h i b i t i o n o f P u r i f i e d ATPase A c t i v i t y by H i g h C o n c e n t r a t i o n s o f C a + 2 i n t h e p r e s e n c e o f M g + 2 . P u r i f i e d ATPase a c t i v i t y was a s s a y e d i n t h e p r e s e n c e o f 5 mM M g + 2 and t h e i n d i c a t e d c o n c e n t r a t i o n s o f C a C l ? . Enzyme a c t i v i t y was d e t e r m i n e d as d e s c r i b e d i n Metnods. The e x p e r i m e n t was p e r f o r m e d o n l y o n c e . 8 6 Z8 CO (B CL > TI (t 1 B 4 O H 1 (u <D ta ci- era D O H- C & rf O 1 < -3 PJ ET O H- H- (D i i i 8 t r <t o . o CD «< uo p a a • ro a . < D < "S ""3 3 T) (-.. p> p) C W pi (R ^ S 1 >-b ct CD H- CD H- >-4 H- O ct M) (II O O D t r H- O O M, O CD ct ct Co CO O ET M > "> T CD i-i CD < X TI S ID O O PI DD T CD CO + ta p- T) CD ro CD CD ct •O Ct P> O p) C I - O 1 1 P S (t P 09 CO H- O ct <! P> CD P" H- + s ci- c+ ro CD O- CT << X H- CD O •a o s: a CD P> 2 PJ T ct W 11 D H- CD + C B o. ropj "s CD • CO H-3 01 ^ ct > (B H-CO i-3 w i< CD CT CD D. 1+ ID (I ft D > ft i a ji H D* CD CO TJ CD CO O P> C 0) CO I—1 + CD ct ro co ro CJ 1 o o ATPase (percent) cn O o oo o to o O o 'Hi ro TABLE XV EFFECT OF VARIOUS CATIONS ON PURIFIED ATPASE ACTIVITY A d d i t i o n 3 A c t i v i t y R e l a t i v e A c t i v i t y (nmol P i / m i n ) {%) none 1 . 1 + 0 . 0 17.7 M g + 2 6 . 2 + 0 . 1 100.0 C a + 2 6.7 + 0.1 108.8 K + 1 . 0 + 0 . 0 16.1 L i + 1 . 2 + 0 . 1 18.7 N a + 1 . 1 + 0 . 1 17.6 Rb + 1 . 1 + 0 . 0 17.6 B a + 2 3 . 1 + 0 . 1 50.0 Z n + 2 1 1 . 5 + 0 . 3 186.3 M n + 2 9 . 2 + 0 . 1 148.6 C u + 2 12.1 + 0.6 195.7 I n a l l c a s e s the c a t i o n c o n c e n t r a t i o n s were 10 mM. The d a t a r e p r e s e n t s t h e means o f t h r e e s e p a r a t e e x p e r i m e n t s + the s t a n d a r d d e v i a t i o n . 88 TABLE XVI EFFECT OF MONOVALENT CATIONS ON M g + 2 OR C a + 2 STIMULATED ATPASE ACTIVITY C a t i o n 3 A c t i v i t y ( % ) b C a + 2 - A T P a s e M g + 2 - A T P a s e No a d d i t i o n 100 100 L i + 90.8 + 3-5 92.0 + 2.2 K + 94.6 + 3.5 9 6 . 7 + 2 . 5 Rb + 104.4 + 1 .9 100.7 + 0.7 Na + 96.0 + 5.4 101 .4 + 0.0 Mo n o v a l e n t c a t i o n c o n c e n t r a t i o n was 50.0 mM. R e a c t i o n m i x t u r e s c o n t a i n e d 10 mM M g + 2 or C a + 2 . R e a c t i o n s were i n i t i a t e d by t h e a d d i t i o n o f 4.0 mM C a + 2 or Mg + 2-ATP. The d a t a shown a r e t h e means o f f o u r i n d e p e n d e n t e x p e r i m e n t s + t h e s t a n d a r d d e v i a t i o n . 89 F i g u r e 33. E f f e c t o f D i v a l e n t C a t i o n s on P u r i f i e d ATPase A c t i v i t y . The p u r i f i e d ATPase was a s s a y e d as d e s c r i b e d i n F i g . 30, i n t h e p r e s e n c e o f t h e i n d i c a t e d c o n c e n t r a t i o n s o f Z n + 2 ( • ) , Cu + 2 ( A ) , M g + 2 ( A ) and M n + 2 ( • ) . The d a t a r e p r e s e n t s a s i n g l e e x p e r i m e n t . 90 TABLE XVI I SUBSTRATE S P E C I F I C I T Y OF THE PURIFIED ATPASE S u b s t r a t e 3 % A c t i v i t y ATP 1 0 0 GTP 33.7 ± 3.4 CTP 57.7 + 2.6 UTP 26.7 + 8.3 ADP 24.5 + 5.5 AMP 0 . 0 pNPP 0 . 0 P P i 0 . 0 The f i n a l s u b s t r a t e c o n c e n t r a t i o n s were 4 mM f o r t h e n u c l e o t i d e s and 1 mM f o r pNPP and P P i . The d a t a a r e means o f t h r e e s e p a r a t e e x p e r i m e n t s +_ t h e s t a n d a r d d e v i a t i o n s . 91 2 0 4 0 6 0 8 0 10 0 ATP (mM) 2 0 40 6 0 80 100 ATP (mM) F i g u r e 3 4 . E f f e c t o f M g + 2 and C a + 2 on ATPase A c t i v i t y D e t e r m i n e d as a F u n c t i o n o f the ATP C o n c e n t r a t i o n . P u r i f i e d ATPase a c t i v i t y was measured as a f u n c t i o n o f the ATP c o n c e n t r a t i o n i n the p r e s e n c e and a b s e n c e o f v a r i o u s f i x e d c o n c e n t r a t i o n s o f Mg (A) and C a + 2 ( B ) . Enzyme a c t i v i t y was measured as d e s c r i b e d i n Methods. The v a r i o u s c a t i o n c o n c e n t r a t i o n s were 1 .0 mM ( A . ) , 10 mM ( • ) , 100 mM ( • ) and no c a t i o n ( A ) . The d a t a i s an a v e r a g e o f t h r e e s e p a r a t e e x p e r i m e n t s . S t a n d a r d d e v i a t i o n s have been o m i t t e d f o r t h e sake o f c l a r i t y . 92 ( F i g . 3 4 ) . I n c r e a s i n g the c a t i o n c o n c e n t r a t i o n t o 100 mM r e s u l t e d i n a s l i g h t i n c r e a s e i n the a p p a r e n t ATP a f f i n i t y r e l a t i v e to z e r o c a t i o n but t h e r e a c t i o n v e l o c i t y was d e c r e a s e d ( F i g . 3 4 ) . The f a c t t h a t the enzyme was s t i m u l a t e d by C a + 2 r a i s e d the p o s s i b i l i t y t h a t i t may be a plasma membrane C a + 2 pump. However, the ATPase a c t i v i t y was n o t s t i m u l a t e d by m i c r o m o l a r c o n c e n t r a t i o n s o f C a + 2 and M g + 2 , nor was i t s t i m u l a t e d by C a + 2 / c a l m o d u l i n ( T a b l e X V I I I ) . There was no e v i d e n c e f o r a C a + 2 / c a l m o d u l i n s t i m u l a t e d ATPase a c t i v i t y i n the o r i g i n a l p lasma membrane p r e p a r a t i o n s ( T a b l e X V I I I ) , a l t h o u g h t h e s e membranes had been e x t e n s i v e l y washed w i t h e i t h e r EGTA or KC1/EDTA t o e n s u r e c o m p l e t e r e m o v a l o f endogenous c a l m o d u l i n ( J a r r e t t and P e n n i s t o n , 1978). c) P h o s p h o r y l a t i o n o f t h e , P l a s m a Membrane ATPase I n i t i a l a t t e m p t s to p h o s p h o r y l a t e t h e ATPase i n plasma membranes which had been s t o r e d a t -70°C were u n s u c c e s s f u l i n t h a t most o f t h e r a d i o a c t i v e l y l a b e l l e d p r o t e i n s f a i l e d to e n t e r the g e l ( F i g . 3 5 ) . When f r e s h l y p r e p a r e d membranes were used a few p h o s p h o r y l a t e d bands were f a i n t l y v i s i b l e but t h e r e was no a p p a r e n t p h o s p h o r y l a t i o n o f a 64 kDa p r o t e i n . In a d d i t i o n , most o f the r a d i o a c t i v i t y was a g a i n found a t the top o f the g e l ( F i g . 3 6 ) , a phenomenon which c o u l d n o t have been due to p r o t e i n a g g r e g a t i o n as Coomassie s t a i n i n g r e v e a l e d an abundance o f p r o t e i n bands. No 64 kDa 93 TABLE XVIII THE EFFECT OF EXOGENOUS CALMODULIN ON PURIFIED AND PLASMA MEMBRANE BOUND ATPASE ACTIVITY + 2 Ca -ATPase A c t i v i t y (nmol Pi/min/mg p r o t e i n ) C a + 2 C o n c e n t r a t i o n P u r i f i e d EGTA Washed KC1/EDTA Washed a Ii (yUM) ATPase Membranes Membranes  + C a l . c - C a l . + C a l . c - C a l . + C a l . c - C a l . 0.01 - 54.0 51.0 5.9 5.0 5.2 5.5 0.05 46.0 48.0 5.2 5.5 5.7 5.7 0.10 36.0 48.0 5.2 5.5 5.7 6.1 0.50 44.0 50.0 5.9 5.5 6.1 7.0 1.00 40.0 45.0 6.2 5.2 5.9 7.3 10.00 44.0 48.0 5.2 5.7 5.9 7.5 a P l a s m a membranes were washed once w i t h lOmM T r i s - C l , pH 7.5 c o n t a i n i n g 2mM EGTA and t w i c e w i t h lOmM T r i s - C l , pH 7.5. b Plasma membranes were washed as described i n the methods. 5 yug bovine brain calmodulin was added. 207 K — 1 1 6 K — * 9 7 K — 6 6 K — 45 K— F i g u r e 35. A u t o r a d i o g r a p h y o f 3 P - l a b e l e d D e t e r g e n t S o l u b i l i z e d Plasma Membranes. Plasma membranes w h i c h had been s t o r e d a t -70°C f o r s e v e r a l days were s o l u b i l i z e d w i t h 1$ o c t y l g l u c o s i d e as d e s c r i b e d i n Methods. S o l u b i l i z e d p r o t e i n s were l a b e l e d f o r 5 ( Lanes A ) , 10 ( L a n e s B) o r 15 (Lanes C) s e c o n d s i n r e a c t i o n m i x t u r e s c o n t a i n i n g , i n a f i n a l volume o f 0.05 ml, 10 mM M E S / T r i s pH 6.8,0.02 mg p r o t e i n . 10 mM MgCl and (2) 0.03, (3) 0.3 o r (4) 3.0 mM [Y - 3 2 P ] - A T P ( s p e c i f i c a c t i v i t i e s 3.3 mCi/umol, 0.33 mCi/umol and 0.033 mCi/umol, r e s p e c t i v e l y ) . Lane 1 r e p r e s e n t s a z e r o time c o n t r o l . The r e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n o f 0.05 ml e l e c t r o p h o r e s i s sample b u f f e r c o n t a i n i n g 0.083 M T r i s - C l pH 6.8, g l y c e r o l , d e - i o n i z e d water (1:1:6, v / v / v ) , 0.05 M 2 - m e r c a p t o e t h a n o l and 5% (w/v) SDS. The samples were s u b j e c t e d t o e l e c t r o p h o r e s i s a t 4°C as d e s c r i b e d i n Methods. The g e l was t h e n soaked f o r 12 h r s , w i t h two c h a n g e s , i n m e t h a n o l / a c e t i c a c i d / d e - i o n i z e d water (0.8:0.2:1, v / v / v ) , d r i e d and p l a c e d a g a i n s t r a d i o g r a p h i c X - r a y f i l m f o r 1 week b e f o r e b e i n g d e v e l o p e d . 95 1 2 3 4 I II II 6 6 K — 4 5 K — F i g u r e 36. A u t o r a d i o g r a p h y o f 3 2 p _ l a b e l e d c r u ( j e membranes. Crude membranes were p r e p a r e d a c c o r d i n g t o the m o d i f i e d p r o c e d u r e o f Das and Henderson (1983) as d e s c r i b e d i n Methods and were p h o s p h o r y l a t e d i m m e d i a t e l y a f t e r p r e p a r a t i o n . R e a c t i o n m i x t u r e s were as d e s c r i b e d i n F i g . 34 and the i n c u b a t i o n s were p e r f o r m e d i n d u p l i c a t e . The f i n a l c o n c e n t r a t i o n s o f [ V 3 2 P ] - A T P were (2) 0.03, (3) 0.3 and (4) 3.0 mM. Lane 1 r e p r e s e n t s a z e r o t i m e p o i n t . R e a c t i o n s were t e r m i n a t e d and g e l s r u n and t r e a t e d as d e s c r i b e d i n F i g . 33. 96 p h o s p h o p r o t e i n was d e t e c t e d when s i m i l a r e x p e r i m e n t s were p e r f o r m e d w i t h p u r i f i e d enzyme ( d a t a n o t shown). d) E f f e c t o f DIF on ATPase A c t i v i t y I t has been p r o p o s e d t h a t DIF e f f e c t s s t a l k c e l l d e v elopment by i n t e r a c t i n g d i r e c t l y w i t h a c e l l s u r f a c e p r o t o n pumping ATPase ( G r o s s e t a l . , 1 9 8 3 ) . S i n c e i t was p o s s i b l e t h a t a t l e a s t one o f the ATPases d e s c r i b e d i n t h i s t h e s i s was i n v o l v e d i n p r o t o n pumping the e f f e c t s o f DIF on enzyme a c t i v i t y were s t u d i e d . As shown i n T a b l e XIX p a r t i a l l y p u r i f i e d DIF (Kay e t a l . , 1 9 8 3 ) had no e f f e c t on e i t h e r t o t a l p l a s m a membrane bound o r p a r t i a l l y p u r i f i e d peaks I and I I ATPase a c t i v i t y . 97 TABLE XIX EFFECT OF DIF ON ATPASE ACTIVITY T r e a t m e n t 3 R e l a t i v e A c t i v i t y ($) Plasma Membranes Peak I Peak I I No a d d i t i o n 100 100 100 5 Jul DIF 93 - 93 10 j i l DIF 98 102 97 20 j i l DIF - - 99 10 j u l DIF - - 9 8 s o n i c a t e d 10 )il E t h a n o l - - 100 s o n i c a t e d 5 j i l DIF i n d u c e d 64$ s t a l k c e l l d i f f e r e n t i a t i o n i n a s t a n d a r d DIF a s s a y ( S o b o l e w s k i e t a l . , 1983). DIF was added as an e t h a n o l i c s o l u t i o n . Where i n d i c a t e d t h e DIF s o l u t i o n was s o n i c a t e d a l o n g w i t h the ATPase f o r a t o t a l o f 30 s e c o n d s i n 10 s e c o n d b u r s t s . Enzyme a c t i v i t y was d e t e r m i n e d as d e s c r i b e d i n Methods. F o r peaks I and I I 40 ug p r o t e i n was used and f o r t h e plasma membranes 95 ug p r o t e i n was u s e d . 98 DISCUSSION P l a s m a Membrane P u r i f i c a t i o n At t h e o u t s e t o f t h i s work o n l y two r e p o r t s e x i s t e d d e s c r i b i n g p l a s m a membrane A T P a s e s i n D. d i s c o i d e u m . P a r i s h and W i e b e l (1980) o b s e r v e d t h a t i n t a c t c e l l s h y d r o l y z e d e x t r a c e l l u l a r [' C ] - A T P , i n d i c a t i n g t h e p r e s e n c e o f an e c t o -A T P a s e . T h i s enzyme was i n h i b i t e d by t h e N a + , K + - A T P a s e i n h i b i t o r s u r a m i n b u t n o t by o l i g o m y c i n o r o u a b a i n . M o r e o v e r , P a r i s h and W i e b e l (1980) a l s o s u g g e s t e d t h a t t h e enzyme may be c o u p l e d t o C a + 2 u p t a k e i n t h a t t h e a d d i t i o n o f ATP t o a c e l l s u s p e n s i o n l e d t o an e n h a n c e d u p t a k e o f ^ C a + 2 . The ^ C a + ^ u p t a k e was r e d u c e d when c e l l s were i n c u b a t e d w i t h u n c o u p l e r s , s u r a m i n o r n o n - h y d r o l y z a b l e ATP a n a l o g u e s , a l t h o u g h i n most c a s e s t h e r e d u c t i o n was l e s s t h a n 50%. The ATPase had a c o m p a r a t i v e l y h i g h a f f i n i t y f o r ATP w i t h a Kffl 0 f between 1 t o 25 jiM and had a r e q u i r e m e n t f o r Mg + 2 , I n a d d i t i o n , M g + 2 c o u l d n o t be r e p l a c e d by any o t h e r i o n . In t h e o t h e r r e p o r t , B l a n c o (1982) d e s c r i b e d t h e e x i s t e n c e o f a N a + , K + s t i m u l a t e d , o u a b a i n r e s i s t a n t ATPase a c t i v i t y i n D. d i s c o i d e u m p l a s m a membranes p r e p a r e d u s i n g t h e two phase s y s t e m o f B r u n e t t e and T i l l ( 1 9 7 1 ) . T h i s ATPase was a l s o s t r o n g l y i n h i b i t e d by a z i d e and t h i m e r o s a l . No d a t a was i n c l u d e d w i t h r e g a r d t o t h e p u r i t y o f t h e p l a s m a membranes. 99 A wide v a r i e t y o f p l a s m a membrane p u r i f i c a t i o n methods f o r I) . d i s c o i d e u m have been p u b l i s h e d and c o n s i d e r a b l e e f f o r t was i n i t i a l l y d i r e c t e d t o w a r d f i n d i n g t h e method most a p p r o p r i a t e f o r ATPase c h a r a c t e r i z a t i o n . P a r i s h and M u l l e r (1976) and C o n d e e l i s (1978) s t a b i l i z e d p l a s m a membranes w i t h ConA b e f o r e d i s r u p t i n g t h e c e l l s w i t h T r i t o n X-100. P l a s m a membranes were t h e n i s o l a t e d f o l l o w i n g low s p e e d c e n t r i f u g a t i o n and ConA was removed by w a s h i n g w i t h m e t h y l m a n n o s i d e . A d i s a d v a n t a g e o f t h i s t e c h n i q u e was t h a t a c o n s i d e r a b l e l o s s o f p l a s m a membrane a l k a l i n e p h o s p h a t a s e , and p o s s i b l y o t h e r p r o t e i n s , o c c u r r e d , p r e s u m a b l y t h r o u g h d e t e r g e n t s o l u b i l i z a t i o n ( P a r i s h and M u l l e r , 1 9 7 6 ) . I n view o f t h i s drawback t h i s t e c h n i q u e was n o t u s e d . The two phase p o l y m e r p a r t i t i o n i n g s y s t e m o f B r u n e t t e and T i l l ( 1 9 7 1 ) , t h a t had been a p p l i e d p r e v i o u s l y t o D. d i s c o i d e u m ( S i u e_t a l . , 1977; Ono e t a l . , 1978; B l a n c o , 1 9 8 2 ) , was a l s o t r i e d b u t a c o n s i d e r a b l e amount o f m i t o c h o n d r i a l c o n t a m i n a t i o n p e r s i s t e d i n t h e p l a s m a membrane f r a c t i o n ( d a t a n o t shown). J a c o b s o n ( 1980 ) and Chaney and J a c o b s o n ( 19 8 3) i s o l a t e d membranes by c o a t i n g t h e c e l l s w i t h e i t h e r p o l y c a t i o n i c b e a d s o r c o l l o i d a l s i l i c a , t h e n b r e a k i n g t h e c e l l s by m e c h a n i c a l a g i t a t i o n ( J a c o b s o n , 1 9 8 0 ) o r e x p l o s i v e d e c o m p r e s s i o n (Chaney and J a c o b s o n , 19 83) . When t h i s t e c h n i q u e was u s e d i t was f o u n d t h a t o n l y v e r y s m a l l amounts o f m a t e r i a l c o u l d be p r o c e s s e d and c o n s e q u e n t l y v e r y l i t t l e A TPase a c t i v i t y was o b t a i n e d ( d a t a n o t s h o w n ) . 1 00 D i f f e r e n t i a l c e n t r i f u g a t i o n was a l s o a t t e m p t e d w i t h D i c t y o s t e l i u m membranes, but r e s u l t e d i n o n l y m a r g i n a l e n r i c h m e n t o f p l a s m a membranes ( d a t a n o t shown). Such a t e c h n i q u e had been u t i l i z e d t o o b t a i n a 3 5 - f o l d e n r i c h m e n t o f plasma membrane ATPase a c t i v i t y from N e u r o s p o r a c r a s s a (Bowman e t a l . , 1981b). A somewhat l a b o r i o u s c e n t r i f u g a t i o n o f c r u d e membranes t h r o u g h s u c r o s e g r a d i e n t s ( G i l k e s and Weeks, 1977b) r e s u l t e d i n r e l a t i v e l y p u r e plasma membrane p r e p a r a t i o n s w h i c h were f r e e o f m i t o c h o n d r i a l c o n t a m i n a t i o n , as a s s e s s e d by s u c c i n a t e d e h y d r o g e n a s e a c t i v i t y , and c o n t a i n e d a h i g h l e v e l o f ATPase a c t i v i t y ( T a b l e I ) . M o r e o v e r , the ATPase a c t i v i t y was r e f r a c t o r y t o the u s u a l m i t o c h o n d r i a l ATPase i n h i b i t o r s a z i d e and o l i g o m y c i n , c o n f i r m i n g the a b s e n c e o f m i t o c h o n d r i a ( F i g u r e 2 ) . The more r e c e n t l y d e s c r i b e d s u c r o s e d e n s i t y g r a d i e n t p r o c e d u r e o f Das and H enderson (1983) was a l s o used d u r i n g t h e c o u r s e o f t h i s work and y i e l d e d plasma membranes f r e e o f m i t o c h o n d r i a l c o n t a m i n a t i o n . However, t h i s p r o c e d u r e was no b e t t e r t h a n th e G i l k e s and Weeks (1977b) method and i t was f o u n d t h a t t h e the ATPase a c t i v i t y was l o s t upon f r a c t i o n a t i o n o f t h e s o l u b i l i z e d membranes a phenomenon t h a t was a p p a r e n t l y due t o t h e 50 mM g l y c i n e pH 8.5 b u f f e r used t h r o u g h o u t ( d a t a not shown). I d e n t i f i c a t i o n of plasma membrane ATPase a c t i v i t i e s The plasma membrane ATPase a c t i v i t y was not s t i m u l a t e d by e q u i m o l a r N a + and K + ( F i g . 5) and i t was i n s e n s i t i v e t o o u a b a i n ( T a b l e I I I ) , i n d i c a t i n g t h a t t h e enzyme was n o t a 101 N a + ,K + - A T F a s e . T h i s r e s u l t s u g g e s t e d t h a t t h e a z i d e s e n s i t i v e N a + , K + - A T P a s e i n £ . d i s c o i d e u m d e s c r i b e d e a r l i e r ( B l a n c o , 1982) may have been due t o m i t o c h o n d r i a l c o n t a m i n a t i o n , a l t h o u g h t h e m i t o c h o n d r i a l ATPase f r o m D_. d i s c o i d e u m has n e v e r been c h a r a c t e r i z e d . S o l u b i l i z a t i o n o f p l a s m a membranes r e v e a l e d t h e e x i s t e n c e o f two d i s t i n c t ATPase a c t i v i t i e s , one b e i n g s e n s i t i v e and t h e o t h e r i n s e n s i t i v e t o DES ( F i g s . 12 and 1 3 ) . The DES s e n s i t i v e ATPase was u n s t a b l e when removed f r o m i t s n a t i v e membrane bound s t a t e and f r a c t i o n a t i o n o f t h e s o l u b i l i z e d membranes r e s u l t e d i n a c o m p l e t e l o s s o f a c t i v i t y . I n c o n t r a s t , t h e DES i n s e n s i t i v e a c t i v i t y was s t a b l e t o d e t e r g e n t s o l u b i l i z a t i o n and c o u l d be f r a c t i o n a t e d . S o l u b i l i z a t i o n o f some p l a s m a membrane DES s e n s i t i v e H + - A T P a s e s has r e s u l t e d i n a l o s s o f a c t i v i t y w h i c h c o u l d be r e s t o r e d by t h e r e c o n s t i t u t i o n o f t h e enzyme w i t h e x o g e n o u s p h o s p h o l i p i d s (Bowman e t a_l. , 1981a; V a r a and S e r r a n o , 1982; C o c u c c i and M a r r e , 1 9 8 4 ) . I n t h e c a s e o f t h e D. d i s c o i d e u m DES s e n s i t i v e A T P a s e , however, a d d i t i o n o f p h o s p h a t i d y l c h o l i n e t o c 1 2 E q s o l u b i l i z e d p l a s m a membranes f a i l e d t o r e c o n s t i t u t e a DES s e n s i t i v e ATPase ( T a b l e V I ) . M o r e o v e r , l o s s o f DES s e n s i t i v e enzyme a c t i v i t y o c c u r r e d e ven when p l a s m a membranes were s o l u b i l i z e d w i t h l y s o l e c i t h i n ( F i g . 1 3 a ) . T h u s , t h e i n a c t i v a t i o n o f DES s e n s i t i v e ATPase was p r o b a b l y n o t due t o d e l i p i d a t i o n . I n a d d i t i o n t o t h e i r d i s s i m i l a r s t a b i l i t i e s t h e DES s e n s i t i v e and i n s e n s i t i v e a c t i v i t i e s had o t h e r d i s t i n c t i v e 102 p r o p e r t i e s . The DES s e n s i t i v e A TPase d i s p l a y e d a pH optimum between pH 7.0 t o 7.5 w h i l e t h e DES i n s e n s i t i v e ATPase had a b r o a d optimum between pH 6.0 and 7.5 ( F i g . 1 8 ) . F u r t h e r m o r e , w h i l e t h e DES i n s e n s i t i v e ATPase e x p r e s s e d s i m i l a r a c t i v i t i e s i n t h e p r e s e n c e o f e i t h e r M g + 2 o r Ca + 2 , t h e DES s e n s i t i v e enzyme d i s p l a y e d a d i s t i n c t p r e f e r e n c e f o r M g + 2 ( F i g s . 14 and 1 5 ) . I n t e r e s t i n g l y , r e p l a c e m e n t o f Mg + 2 w i t h C a + 2 d i d n o t r e s u l t i n a change i n t h e a p p a r e n t Km (Km = 0.42 mM) o f t h e DES s e n s i t i v e ATPase f o r s u b s t r a t e b u t r a t h e r a f f e c t e d o n l y t h e v e l o c i t y o f t h e r e a c t i o n ( F i g . 1 6 ) . D o u b l e r e c i p r o c a l p l o t s o f ATP c o n c e n t r a t i o n a g a i n s t v e l o c i t y f o r t h e DES i n s e n s i t i v e ATPase d i d n o t y i e l d l i n e a r r e l a t i o n s h i p s ( F i g . 1 7 ) , i n d i c a t i n g t h a t t h i s enzyme d i d n o t d i s p l a y M i c h a e l i s - M e n t e n k i n e t i c s . C o m p a r i s o n o f ATPase t o P r e v i o u s l y I d e n t i f i e d P l a s m a  Membrane A T p a s e s f r o m D. d i s c o i d e u m W h i l e t h i s work was i n p r o g r e s s Pogge-von Strandmann e t a l . (1984) a l s o d e s c r i b e d a DES s e n s i t i v e p l a s m a membrane A T P a s e , i n D. d i s c o i d e u m . t h a t was s o l u b i l i z e d by l y s o l e c i t h i n b u t n o t by o t h e r d e t e r g e n t s . The enzyme was f r a c t i o n a t e d o v e r s u c r o s e g r a d i e n t s and DES s e n s i t i v e p r o t o n t r a n s l o c a t i o n was d e m o n s t r a t e d i n r e c o n s t i t u t e d p h o s p h a t i d y l c h o l i n e v e s i c l e s . I n a d d i t i o n , t h i s enzyme was r e p o r t e d t o have a b r o a d pH optimum a r o u n d pH 6.8 w i t h l e s s t h a n 50? o f t h e a c t i v i t y b e l o w pH 5.7 and above pH 8.0. The a c t i v i t y was a l s o s e n s i t i v e t o t h e s u l f h y d r y l g r o u p r e a g e n t s C u + 2 and H g + 2 . Pogge-von Strandmann e t a l . (1984) a l s o 103 r e p o r t e d a Km o f 0.9 mM f o r M g + 2 - A T P h y d r o l y s i s measured by p h o s p h a t e r e l e a s e and 0.5 t o 1.5 mM f o r M g + 2 - A T P h y d r o l y s i s m easured by p r o t o n t r a n s l o c a t i o n . I t i s p o s s i b l e t h a t t h e DES s e n s i t i v e ATPase d e s c r i b e d i n t h i s t h e s i s i s t h e same enzyme t h a t was d e s c r i b e d by Pogge-von Strandmann e t a l . (1984) b u t a l l a t t e m p t s t o d e m o n s t r a t e p r o t o n pumping i n e i t h e r membrane v e s i c l e s o r r e c o n s t i t u t e d v e s i c l e s were u n s u c c e s s f u l ( d a t a n o t shown). I t was n o t c l e a r i f Pogge-von Strandmann e_t al. ( 1 984 ) were m e a s u r i n g a c o m b i n a t i o n o f t h e DES s e n s i t i v e and DES i n s e n s i t i v e a c t i v i t i e s i n t h e i r s t u d y , as no r e f e r e n c e was made t o a DES i n s e n s i t i v e A T P a s e . I n a d d i t i o n , o n l y d a t a p e r t a i n i n g t o DES s e n s i t i v e p r o t o n pumping as o p p o s e d t o A TPase a c t i v i t y was p r e s e n t e d . I t i s s u r p r i s i n g t h a t t h e y o b t a i n e d s u c h p o o r s o l u b i l i z a t i o n w i t h d e t e r g e n t s s u c h as CHAPS and T r i t o n X-100 and s u c c e s s f u l s o l u b i l i z a t i o n w i t h l y s o l e c i t h i n , c o n s i d e r i n g t h e f a c t t h a t i n t h e p r e s e n t s t u d y CHAPS and T r i t o n X-100 were o n l y s l i g h t l y l e s s e f f i c i e n t t h a n l y s o l e c i t h i n . S e r r a n o e_t a l . ( 1985) have a l s o r e c e n t l y r e p o r t e d an ATPase a c t i v i t y w h i c h was i n h i b i t e d 75$ by 150 u^M DES i n D_. d i s c o i d e u m p l a s m a membranes. T h i s a c t i v i t y was a l s o i n h i b i t e d by DCCD , v a n a d a t e and s u l f h y d r y l r e a g e n t s s u c h as C u + 2 and p - c h l o r o m e r c u r i p h e n y l s u l f o n a t e . The pH optimum f o r t h i s enzyme was r e p o r t e d a t pH 6.5 w i t h a 50$ r e d u c t i o n o f a c t i v i t y a t pH 7.5. The enzyme had a r e q u i r e m e n t f o r M g + 2 b u t was i n a c t i v e w i t h C a + 2 . The ATPase e x p r e s s e d h y p e r b o l i c 104 k i n e t i c s w i t h r e s p e c t t o Mg + 2_ATP w i t h an a p p a r e n t Km o f 0.1 mM. No a t t e m p t was made t o s o l u b i l i z e t h e a c t i v i t y o r d e m o n s t r a t e p r o t o n t r a n s l o c a t i o n and no r e f e r e n c e was made t o a DES i n s e n s i t i v e component. A l t h o u g h t h e DES s e n s i t i v e ATPase d e s c r i b e d i n t h i s t h e s i s i s p o s s i b l y a n a l o g o u s t o t h e w e l l c h a r a c t e r i z e d e l e c t r o g e n i c p r o t o n pumps i n t h e p l a s m a membrane o f N e u r o s p o r a and y e a s t , a t l e a s t two d i f f e r e n c e s e x i s t . One o f t h e s e d i f f e r e n c e s , n o t e d e a r l i e r , i s t h e i r r e v e r s i b l e i n s t a b i l i t y o f t h e D i c t y o s t e l i u m DES s e n s i t i v e ATPase upon s o l u b i l i z a t i o n . The s e c o n d d i f f e r e n c e i s t h a t when DES s e n s i t i v e ATPase a c t i v i t y was measured as a f u n c t i o n o f t h e Mg + 2 c o n c e n t r a t i o n , t h e amount o f ATP h y d r o l y s i s i n c r e a s e d w i t h i n c r e a s i n g M g + 2 t o a p o i n t a t w h i c h t h e r e was a 1 6 - f o l d e x c e s s i n t h e l e v e l o f M g + 2 r e l a t i v e t o t h e ATP c o n c e n t r a t i o n ( F i g . 1 4 ) . As t h e M g + 2 c o n c e n t r a t i o n was i n c r e a s e d f u r t h e r t h e enzyme a c t i v i t y d e c r e a s e d . I n c o n t r a s t t o t h e D. d i s c o i d e u m r e s u l t s , B r o o k e r and Slayman (1983b, 1983c) c h a r a c t e r i z e d t h e k i n e t i c s o f ATP h y d r o l y s i s w i t h r e g a r d t o d i v a l e n t c a t i o n r e q u i r e m e n t s f o r t h e N e u r o s p o r a DES s e n s i t i v e H + - A T P a s e , and f o u n d t h a t t h e enzyme f u n c t i o n e d o p t i m a l l y i n t h e p r e s e n c e o f e q u i m o l a r c o n c e n t r a t i o n s o f M g + 2 and ATP. E x c e s s M g + 2 was a l s o f o u n d t o i n h i b i t t h e N e u r o s p o r a enzyme ( B r o o k e r and Sl a y m a n , 1 9 8 3 c ) , b u t a t much l o w e r c o n c e n t r a t i o n s t h a n t h o s e o b s e r v e d f o r t h e D i c t y o s t e l i u m DES s e n s i t i v e A T P a s e . The 105 D i o t y o s t e l i u m and N e u r o s p o r a DES s e n s i t i v e A T P a s e s were s i m i l a r i n t h a t Mg + 2 was p r e f e r r e d o v e r C a + 2 . The f o r m e r enzyme, however, d i d e x p r e s s some enzyme a c t i v i t y i n t h e p r e s e n c e o f C a + 2 , whereas t h e N e u r o s p o r a ATPase was c o m p l e t e l y i n a c t i v e when C a + 2 s e r v e d as t h e c a t i o n (Bowman e t a l . , 1981; B r o o k e r and S l a y m a n , 1983b). D e v e l o p m e n t a l R o l e o f t h e DES S e n s i t i v e ATPase The p o t e n t i a l d e v e l o p m e n t a l r o l e o f t h e DES s e n s i t i v e ATPase r e m a i n s t o be e s t a b l i s h e d . G r o s s e t a l . (1983) s u g g e s t e d t h a t i n t r a c e l l u l a r pH c h a n g e s , e f f e c t e d by an i n h i b i t o r y i n t e r a c t i o n between t h e s t a l k c e l l d i f f e r e n t i a t i o n i n d u c i n g f a c t o r ( D I F ) and a c e l l s u r f a c e p r o t o n pump, p l a y an i m p o r t a n t d e t e r m i n a t i v e r o l e i n s t a l k c e l l d e v e l o p m e n t . C o m p e l l i n g e v i d e n c e s u p p o r t i n g t h i s h y p o t h e s i s was o b t a i n e d when i t was o b s e r v e d t h a t DES m i m i c k e d t h e e f f e c t o f DIF i n i n d u c i n g s t a l k c e l l d e v e l o p m e n t i n low d e n s i t y m o n o l a y e r s ( G r o s s e t a l _ . , 1983). However, p a r t i a l l y p u r i f i e d DIF had no e f f e c t w h a t s o e v e r on ATPase a c t i v i t y ( T a b l e X I X ) . I n a d d i t i o n , r e c e n t work by Kay e t a l . (1986) f a i l e d t o show a d e c r e a s e i n i n t r a c e l l u l a r pH, as d e t e r m i n e d by ^ P-NMR s p e c t r o s c o p y , when d i f f e r e n t i a t i n g c e l l s were e x p o s e d t o DIF o r DES. A l t h o u g h i t a p p e a r s t h a t DIF and DES p r o b a b l y e f f e c t d i f f e r e n t i a t i o n by some mechanism o t h e r t h a n i n f l u e n c i n g i n t r a c e l l u l a r pH i t s h o u l d be n o t e d t h a t t h e f i n d i n g s o f Kay e t a l . (1986) and o t h e r s ( J e n t o f t and Town, 1985; R a t n e r , 1986) w h i c h f a i l t o show q u a n t i t a t i v e c h a n g e s i n 106 i n t r a c e l l u l a r pH do n o t n e c e s s a r i l y mean t h a t pH c h a n g e s do n o t o c c u r . I t i s e n t i r e l y p o s s i b l e t h a t any pH change may be l o c a l i z e d a t a s p e c i f i c p o i n t e g . t h e i n n e r s u r f a c e o f t h e p l a s m a membrane, s i n c e i f t h i s were t h e c a s e i t i s u n l i k e l y t h a t s u c h a pH change would be d e t e c t e d . I n a d d i t i o n , Kay e_t a l _ . ( 1986) d i d n o t examine t h e e f f e c t s o f DIF on ATPase a c t i v i t y and i t i s p o s s i b l e t h a t t h e o b s e r v a t i o n made i n t h i s t h e s i s , s h o w i n g t h a t DIF had no e f f e c t on ATPase a c t i v i t y ( T a b l e X I X ) , may have been due t o t h e r e m o v a l o f a f a c t o r n e c e s s a r y t o c o n f e r DIF s e n s i t i v i t y t o t h e enzyme. C l e a r l y , p r o t o n f l u x p l a y s some r o l e i n D_. d i s c o i d e u m d e v e l o p m e n t s i n c e G r o s s e_t a_l. (1983) o b s e r v e d t h a t weak a c i d s o r m i l d l y a c i d i c c o n d i t i o n s i n f l u e n c e d s t a l k c e l l d i f f e r e n t i a t i o n whereas n e u t r a l o r m i l d l y a l k a l i n e c o n d i t i o n s f a v o u r e d s p o r e s when c e l l s were p l a t e d o u t i n submerged m o n o l a y e r s , b u t i t r e m a i n s t o be e s t a b l i s h e d w h e t h e r t h e DES s e n s i t i v e ATPase p l a y s any r o l e i n t h e s e p r o t o n movements. J a m i e s o n e t a l . (1984) have p r e s e n t e d e v i d e n c e s u g g e s t i n g a p o s s i b l e r o l e o f i n c r e a s e d c e l l u l a r pH i n t h e i n i t i a t i o n o f D_. d i s c o i d e u m d e v e l o p m e n t , m e d i a t e d t h r o u g h a N a + / H + a n t i p o r t e r , r a t h e r t h a n a DES s e n s i t i v e H + - A T P a s e . They f o u n d t h a t a t r a n s i e n t (10 min) i n t r a c e l l u l a r a l k a l i n i z a t i o n i n c e l l s w h i c h had been s t a r v e d f o r two h o u r s was a b o l i s h e d when t h e c e l l s were e x p o s e d t o t h e N a + / H + a n t i p o r t a n t a g o n i s t a m i l o r i d e . F u r t h e r e v i d e n c e s u p p o r t i n g t h e r o l e o f a p u t a t i v e a n t i p o r t e r was o b t a i n e d when i t was 107 o b s e r v e d t h a t a g g r e g a t i o n was d e l a y e d when c e l l s were p l a t e d o u t i n t h e p r e s e n c e o f a r a i l o r i d e ( J a m i e s o n e t a ^ . , 1 9 8 4 ) . P r e v i o u s l y i t had been n o t e d t h a t a low e x t e r n a l N a + c o n c e n t r a t i o n a l s o d e l a y e d a g g r e g a t i o n ( M a r i n and Rothman, 1980. Malchow e t a_l. ( 1978a,b) had p r e v i o u s l y o b s e r v e d e x t r a c e l l u l a r pH o s c i l l a t i o n s i n s u s p e n s i o n s o f a g g r e g a t i o n c o m p e t e n t c e l l s . M o r e o v e r , t h e s e pH p u l s e s were shown t o be d e p e n d e n t on cAMP s i g n a l s (Malchow e t a l . , 1978b, Gottman and W e i j e r , 1 9 8 6 ) . The e x a c t n a t u r e o f t h e s e o s c i l l a t i o n s i s n o t known. They a r e p o s s i b l y due i n p a r t t o t h e h y d r o l y s i s o f e x t r a c e l l u l a r cAMP by p h o s p h o d i e s t e r a s e b u t t h e m a g n i t u d e o f t h e p r o t o n p u l s e was c o n s i d e r a b l y h i g h e r t h a n t h a t o f t h e cAMP p u l s e (Malchow e t a l . . , 1978b) and weak a c i d and CO2 e x t r u s i o n were a l s o c i t e d as p o s s i b l e a l t e r n a t i v e s o u r c e s f o r t h e pH change (Malchow e t a l _ . , 1978a, 1 9 7 8 b ) . I t i s p o s s i b l e t h a t t h e pH o s c i l l a t i o n s a r e m e d i a t e d by p r o t o n e x t r u s i o n t h r o u g h a p u t a t i v e N a + / H + a n t i p o r t e r . I f t h i s were t h e c a s e i t s h o u l d be p o s s i b l e t o a b o l i s h t h e pH p u l s e s by t h e a d d i t i o n o f a m i l o r i d e . S i m i l a r l y , i f t h e p u t a t i v e p r o t o n pump p l a y s a r o l e i n t h e r e g u l a t i o n o f t h e s e pH o s c i l l a t i o n s i t s h o u l d be p o s s i b l e t o a b o l i s h t h e p u l s e s by t h e a d d i t i o n o f DES. C o m p a r i s o n t o P y r o p h o s p h a t a s e When s o l u b i l i z e d p l a s m a membranes were f r a c t i o n a t e d o v e r an i o n e x c h a n g e column two d i s t i n c t DES i n s e n s i t i v e a c t i v i t i e s ( Peaks I and I I ) were d e t e c t e d ( F i g . 1 9 ) . 108 P y r o p h o s p h a t e was h y d r o l y z e d more r a p i d l y t h a n n u c l e o s i d e t r i p h o s p h a t e s by peak I enzyme ( T a b l e X I I I ) , s u g g e s t i n g t h a t t h i s enzyme might be an i n o r g a n i c p y r o p h o s p h a t a s e . The enzyme a l s o had a d i s t i n c t p r e f e r e n c e f o r M g + 2 when p y r o p h o s p h a t e was t h e s u b s t r a t e and a c t i v i t y was g r e a t e r a t a l k a l i n e pH ( F i g . 2 7 ) . T h i s enzyme was a l s o i n s e n s i t i v e t o va n a d a t e ( T a b l e X I I I ) . Enzymes w i t h c h a r a c t e r i s t i c s s i m i l a r t o the p y r o p h o s p h a t e h y d r o l y z i n g a c t i v i t y d e s c r i b e d above have been i s o l a t e d from t h e t o n o p l a s t membrane o f o a t r o o t s (Wang ejt a l . , 1986) and b e e t s ( K a r l s s o n , 1975; Walk e r and L e i g h , 1981b; Rea and P o o l e , 1985, 1986; T a b l e X X ) . These enzymes were found t o be v a n a d a t e i n s e n s i t i v e and were s t i m u l a t e d c o n s i d e r a b l y by K + ( K a r l s s o n , 1975 ; W a l k e r and L e i g h , 1981b; Wang e t a l . , 1986) and to a l e s s e r e x t e n t by Rb + ( K a r l s s o n , 1975) and C s + (Wang e_t a l . , 19 86 ) . The p y r o p h o s p h a t a s e s a l s o e x p r e s s e d a d i v a l e n t c a t i o n r e q u i r e m e n t f o r M g + 2 w i t h a s m a l l amount o f a c t i v i t y w i t h M n + 2 but none w i t h C a + 2 (Walker and L e i g h , 1981; Rea and P o o l e , 1986 ; Wang e_t a l . , 1986) and enzyme a c t i v i t y was g r e a t e s t a t a l k a l i n e pH (Rea and P o o l e , 1985 ) . The t o n o p l a s t i n o r g a n i c p y r o p h o s p h a t a s e was a l s o shown to t r a n s l o c a t e p r o t o n s (Rea and P o o l e , 1986 ; Wang e t a l . . 1986), a phenomenon w h i c h was s e n s i t i v e t o f l u o r i d e , NEM and DCCD ( T a b l e XX), but was i n s e n s i t i v e t o 4,4-d i i s o t h i o c y a n o s t i l b e n e - 2 , 2 - d i s u l f o n a t e (DIDS) and KNC^ -(Wang e t a l . , 1 986 ) . Rea and P o o l e (1985, 1986) a l s o 109 TABLE XX COMPARISON OF THE PURIFIED D. DISCOIDEUM PLASMA MEMBRANE ATPASE WITH SEVERAL OTHER MEMBRANE BOUND PHOSPHATASES ATPase D. discoideum DES Insensitive H +-ATPase ATPase Tonoplast (Ca + 2:Mg + 2)-ATPase C a + 2 o r Mg + 2-ATPase ATPase Pyrophosphatase Inhibitor vanadate DES DCCD fluo r i d e ouabain NEM + N.D. + + + N.D. + N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. + + N.D. + + Membrane o r i g i n P.M. P.M. P.M. P.M. Vacuolar membrane Vacoular membrane Molecular weight 64 kDa 104 kDa 130-140 kDa N.D. Several subunlts 20 - 90 kDa N.D. N.D.= not determined; P.M.= plasma membrane. For references see the appropriate sections of the text. r e p o r t e d a c o m p l e t e l a c k o f enzyme a c t i v i t y when t h e p y r o p h o s p h a t a s e o f b e e t r o o t was a s s a y e d w i t h n u c l e o t i d e s . I t i s p o s s i b l e t h a t t h e J). d i s c o i d e u m p y r o p h o s p h a t e h y d r o l y z i n g a c t i v i t y a l s o f u n c t i o n s as a p r o t o n pump. E n r i c h m e n t o f p y r o p h o s p h a t a s e a c t i v i t y was n o t f o l l o w e d d u r i n g t h e c o u r s e o f t h i s t h e s i s and i t i s c o n c e i v a b l e t h a t t h e a p p a r e n t c e l l s u r f a c e l o c a l i z a t i o n o f t h e p y r o p h o s p h a t e h y d r o l y z i n g a c t i v i t y was due t o c o n t a m i n a t i o n o f t h e p l a s m a membrane p r e p a r a t i o n w i t h c o n t r a c t i l e v a c u o l a r o r l y s o s o m a l membranes. V a c u o l a r membranes f r o m D i c t y o s t e l i u m have n o t been p u r i f i e d , p o s s i b l y due t o t h e l a c k o f k n o w l e d g e r e g a r d i n g s p e c i f i c membrane m a r k e r s . Q u i v i g e r e_t a l . . (1978, 1980) and Glomp e t a l . (1985) have p r o v i d e d h i s t o c h e m i c a l e v i d e n c e f o r a l k a l i n e p h o s p h a t a s e on D_. d i s c o i d e u m c o n t r a c t i l e v a c u o l a r membranes o f and, s i n c e t h e v a c u o l a r and p l a s m a membranes f u s e a t t h e t e r m i n a l s t a g e o f t h e c o n t r a c t i l e p r o c e s s ( M u r r a y , 1 9 8 2 ) , i t i s p o s s i b l e t h a t t h e two membrane t y p e s a r e s i m i l a r i n terms o f t h e i r m o l e c u l a r c o m p o s i t i o n . Peak I I a c t i v i t y was v a n a d a t e s e n s i t i v e and was p u r i f i e d v i a two d i f f e r e n t p r o c e d u r e s . The f i r s t method i n v o l v e d s e p a r a t i o n on an i o n e x c h a n g e c o l u m n , f o l l o w e d by c e n t r i f u g a t i o n t h r o u g h a g l y c e r o l g r a d i e n t . T h i s p r o c e d u r e r e s u l t e d i n a r e l a t i v e l y homogeneous p r e p a r a t i o n w i t h a s i n g l e m a j o r p r o t e i n band o f 64 kDa, as shown by SDS-PAGE ( F i g . 2 0 ) , b u t t h e r e c o v e r y o f enzyme was low ( T a b l e I X ) . A s e c o n d method w h i c h u s e d an i n i t i a l s e p a r a t i o n by g e l 11 1 f i l t r a t i o n on S e p h a c r y l S-300 i n t h e p r e s e n c e o f 0.5$ CHAPS, f o l l o w e d by i o n e x c h a n g e c h r o m a t o g r a p h y , gave t h e same 64 kDa p r o t e i n ( F i g . 23) w i t h a t w o - f o l d h i g h e r y i e l d ( T a b l e X I I I ) . A c o m p a r i s o n o f t h e s e d i m e n t a t i o n v e l o c i t i e s o f s e v e r a l p r o t e i n s o f known m o l e c u l a r w e i g h t s r e v e a l e d 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 o f t h e a c t i v e ATPase t o be between 65 and 77 kDa. In o r d e r t o c o n f i r m t h a t t h e 64 kDa band c o r r e s p o n d e d t o t h e A T P a s e , a t t e m p t s were made t o p h o s p h o r y l a t e t h e p r o t e i n , s i n c e p h o s p h o p r o t e i n i n t e r m e d i a t e s have been o b s e r v e d f o r many o t h e r p l a s m a membrane A T P a s e s (Lane e_t a l . , 1973; W a l l i c k e t a l . , 1978; Dame and S c a r b o r o u g h , 1980; Amory e t a l . , 1980; B r i s k e n and P o o l e , 1 9 8 3 ) . I n i t i a l r e s u l t s u s i n g membranes w h i c h had been s t o r e d a t -70°C r e v e a l e d t h a t t h e p h o s p h o r y l a t e d p r o t e i n s f a i l e d t o e n t e r t h e g e l ( F i g . 3 5 ) , a p r o b l e m w h i c h had p r e v i o u s l y been e n c o u n t e r e d w i t h p h o s p h o r y l a t e d a l k a l i n e p h o s p h a t a s e f r o m D_. d i s c o i d e u m ( P . B h a n o t , 1 9 8 6 ) . S p e c i f i c p h o s p h o r y l a t e d bands were f a i n t l y d e t e c t e d when f r e s h l y p r e p a r e d membranes were u s e d , i n d i c a t i n g t h a t some p h o s p h o p r o t e i n s were e n t e r i n g t h e g e l , b u t t h e r e was no p h o s p h o r y l a t i o n o f t h e 64 kDa p r o t e i n ( F i g . 3 6 ) . No p h o s p h o r y l a t i o n was o b s e r v e d w i t h t h e p u r i f i e d p r o t e i n . I t must be s t r e s s e d t h a t i f t h e ATPase formed a - a s p a r t y l p h o s p h a t e i n t e r m e d i a t e i t m i g h t be s u s c e p t i b l e t o h y d r o l y s i s u n d e r t h e e l e c t r o p h o r e t i c c o n d i t i o n s u s e d h e r e . The same e x p e r i m e n t s were a t t e m p t e d u s i n g a c i d g e l s (Amory e t a l . , 1 9 8 0 ) , b u t t h e p r o t e i n s f a i l e d t o m i g r a t e as 1 12 d i s c r e t e bands and no p h o s p h o p r o t e i n s were d e t e c t e d ( d a t a n o t shown). I t i s l i k e l y t h a t t h i s ATPase does form a p h o s p h o p r o t e i n i n t e r m e d i a t e s i n c e the enzyme i s v a n a d a t e s e n s i t i v e ( F i g . 2 9 ) . V a n a date s e n s i t i v i t y a p p e a r s t o be a common p r o p e r t y among p h o s p h o h y d r o l a s e s w h i c h form c o v a l e n t a t t a c h m e n t s w i t h p h o s p h a t e d u r i n g t h e c o u r s e o f t h e i r r e a c t i o n c y c l e s ( S z e , 1985). I t i s b e l i e v e d t h a t t h e i n h i b i t o r y e f f e c t o f v a n a d a t e i s due t o i t s c o m p e t i t i o n w i t h p h o s p h a t e f o r b i n d i n g , t h u s p r e v e n t i n g the r a p i d t u r n o v e r o f t h e enzyme ( S z e , 1985). N e i t h e r t h e m i t o c h o n d r i a l nor t h e t o n o p l a s t ATPases form p h o s p h o p r o t e i n i n t e r m e d i a t e s and n e i t h e r o f t h e s e enzymes a r e s e n s i t i v e t o v a n a d a t e (Amzel e_t a l . , 1983; S z e , 1985). C h a r a c t e r i z a t i o n , of jLha p u r i f i e d PES i n s e n s i t i v e ATPase from D. d i s c o i d e u m The p u r i f i e d DES i n s e n s i t i v e ATPase i s u n i q u e i n a number o f ways when compared t o o t h e r w e l l c h a r a c t e r i z e d c e l l s u r f a c e A TPases ( T a b l e X X ) . The 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 the enzyme was c o n s i d e r a b l y l e s s t h a n t h a t o b s e r v e d f o r t h e H + - A T P a s e s ( 104 kDa) o f N e u r o s p o r a (Bowman e_t a l . , 1981a; A d d i s o n and S c a r b o r o u g h , 1981), y e a s t ( D u f o u r and G o f f e a u , 1978) and p l a n t s ( S z e , 1985), f o r the ( C a + 2 : M g + 2 ) - A T P a s e s ( 130 t o 140 kDa; De Smedt e t a l . , 1983; D e b e t t o and C a n t l e y , 1984; Ansah e t a l _ . , 1984) and f o r t h e c a t a l y t i c s u b u n i t o f t h e N a + , K + - A T P a s e s ( a p p r o x i m a t e l y 121 kDa; C r a i g and K y t e , 1980). The p u r i f i e d D i c t y o s t e l i u m ATPase was a l s o s t i m u l a t e d by d i v a l e n t c a t i o n s , i n c l u d i n g 113 Z n + 2 and C u + , but not by monovalent c a t i o n s ( F i g . 33, T a b l e XVI). In c o n t r a s t , the H +-ATPases o f N e u r o s p o r a and p l a n t s were n o t s t i m u l a t e d by Ca+ 2 o r Z n + 2 (Bowman e t aJL., 1981a; O ' N e i l l and S p a n s w i c k , 1984b), a l t h o u g h a c e r t a i n d e g r e e o f a c t i v i t y was o b s e r v e d w i t h M n + 2 ( T a b l e XX), but were s t i m u l a t e d by m o n o v a l e n t c a t i o n s . The H +-ATPase o f r a d i s h ( C o c u c c i and M a r r e , 1984) and t h e C a + 2 - A T P a s e (Ansah e t a l . , 1984) o f p a n c r e a t i c a c i n a r c e l l plasma membranes were a l s o c o m p a r a t i v e l y a c t i v e w i t h Z n + 2 as the c a t i o n , a l t h o u g h i n b o t h c a s e s t h e a c t i v i t y was l o w e r than t h a t seen w i t h M g + 2 . The p u r i f i e d D . d i s c o i d e u m ATPase was a l s o i n s e n s i t i v e t o NEM ( F i g . 30) s u g g e s t i n g t h a t t h e NEM i n h i b i t i o n o f plasma membrane bound ATPase a c t i v i t y ( F i g . 6) was due t o i n a c t i v a t i o n o f t h e DES s e n s i t i v e ATPase. A wide v a r i e t y o f ATPases ( T a b l e XX), i n c l u d i n g the N a + , K + - A T P a s e ( W a l l i c k e t a l . , 1978), a r e s e n s i t i v e t o NEM. Walderhaug e t a l . (1985) have d e t e r m i n e d t h e amino a c i d sequence o f t h e a c t i v e s i t e s o f t h e N a + , K + - A T P a s e , the K + , H + - A T P a s e and t h e H +-ATPase from c o r n r o o t p l a s m a membranes t o be - c y s - ( s e r / t h r ) - a s p -l y s - and i t i s b e l i e v e d t h a t NEM i n h i b i t i o n i s due t o t h e c o v a l e n t m o d i f i c a t i o n o f the c y s t e i n e r e s i d u e ( B r o o k e r and Slayman, 1982, 19 8 3 a , b ) . These d a t a s u g g e s t a f u n d a m e n t a l d i f f e r e n c e i n the a c t i v e s i t e between t h e s e ATPases and the p u r i f i e d _D. d i s c o i d e u m ATPase. A l t h o u g h t h e p u r i f i e d ATPase was s t i m u l a t e d by M g + 2 o r C a + 2 , enzyme a c t i v i t y was i n c r e a s i n g l y i n h i b i t e d as t h e c o n c e n t r a t i o n o f e i t h e r o f t h e s e c a t i o n s was i n c r e a s e e d 114 above 10 mM ( F i g . 32 ) . The enzyme was a l s o i n h i b i t e d by m i l l i m o l a r c o n c e n t r a t i o n s o f M g + 2 and C a + 2 when b o t h i o n s were p r e s e n t ( F i g . 31) a l t h o u g h a t l o w e r t o t a l c a t i o n c o n c e n t r a t i o n s t h a n t h a t o b s e r v e d when t h e i o n s were p r e s e n t i n d i v i d u a l l y . The plasma membrane H + - A T P a s e s o f N e u r o s p o r a ( B r o o k e r and Slayman, 1983b) and r a d i s h e s ( C o c c u c i and M a r r e , 1984) were a l s o shown t o be i n h i b i t e d by C a + 2 i n the p r e s e n c e o f M g + 2 . In t h e c a s e o f t h e N e u r o s p o r a enzyme i t was s u g g e s t e d t h a t C a + 2 i n h i b i t e d ATPase a c t i v i t y by c o m p e t i n g w i t h M g + 2 a t a h i g h a f f i n i t y b i n d i n g s i t e a t which M g + 2 b i n d i n g was n e c e s s a r y f o r the a c t i v a t i o n o f the enzyme ( B r o o k e r and Slayman, 1983b). Such a mechanism i s , however, i n s u f f i c i e n t t o e x p l a i n t h e combined C a + 2 / M g + 2 i n d u c e d i n h i b i t i o n o f t h e p u r i f i e d D. d i s c o i d e u m ATPase as t h i s enzyme was s t i m u l a t e d e q u a l l y be e i t h e r M g + 2 or C a + 2 a l o n e ( F i g . 32, T a b l e XV ). N e i t h e r the N e u r o s p o r a nor the r a d i s h H +-ATPase d i s p l a y e d any a c t i v i t y when M g + 2 was r e p l a c e d w i t h C a + 2 . A s u b s t a n t i a l i n c r e a s e i n s u b s t r a t e a f f i n i t y was o b s e r v e d w i t h a M g + 2 o r C a + 2 c o n c e n t r a t i o n (1 mM) a t which most o f the ATP would be uncomplexed w i t h t h e d i v a l e n t c a t i o n ( F i g . 3 4 ) . A t e n - f o l d i n c r e a s e i n t h e M g + 2 or C a + 2 . c o n c e n t r a t i o n d i d n o t r e s u l t i n a s u b s t a n t i a l i n c r e a s e i n s u b s t r a t e a f f i n i t y o v e r and above t h a t o b s e r v e d a t 1 mM c a t i o n ( F i g . 3 4 ) . Thus, the i n c r e a s e i n s u b s t r a t e a f f i n i t y i n t h e p r e s e n c e o f M g + 2 or C a + 2 was n o t due t o t h e enzyme e x p r e s s i n g a g r e a t e r a f f i n i t y f o r the A T P - d i v a l e n t c a t i o n 115 complex. From t h e s e d a t a i t i s p o s s i b l e to p o s t u l a t e t h e p r e s e n c e o f a t l e a s t one d i s t i n c t d i v a l e n t c a t i o n b i n d i n g s i t e a t wh i c h b i n d i n g o f M g + 2 0 r C a + 2 s e r v e s t o a c t i v a t e t h e enzyme by i n c r e a s i n g i t s a f f i n i t y f o r s u b s t r a t e . The r e d u c t i o n i n v e l o c i t y o b s e r v e d w i t h 100 mM M g + 2 o r C a + 2 may i n d i c a t e an a d d i t i o n a l d i v a l e n t c a t i o n b i n d i n g s i t e , o f l o w e r a f f i n i t y t h a n t h e f i r s t , a t w h i c h M g + 2 o r C a + 2 b i n d i n g i n h i b i t s , r a t h e r t h a n a c t i v a t e s , t h e enzyme. The i n h i b i t i o n i n d u c e d u n d e r c o n d i t i o n s o f e x c e s s d i v a l e n t c a t i o n ( F i g s . 32 and 34) was n o t s i m p l y an i o n i c e f f e c t , as enzyme a c t i v i t y was u n a f f e c t e d by h i g h c o n c e n t r a t i o n s o f N a C l o r KC1. P h y s i o l o g i c a l R o l e o f t h e D. d i s c o i d e u m DES I n s e n s i t i v e  ATPase The p h y s i o l o g i c a l r o l e o f t h e p u r i f i e d J). d i s c o i d e u m ATPase i s n o t known. I t i s u n l i k e l y t h a t i t c o r r e s p o n d s t o th e D. d i s c o i d e u m p l a s m a membrane A T P - p y r o p h o s p h o h y d r o l a s e d e s c r i b e d by Rossomando and Hodge - J a h n g e n ( 1 9 8 3 ) , as t h e l a t t e r enzyme was r e p o r t e d t o be v a n a d a t e i n s e n s i t i v e . I t i s a l s o u n l i k e l y t h a t t h e p u r i f i e d ATPase i s a v a c u o l a r ATPase o f t h e t y p e d e s c r i b e d i n p l a n t s ( R a n d a l l and S z e , 1986 ) , y e a s t ( O c h i d a e t a l . , 1985) and N e u r o s p o r a (Bowman, 1983; T a b l e X X ) . The v a c u o l a r A T P a s e s a r e i n s e n s i t i v e t o v a n a d a t e ( C h u r c h i l l and S z e , 1984; S z e , 1985; Bowmann, 1983; O c h i d a e t a l . , 1985) and f l u o r i d e (Wang e t a l . , 1986) but a r e s e n s i t i v e t o DCCD ( W a l k e r and L e i g h , 1981a; Bowman, 1983; O c h i d a e t a l . , 1985; R a n d a l l and S z e , 1986) and DES (W a l k e r and L e i g h , 1981; O c h i d a e t a l . , 1 9 8 5 ) . Bowman 116 ( 1 9 8 3 ) and Bowman e t a_l, ( 1 9 8 5 ) have s u g g e s t e d t h a t the v a c u o l a r ATPase i s s i m i l a r to t h e m i t o c h o n d r i a l F Q p - A T P a s e . A more l i k e l y p o s s i b i l i t y i s t h a t the p u r i f i e d ATPase i s a n o n - s p e c i f i c C a + 2 or M g + 2 - A T P a s e s i m i l a r t o the enzymes t h a t have been i d e n t i f i e d i n t h e pla s m a membranes o f p l a c e n t a (Shami and Radde, 1 9 7 1 ) , k i d n e y ( P a r k i n s o n and Radde, 1 9 7 1 ) , l i v e r ( G a r n e t t and Kemp, 1 9 7 5 ; L o t e r s z t a j n e t a l . , 1 9 8 1 ) , c o r p u s l u t e u m (Verma and P e n n i s t o n , 1 9 8 1 ) and n e u t r o p h i l c e l l s (Ochs and Reed, 1 9 8 4 ) . I n i t i a l work on t h e enzymes from p l a c e n t a and r a t k i d n e y c o r t e x r e v e a l e d t h a t ATPase a c t i v i t y was s t i m u l a t e d by M n + 2 i n a d d i t i o n t o C a + 2 and M g + 2 , but n o t by S r + 2 (Shami and Radde, 1 9 7 1 ; P a r k i n s o n and Radde, 1 9 7 1 ) . S t i m u l a t i o n by M n + 2 d i d n o t exceed t h a t o b s e r v e d w i t h e i t h e r M g + 2 or C a + 2 . Both o f t h e s e enzymes were r e s i s t a n t t o a z i d e and e x p r e s s e d o p t i m a l ATPase a c t i v i t y between pH 7 . 5 t o 9 . 0 , v a l u e s t h a t a r e somewhat h i g h e r t h a n t h o s e o b s e r v e d f o r t h e p u r i f i e d D i c t y o 3 t e l i u m enzyme ( F i g . 2 6 ) . V e r y l i t t l e s u b s e q u e n t work has been done on the n o n s p e c i f i c C a + 2 or M g + 2 - A T P a s e s and t h e i r p h y s i o l o g i c a l r o l e r e m a i n s u n c l e a r , a l t h o u g h A v i s s a r e t a l . ( 1 9 7 5 ) s u g g e s t e d t h e e r y t h r o c y t e p l a s m a membrane C a + 2 o r M g + 2 - A T P a s e t o be a m y o s i n - l i k e ATPase and p o s s i b l y p l a y some r o l e i n t h e c o n t r a c t i l e p r o c e s s o f t h e c e l l , s i n c e t h e a c t i v i t y was s t i m u l a t e d by a c t i n . Thus i t i s c o n c e i v a b l e t h a t the D. d i s c o i d e u m ATPase m i g h t have a f u n c t i o n i n m o t i l i t y . 1 1 7 P e n n i s t o n (1983) has s u g g e s t e d t h a t one o f t h e c r i t e r i a t o be a d d r e s s e d i n o r d e r t o a s c r i b e a C a + 2 pumping f u n c t i o n t o a C a + 2 s t i m u l a t e d ATPase i s t h a t i t s h o u l d be s t i m u l a t e d by f r e e C a + 2 c o n c e n t r a t i o n s _< 10 ^iM i n t h e p r e s e n c e o f c a l m o d u l i n . The p u r i f i e d JD. d i s c o i d e u m ATPase was n o t s t i m u l a t e d by C a + 2 c o n c e n t r a t i o n s u n d e r 1 mM ( F i g . 32) n o r was enzyme a c t i v i t y s t i m u l a t e d by C a + 2 / c a l m o d u l i n ( T a b l e X V I I I ) . However, t h e p o s s i b i l i t y t h a t t h i s ATPase f u n c t i o n s as a h i g h a f f i n i t y C a + 2 pump c a n n o t be r u l e d o u t as i t i s p o s s i b l e t h a t t h e c a l m o d u l i n b i n d i n g s i t e was e i t h e r l o s t o r d e n a t u r e d d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e . I f t h i s were t h e c a s e t h e n t h e low C a + 2 a f f i n i t y c o u l d be e x p l a i n e d as t h e a c t i o n o f c a l m o d u l i n would be t o i n c r e a s e t h e a p p a r e n t a f f i n i t y o f t h e ATPase f o r C a + 2 ( R o u f a g a l i s , 1979; N i g g l i e t a l . , 1981; K o t a g a l e t a l _ . , 1983; P e n n i s t o n , 1983 , Ansah e_t a l . , 1984; C a r a f o l i e t a l . , 1984) P l a s m a membrane bound ATPase f r o m D. d i s c o i d e u m was s i m i l a r l y u n a f f e c t e d by e x o g e n o u s c a l m o d u l i n even t h o u g h t h e membranes had been e x t r a c t e d w i t h EGTA o r KC1/EDTA t o e n s u r e r e m o v a l o f any endogenous c a l m o d u l i n ( J a r r e t t and P e n n i s t o n , 1 9 7 8 ) . A g a i n , t h i s n e g a t i v e r e s u l t c o u l d be due t o t h e r e m o v a l o f an i n t e g r a l membrane component n e c e s s a r y i n c o n f e r r i n g c a l m o d u l i n s e n s i t i v i t y t o a p u t a t i v e C a + 2 pump. I t s h o u l d be n o t e d t h a t t h e s e a s s a y s were n o t p e r f o r m e d i n a C a + 2 / E G T A b u f f e r and t h a t t h e f a i l u r e t o o b s e r v e a c a l m o d u l i n s t i m u l a t e d , h i g h a f f i n i t y Ca + 2 - A T P a s e a c t i v i t y c o u l d be due t o t h e a b s e n c e o f EGTA. I t has been 118 s u g g e s t e d ( P e n n i s t o n , 1983; C a r a f o l i e t a l . . , 1984) t h a t t h e h i g h C a + 2 a f f i n i t y o f t h e ( C a + 2 : M g + 2 ) - A T P a s e s r e q u i r e s t h e p r e s e n c e o f EGTA. I n d e e d , K o t a g a l e t ajL. ( 1983) have o b s e r v e d EGTA t o mimic t h e e f f e c t o f c a l m o d u l i n i n a c t i v a t i n g t h e ( C a + 2 : M g + 2 ) - A T P a s e f r o m i s l e t c e l l p l a s m a membranes . P r e s u m a b l y D_. d i s c o i d e u m p l a s m a membranes do c o n t a i n a C a + 2 t r a n s p o r t e r s i n c e C a + 2 f l u x has been measured d u r i n g cAMP s i g n a l l i n g and ^ C a + 2 u p t a k e has been o b s e r v e d (Wick e t a l . , 1978; Bumann e t a l . , 1984; P a r i s h and W e i b e l , 1980; E u r o p e - F i n n e r and N e w e l l , 1 9 8 5 ) . T h i s p u t a t i v e C a + 2 t r a n s p o r t e r may be a h i g h a f f i n i t y ( C a + 2 - M g + 2 ) - A T P a s e w h i c h has e l u d e d d e t e c t i o n i n t h e p r e s e n t s t u d y and i t i s p o s s i b l e t h a t t h e e c t o - A T P a s e d e s c r i b e d by P a r i s h and W e i b e l (1980) i s a C a + 2 pump. E v i d e n c e f o r a f a c i l i t a t e d d i f f u s i o n mechanism f o r C a + 2 u p t a k e i n JJ. d i s c o i d e u m has been p r e s e n t e d ( E u r o p e - F i n n e r and N e w e l l , 1 9 8 5 a ) , b u t t h e p u t a t i v e t r a n s p o r t e r has n o t y e t been c h a r a c t e r i z e d . I t i s a l s o p o s s i b l e t h a t IK d i s c o i d e u m p l a s m a membranes c o n t a i n a N a + / C a + 2 e x c h a n g e r o f t h e t y p e d e s c r i b e d i n t h e g i a n t axon o f s q u i d , h e a r t t i s s u e and o t h e r e x c i t a b l e c e l l s . ( C a r a f o l i e t a l . , 1984) . F i n a l l y , i t i s p o s s i b l e t h a t t h e p u r i f i e d D. d i s c o i d e u m ATPase i s n o t a p l a s m a membrane enzyme b u t r e p r e s e n t s c o n t a m i n a t i o n w i t h a non m i t o c h o n d r i a l i n t r a c e l l u l a r membrane, and i s i n v o l v e d i n t h e r e g u l a t i o n o f i n t e r n a l Ca + 2 s t o r e s . I n t e r n a l C a + 2 m o b i l i z a t i o n has r e c e n t l y been 119 i m p l i c a t e d i n t h e s y n t h e s i s o f cGMP d u r i n g t h e e a r l y s t a g e s o f J). d i s c o i d e u m d i f f e r e n t i a t i o n ( E u r o p e - F i n n e r and N e w e l l , 1985; E u r o p e - F i n n e r e t a l . . , 1985; S m a l l e t a l . , 1 986; E u r o p e - F i n n e r and N e w e l l , 1 9 8 6 ) . R a b b i t s k e l e t a l m u s c l e t r a n s v e r s e t u b u l e membranes have been shown t o c o n t a i n a C a + 2 o r M g + 2 - A T P a s e w h i c h was c a p a b l e o f t r a n s p o r t i n g C a + 2 ( F e r n a n d e z e_t a _ l . , 1980; H i d a l g o ejt a l . , 1 9 8 3 ) , a l t h o u g h t h e m o l e c u l a r w e i g h t o f t h i s enzyme (112 kDa) was c o n s i d e r a b l y h i g h e r t h a n t h a t o f t h e p u r i f i e d D. d i s c o i d e u m A T P a s e . 120 R E F E R E N C E S A d d i s o n , R. and S c a r b o r o u g h , G.A. ( 1 9 8 1 ) . S o l u b i l i z a t i o n and p u r i f i c a t i o n o f the N e u r o s p o r a plasma membrane ATPase. J. B i o l . Chem. 256:13165-13171. A d d i s o n , R. and S c a r b o r o u g h , G.A. ( 1 9 8 2 ) . C o n f o r m a t i o n a l changes i n t h e N e u r o s p o r a plasma membrane H +-ATPase d u r i n g i t s c a t a l y t i c c y c l e . J . B i o l . Chem 257: 10421 -10426. Ames, B.N. ( 1 9 6 6 ) . A s s a y f o r i n o r g a n i c p h o s p h a t e , t o t a l p h o s p h a t e and p h o s p h a t a s e s . Methods i n Enzy m o l o g y . 8:115-118. Amory, A. F o u r y , F. and G o f f e a u , A. ( 1 9 8 0 ) . The p u r i f i e d plasma membrane ATPase o f t h e y e a s t S c h i z o s a c c h a r o m y c e s  pombe forms a p h o s p h o r y l a t e d i n t e r m e d i a t e . J . B i o l . Chem. 255:9353-9357. Amory, A. and G o f f e a u , A. ( 1 9 8 2 ) . C h a r a c t e r i z a t i o n o f t h e a s p a r t y l p h o s p h a t e i n t e r m e d i a t e formed by t h e H +-t r a n s l o c a t i n g ATPase from t h e y e a s t S c h i z o s a c c h a r o m y c e s  pombe. J . B i o l . Chem. 257:4723-4730. Amzel, L.M. and P e d e r s o n , P.L. ( 1 9 8 3 ) . P r o t o n A T P a s e s : S t r u c t u r e and mechanism. Ann. Rev. Biochem. 52:801-824. Ansah, T-A., M o l l a , A. and K a t z , S. ( 1 9 8 4 ) . C a + 2 _ A T P a s e a c t i v i t y i n p a n c r e a t i c a c i n a r plasma membranes. R e g u l a t i o n by c a l m o d u l i n and a c i d i c p h o s p h o l i p i d s . J . B i o l . Chem. 259:13442-13450. A v i s s a r , N., De V r i e s , A., B e n - S h a u l , Y. and Cohen, I . ( 1 9 7 5 ) . A c t i n a c t i v a t e d ATPase from human e r y t h r o c y t e s . B i o c h i m . B i o p h y s . A c t a . 375:35-43. Bhanot, P. ( 1 9 8 6 ) . S t u d i e s on the p - n i t r o p h e n y l p h o s p h a t e and a d e n o s i n e monophosphate h y d r o l y z i n g a c t i v i t y o f D i e t v o s t e l i u m d i s c o i d e u m . Ph.D. t h e s i s , 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 . B l a n c o , M. ( 1 9 8 2 ) . E v i d e n c e f o r t h e e x i s t e n c e o f a mono v a l e n t c a t i o n s t i m u l a t e d , M g + 2 _ d e p e n d e n t ATPase a c t i v i t y i n t h e i s o l a t e d p lasma membranes o f amoebas o f the 3lime mold D i c t y o s t e l i u m d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 687:94-96. Bonner, J.T. ( 1 9 4 7 ) . E v i d e n c e f o r t h e f o r m a t i o n o f c e l l a g g r e g a t e s by c h e m o t a x i s i n t h e d e v e l o p m e n t o f t h e s l i m e mold D i c t y o s t e l i u m d i s c o i d e u m . J . Exp. Z o o l . 1 0 6 : 1 - 2 6 . 121 B o n t i n g , S.L., Schuurmans S t e k h o v e n , F.M.A.H., S w a r t s , H.G.P. and d e P o n t , J.J.H.H.M. ( 1 9 7 9 ) . The low e n e r g y p h o s p h o r y l a t e d i n t e r m e d i a t e o f Na + -K + -ATPase . in_ Na +K + -ATPase: S t r u c t u r e and K i n e t i c s . S k o r e , J.C. and Norby, J.G. e d s . (Academic P r e s s , New Y o r k ) , pp. 317-330. Bowman, B . J . and Slayman, C.W. (1979). The e f f e c t s o f vanadate on the plasma membrane ATPase o f N e u r o s p o r a  c r a s s a . J . B i o l . Chem. 254:2928-2934 . Bowman, B . J . , B l a s c o , F. and Slayman, C.W. ( 1 9 8 1 a ) . 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 t h e plasma membrane ATPase o f N e u r o s p o r a c r a s s a . J . B i o l . Chem. 256: 1 2343-1 2349 . Bowman, E . J . , Bowman, B . J . and Slayman, C.W. (1981b). 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 pla s m a membranes from w i l d t y p e N e u r o s p o r a c r a s s a . J . B i o l . Chem. 256:12336-12342. Bowman, E . J . (1983). C o m p a r i s o n o f t h e v a c u o l a r membrane ATPase o f N e u r o s p o r a c r a s s a w i t h t h e m i t o c h o n d r i a l and plasma membrane ATPase. J . B i o l . Chem. 258:15238-15244. Bowman, E . J . , Ma n d a l a , S., T a i z , L. and Bowman, B . J . (1986). S t r u c t u r a l s t u d i e s o f t h e v a c u o l a r membrane ATPase from N e u r o s p o r a c r a s s a and c o m p a r i s o n w i t h the t o n o p l a s t membrane ATPase from Zea mays . P r o c . N a t l . A c a d . S c i . U.S.A. 83:48-52. B r a c h e t , P. and K l e i n , P. (1977). C e l l r e s p o n s i v e n e s s t o cAMP d u r i n g the a g g r e g a t i o n phase o f D i c t y o s t e l i u m  d i s c o i d e u m . C o m p a r i s o n between t h e i n h i b i t o r y a c t i o n o f p r o g e s t e r o n e and the s t i m u l a t o r y a c t i o n o f EGTA and i o n o p h o r e A 2 3 1 g 7 . D i f f e r e n t i a t i o n 8_:1-8. B r e i t b a r t , H., S t e r n , B. and R u b e n s t e i n , S. (1983). C a l c i u m t r a n s p o r t and C a + 2 - A T P a s e a c t i v i t y i n ram s p e r m a t o z o a n plasma membrane v e s i c l e s . B i o c h i m . B i o p h y s . A c t a . 728:349-355. B r e n n e r , M. and Thorns, S.D. ( 1 9 8 4 ) . C a f f e i n e b l o c k s a c t i v a t i o n o f cAMP s y n t h e s i s i n D i c t y o s t e l i u m  d i s c o i d e u m . Dev. B i o l . 1 01 : 1 36-1 46 . B r i s k e n , D.P. and P o o l e , R . J . ( 1 9 8 3 ) . E v i d e n c e f o r a a s p a r t y l p h o s p h a t e r e s i d u e i n t h e p h o s p h o r y l a t e d i n t e r m e d i a t e o f t h e r e d b e e t p l a s m a membrane ATPase. P l a n t . P h y s i o l . 72:1133-1135. B r i s k e n , D.P. and P o o l e , R . J . ( 1 9 8 4 ) . C h a r a c t e r i z a t i o n o f the s o l u b i l i z e d p lasma membrane ATPase o f r e d b e e t . P l a n t . P h y s i o l . 76 : 26-30 . 122 B r o o k e r , R.J. and Slayman, C.W. ( 1 9 8 2 ) . I n h i b i t i o n o f the plasma membrane H +-ATPase o f N e u r o s p o r a c r a s s a by N-e t h y l m a l e i m i d e . P r o t e c t i o n by n u c l e o t i d e s . J . B i o l . Chem. 257 : 12051-12055 . B r o o k e r , R.J. and Slayman, C.W. ( 1 9 8 3 a ) . [ 1 i*C]-N-e t h y l m a l e i m i d e l a b e l i n g o f t h e p l a s m a membrane H +-ATPase o f N e u r o s p o r a c r a s s a . J . B i o l . Chem. 258:222-226 . B r o o k e r , J . J . and Slayman, C.W. ( 1 9 8 3 b ) . E f f e c t s o f M g + 2 i o n s on t h e plasma membrane H +-ATPase o f Mueurospora  c r a s s a . I . I n h i b i t i o n by N - e t h y l m a l e i m i d e and t r y p s i n . J . B i o l . Chem. 25 8: 8827-8832 . B r o o k e r , R.J. and Slayman, C.W. ( 1 9 8 3 c ) . E f f e c t s o f Mg +^ i o n s on t h e plasma membrane H +-ATPase o f N e u r o s p o r a  c r a s s a . I I . K i n e t i c s t u d i e s . J . B i o l . Chem. 258:8833-8838 . B r u n e t t e , R.W. and T i l l , J . E . ( 1 9 7 1 ) . A r a p i d method f o r t h e i s o l a t i o n o f L - c e l l s u r f a c e membranes u s i n g an aqueous two phase polymer s y s t e m . J . Membrane B i o l . 5_:212-224. Bumann, J . , Wuster, B. and Malchow, D. ( 1 9 8 4 ) . A t t r a c t a n t i n d u c e d changes and o s c i l l a t i o n s o f t h e e x t r a c e l l u l a r C a + 2 c o n c e n t r a t i o n i n s u s p e n s i o n s o f d i f f e r e n t i a t i n g D i c t y o s t e l i u m c e l l s . J . C e l l B i o l . 98:173-178. C a r a f o l i , E., I n e s i , G. and Rosen, G. ( 1984). in_ M e t a l i o n s i n b i o l o g i c a l s y s t e m s , v o l . 17. S i g e l , H., ed. ( M a r c e l Dekker I n c . , New Y o r k , B a s e l ) , pp. 99-149. C a r o n i , P. and C a r a f o l i , E. ( 1 9 8 3 ) . The r e g u l a t i o n o f the N a + , C a + e x c h a n g e r o f h e a r t s a r c o l e m m a . E u r . J . Biochem. 132:451-560. C a r o n i , P. Z u r i n i , M., C l a r k , A. and C a r a f o l i , E. ( 1 9 8 3 ) . F u r t h e r c h a r a c t e r i z a t i o n and r e c o n s t i t u t i o n o f t h e p u r i f i e d C a + 2 pumping ATPase o f h e a r t sarcolemma. J . B i o l . Chem. 25 8:7305-7310• Chaney, L. and J a c o b s o n , B.S. ( 1 9 8 3 ) . C o a t i n g c e l l s w i t h c o l l o i d a l s i l i c a f o r h i g h y i e l d i s o l a t i o n o f p l a s m a membrane s h e e t s and i d e n t i f i c a t i o n o f transmembrane p r o t e i n s . J . B i o l . Chem. 25 8:10072-10083. Ch a r l e m a g n e , D., M a i x e n , J-M., P r e t e s e i l l e , M. and L e l i e v r e , L.G. ( 1 9 8 6 ) . O u a b a i n b i n d i n g s i t e s and N a + , K + - A T P a s e a c t i v i t y i n r a t c a r d i a c h y p e r t r o p h y . E x p r e s s i o n o f n e o n a t a l f o r m s . J . B i o l . Chem. 261:1 85-189 . i 2 3 C h u r c h i l l , K.A. and S z e , H. ( 1 9 8 4 ) . A n i o n s e n s i t i v e H + -pumping ATPase from o a t r o o t s : d i r e c t e f f e c t s o f C l ~ , NO - and a d i s u l f o n i c s t i l b e n e . P l a n t P h y s i o l . 76:490-497 . C o c u c c i , M.C. and Marre , E. ( 1 984 ) . L y s o p h o s p h a t i d y l c h o l i n e a c t i v a t e d , v a n a d a t e s e n s i t i v e M g + 2 - A T P a s e from r a d i s h m i c r o s o m e s . B i o c h i m . B i o p h y s . A c t a . 771:42-52. C o l l , R . J . and Murphy, A . J . ( 1 9 8 4 ) . P u r i f i c a t i o n o f t h e C a + 2 ATPase o f s a r c o p l a s m a r e t i c u l u m by a f f i n i t y c h r o m a t o g r a p h y . J . B i o l . Chem. 259 : 1 4249-1 4254 . C o n d e e l i s , J . ( 1 9 7 9 ) . I s o l a t i o n o f ConA c a p s d u r i n g v a r i o u s s t a g e s o f f o r m a t i o n and t h e i r a s s o c i a t i o n w i t h a c t i n and m y o s i n . J . C e l l B i o l . 80:7 51-758. C r a i g , W.S. and K y t e , J . ( 1 9 8 0 ) . S t o i c h i o m e t r y and m o l e c u l a r weight o f the minimum asy m m e t r i c u n i t o f c a n i n e r e n a l N a + , K + - A T P a s e . J . B i o l . Chem. 255 : 6262-6269 . Dame, J.B. and S c a r b o r o u g h , G.A. ( 1 9 8 0 ) . I d e n t i f i c a t i o n o f the h y d r o l y t i c m o i e t y o f the N e u r o s p o r a p l a s m a membrane H +-ATPase and d e m o n s t r a t i o n o f a p h o s p h o r y l - e n z y m e i n t e r m e d i a t e i n i t s c a t a l y t i c mechanism. B i o c h e m i s t r y 1 9 ;2931-2937 . Dame, J.B. and S c a r b o r o u g h , G.A. ( 1 9 8 1 ) . I d e n t i f i c a t i o n o f the p h o s p h o r y l a t e d i n t e r m e d i a t e o f t h e N e u r o s p o r a plasma membrane H +-ATPase a /3 - a s p a r t y l p h o s p h a t e . J . B i o l . Chem. 256:10724-10730. Darmon, M., B r a c h e t , P. and Da S i l v a , L.H. ( 1 9 7 5 ) . C n e m o t a c t i c s i g n a l s i n d u c e c e l l d i f f e r e n t i a t i o n i n D i c t y o s t e l i u m d i s c o i d e u m . P r o c . N a t l . A c a d . S c i . U.S.A. 72:3162-3166. Das, O.P. and H e n d e r s o n , E . J . ( 1 9 8 3 ) . A n o v e l t e c h n i q u e f o r g e n t l e l y s i s o f e u k a r y o t i c c e l l s . I s o l a t i o n o f plasma membranes from D i c t y o s t e l i u m d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 736:45-56. D e b e t t o , P. and C a n t l e y , L.C. ( 1 9 8 4 ) . C h a r a c t e r i z a t i o n o f a C a + 2 s t i m u l a t e d Mg + 2 dependent ATPase i n F r i e n d murine e r y t h r o l e u k e m i a c e l l plasma membranes. J . B i o l . Chem. 259:13824-13831• De Smedt, H. P a r y s , J.B., B o r g h g r u e f , R. and Wrytak, F. ( 1 9 8 3 ) . P h o s p h o r y l a t e d i n t e r m e d i a t e s o f (Ca + 2 + M g + 2 ) -ATPase and a l k a l i n e p h o s p h a t a s e i n r e n a l p l a s m a membranes. B i o c h i m . B i o p h y s . A c t a . 728:409-418. 1 2 4 D i x o n , J . F . and H o k i n , L . E . ( 1 9 7 4 ) . S t u d i e s on the c h a r a c t e r i z a t i o n o f the N a + , K + - t r a n s p o r t ATPase. P u r i f i c a t i o n o f the enzyme from e l e c t r i c o r g a n o f E l e c t r o p h o r u s e l e c t r i c u s . A r c h . Biochem. B i o p h y s . 163:749-758. D i x o n , J . F . and H o k i n , L.W. ( 1 9 7 8 ) . A s i m p l e p r o c e d u r e f o r the p r e p a r a t i o n o f h i g h l y p u r i f i e d N a + , K + - A T P a s e from the r e c t a l s a l t g l a n d o f S q u a l u s a c a n t h i a s and t h e e l e c t r i c o r g a n o f e l e c t r o p h o r u s e l e c t r i c u s . A n a l . Biochem. 86: 378-385 • D u f o u r , J-P. and G o f f e a u , A. ( 1 9 7 8 ) . S o l u b i l i z a t i o n by l y s o l e c i t h i n and p u r i f i c a t i o n o f t h e p l a s m a membrane ATPase o f the y e a s t S c h i z o s a c c h a r o m y c e s pombe. J . B i o l . Chem. 253:7026-7032. D u f o u r , J-P., G o f f e a u , A. and T s o n g , T.Y. ( 1 9 8 2 ) . A c t i v e p r o t o n u p t a k e i n l i p i d v e s i c l e s r e c o n s t i t u t e d w i t h t h e p u r i f i e d y e a s t p l a s m a membrane ATPase. F l u o r e s c e n c e q u e n c h i n g o f 9 - a m i n o - 6 - c h l o r o - 2 - m e t h o x y a c r i d i n e . J . B i o l . Chem. 257: 9365-9371 . Esmann, M., Skou, J.C. and C h r i s t i a n s e n , C . ( 1979). S o l u b i l i z a t i o n and 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 o f the N a + , K + - A T P a s e from r e c t a l g l a n d o f S q u a l u s  a c a n t h i a s . B i o c h i m . B i o p h y s . A c t a . 567:410-420. E u r o p e - F i n n e r , G.N. and N e w e l l , P.C. ( 1 9 8 4 ) . I n h i b i t i o n o f cGMP f o r m a t i o n and a g g r e g a t i o n i n D i c t y o s t e l i u m by t h e i n t r a c e l l u l a r C a + 2 a n t a g o n i s t TMB-8. FEBS L e t t s . 171:315-319. E u r o p e - F i n n e r , G.N. and N e w e l l , P.C. ( 1 9 8 5 a ) . C a l c i u m t r a n s p o r t i n t h e c e l l u l a r s l i m e mould D i c t y o s t e l i u m  d i s c o i d e u m . FEBS L e t t . 1 86:70-74 . E u r o p e - F i n n e r , G.N. and N e w e l l , P.C. ( 1 9 8 5 b ) . I n o s i t o l 1 , 4 , 5 - t r i s p h o s p h a t e i n d u c e s cGMP f o r m a t i o n i n D i c t y o s t e l i u m d i s c o i d e u m . Biochem. B i o p h y s . Res. Comm. 130:1115-1122. E u r o p e - F i n n e r , G.N. and N e w e l l , P.C. ( 1 9 8 6 ) . I n o s i t o l 1,4,5-t r i s p h o s p h a t e i n d u c e s Ca r e l e a s e from a n o n m i t o c h o n d r i a l p o o l i n amoebae o f D i c t y o s t e l i u m d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 887: 335-340. E u r o p e - F i n n e r , G.N., M c C l u e , S . J . and N e w e l l , P.C. ( 1 9 8 4 ) . I n h i b i t i o n of. a g g r e g a t i o n i n D i c t y o s t e l i u m by EGTA i n d u c e d d e p l e t i o n o f Ca + 2 . FEMS L e t t s . 21:21-25 . E u r o p e - F i n n e r , G.N., T i l l i n g h a s t J r . , H.S., McRobbie, S . J . and N e w e l l , P.C. ( 1 9 8 5 ) . TMB-8 i n h i b i t s r e s p i r a t i o n and cGMP f o r m a t i o n i n D. d i s c o i d e u m . J . C e l l . S c i . 79:151-160. 125 F a r n s w o r t h , P. ( 1 9 7 3 ) . M o r p h o g e n e s i s i n the c e l l u l a r s l i m e mold D i c t y o s t e l i u m d i s c o i d e u m ; the f o r m a t i o n and r e g u l a t i o n o f a g g r e g a t e t y p e s and the s p e c i f i c a t i o n o f d e v e l o p m e n t a l a x e s . J . E m b r y o l . Exp. Morph. 29:253-266 . F e r n a n d e z , J . L . , R o s e n b l a t t , M. and H i d a l g o , C. ( 1 9 8 0 ) . H i g h l y p u r i f i e d s a r c o p l a s m i c r e t i c u l u m v e s i c l e s a r e d e v o i d o f C a + 2 i n d e p e n d e n t ( B a s a l ) ATPase a c t i v i t y . B i o c h i m . B i o p h y s . A c t a . 599: 552-568 . G a r n e t t , H.M. and Kemp, R.B. ( 1 9 7 5 ) . ( C a + 2 + M g + 2 ) a c t i v a t e d ATPase i n t h e p l a s m a membranes o f mouse l i v e r c e l l s . B i o c h i m . B i o p h y s . A c t a . 382: 526-533 . G e r i s c h , G., H u l s e r , D., Malchow, D. and Wick, V. ( 1 9 7 5 ) . C e l l c o m m u n i c a t i o n by p e r i o d i c cAMP p u l s e s . P h i l . T r a n s . R. Soc. Lond . 272:181-1 92. G i l k e s , N.R. and Weeks, G. ( 1 9 7 7 a ) . The 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 D i c t y o s t e l i u m d i s c o i d e u m p l a s m a membranes. B i o c h i m . B i o p h y s . A c t a . 464:142-156. G i l k e s , N.R. and Weeks, G. ( 1 9 7 7 b ) . An i m p r o v e d method f o r the p u r i f i c a t i o n o f t h e plasma membrane from D_. d i s c o i d e u m . Can J . Biochem. 55:1233-1236. Glomp, I . , S c h a f e r , D. and Hess, B. ( 1 9 8 5 ) . C y t o c h e m i c a l l o c a l i z a t i o n o f a l k a l i n e p h o s p h a t a s e i n t h e c e l l membrane o f D i c t y o s t e l i u m d i s c o i d e u m amoebae. H i s t o c h e m . 8_3_:251-255. Gmaj, P., Z u r i n i , M. M u r e r , H. and C a r a f o l i , E. ( 1 9 8 3 ) . A h i g h a f f i n i t y , c a l m o d u l i n d e p e n d e n t C a + 2 pump i n the b a s a l l a t e r a l p l a s m a membrane o f k i d n e y c o r t e x . E u r . J . Biochem. 136:71-76. Green, A.A. and N e w e l l , P.C. ( 1 9 7 4 ) . The i s o l a t i o n and s u b f r a c t i o n a t i o n o f p l a s m a membranes f r o m t h e c e l l u l a r s l i m e mold D i c t y o s t e l i u m d i s c o i d e u m . Biochem. J . 140:313-322. G r o s s , J.D., B r a d b u r y , J . , Kay, R.R. and P e a c y , J . J . ( 1 9 8 3 ) . I n t r a c e l l u l a r pH and the c o n t r o l o f c e l l d i f f e r e n t i a t i o n i n D i c t y o s t e l i u m d i s c o i d e u m . N a t u r e ( L o n d o n ) . 303:244-245. Gottmann, K. and W e i j e r , C . J . ( 1 9 8 6 ) . I n s i t u measurements o f e x t e r n a l pH and o p t i c a l d e n s i t y o s c i l l a t i o n s i n D i c t y o s t e l i u m d i s c o i d e u m a g g r e g a t e s . J . C e l l B i o l . 102:1623-1629. H a s t i n g s , D.F. and R e y n o l d s , J.A. ( 1 9 7 9 ) . M o l e c u l a r w e i g h t o f N a + , K + - A T P a s e from s h a r k r e c t a l g l a n d . B i o c h e m i s t r y 18:817-821. 126 H i d a l g o , C., G o n z a l e z , M.E. and L a g o s , R. ( 1 9 8 3 ) . C h a r a c t e r i z a t i o n o f the C a + 2 or M g + 2 - A T P a s e o f t r a n s v e r s e t u b u l e membranes i s o l a t e d from r a b b i t s k e l e t a l m u s c l e . J . B i o l . Chem. 258:13937-139^5 . H o k i n , L . E . , D u h l , J . L . Deupree, J.D., D i x o n , J . F . , Hackney, J . F . and P e r d u e , J . F . ( 1 9 7 3 ) . S t u d i e s on the c h a r a c t e r i z a t i o n o f the N a + , K + t r a n s p o r t ATPase. X. P u r i f i c a t i o n o f the enzyme from t h e r e c t a l g l a n d o f S q u a l u s a c a n t h i a s . J . B i o l . Chem. 248: 2593-2605 . I m b r i e , C.W. and Murphy, T.M. ( 1 9 8 4 ) . P r o t o n pumping by v e s i c l e s r e c o n s t i t u t e d f r o m two f r a c t i o n s o f s o l u b i l i z e d r o s e c e l l p lasma membrane ATPase. B i o c h i m . B i o p h y s . A c t a . 778:229-232. J a c o b s o n , B.S. ( 1 9 8 0 ) . Improved method f o r i s o l a t i o n o f plasma membranes on c a t i o n i c b e a d s . Membranes f r o m D i c t y o s t e l i u m d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 600:769-780. Jamieson J r , G.A., F r a z i e r , W.A. and S c h l e s i n g e r , P.H. ( 1 9 8 4 ) . T r a n s i e n t i n c r e a s e i n i n t r a c e l l u l a r pH d u r i n g D i c t y o s t e l i u m d i f f e r e n t i a t i o n . ^ . C e l l B i o l . 99:1883-1887. J a r r e t , H.W. and P e n n i s t o n , J.T. ( 1 9 7 8 ) . P u r i f i c a t i o n o f t h e Ca s t i m u l a t e d ATPase a c t i v a t o r f r o m human e r y t h r o c y t e s : I t s membership i n t h e c l a s s o f Ca b i n d i n g m o d u l a t o r p r o t e i n s . J . B i o l . Chem. 253:4676-4682. J e n t o f t , J . E . and Town, C.D. ( 1 9 8 5 ) . I n t r a c e l l u l a r pH i n 3 1 D i c t y o s t e l i u m d i s c o i d e u m : a J p NMR s t u d y . J . C e l l B i o l . 101 :778-784. J o r g e n s e n , P.L. ( 1 9 7 4 ) . 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 N a + , K + - A T P a s e . I I I . P u r i f i c a t i o n f r o m t h e o u t e r m e d u l l a o f mammalian k i d n e y a f t e r s e l e c t i v e r e m o v a l o f membrane components by SDS. B i o c h i m . B i o p h y s . A c t a . 356:36-52. J o r g e n s e n , P.L. and A n d e r s e n , J.P. ( 1 9 8 6 ) . Thermo-i n a c t i v a t i o n and a g g r e g a t i o n o f °C/3 u n i t s i n s o l u b l e and membrane bound N a + , K + - A T P a s e . B i o c h e m i s t r y 25:2889-2897. J u l i a n i , M.H. and K l e i n , C. ( 1 9 7 7 ) . C a l c i u m i o n e f f e c t s on cAMP b i n d i n g t o t h e plasma membrane o f D i c t y o s t e l i u m  d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 497: 369-376 . K a r l s s o n , J . ( 1 9 7 5 ) . Membrane bound K + and M g + 2 s t i m u l a t e d i n o r g a n i c p y r o p h o s p h a t a s e from r o o t s and c o t y l e d o n s o f s u g a r b e e t s . ( B e t a v u l g a r i s L_. ) . B i o c h i m . B i o p h y s . A c t a . 399; 3 5 6 - 3 T T 7 127 Kay, R.R., D h o k i a , B. and Jermyn, K.A. (1983 ) . P u r i f i c a t i o n o f s t a l k c e l l i n d u c i n g morphogens from D i c t y o s t e l i u m  d i s c o i d e u m . E u r . J . Biochem. 136:51—56. Kay, R.R., G a d i a n , D.G. and W i l l i a m s , S.R. ( 1 986 ) . I n t r a c e l l u l a r pH i n D i c t y o s t e l i u m : a 31p NMR s t u d y o f i t s r e g u l a t i o n and p o s s i b l e r o l e i n c o n t r o l l i n g c e l l d i f f e r e n t i a t i o n . J . C e l l S c i . 83:165-179. Kogawa, U. and R a c k e r , E. ( 1 9 7 1 ) . P a r t i a l r e s o l u t i o n o f the enzymes 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 . XXV. R e c o n s t i t u t i o n o f v e s i c l e s c a t a l y z i n g 3 2 p _ a ( j e n o s ^ n e . t r i p h o s p h a t e e x c h a n g e . J . B i o l . Chem. 246:4157-4165. K o t a g a l , N. , C o l c a , J.R. and M c D a n i e l , M.L. (19 83 ) . A c t i v a t i o n o f an i s l e t c e l l p l a s m a membrane ( C a + 2 , M g + 2 ) - A T P a s e by c a l m o d u l i n and EGTA. J . B i o l . Chem. 258:4808-4813. K y t e , J . ( 1 9 7 1 ) . P u r i f i c a t i o n o f t h e N a + , K + d e p e n d e n t ATPase from c a n i n e r e n a l m e d u l l a . J . B i o l . Chem. 246:4157-4165. K y t e , J . ( 1 9 7 4 ) . The r e a c t i o n s o f N a + , K + a c t i v a t e d ATPase w i t h s p e c i f i c a n t i b o d i e s . I m p l i c a t i o n s f o r t h e mechanism o f a c t i v e t r a n s p o r t . J . B i o l . Chem. 249:3652-3660 . Laemmli, U.K. ( 1 9 7 0 ) . C l e a v a g e o f s t r u c t u r a l p r o t e i n s d u r i n g the a s s e m b l y o f t h e head o f b a c t e r i o p h a g e T4 . N a t u r e ( L o n d o n ) . 227:680-685. Lane, L.K., Cop e n h a v e r J r . , J.H., L i n d e n m a y e r , G.E. and S c h w a r t z , A. ( 1 9 7 3 ) . 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 and [^H]- o u a b a i n b i n d i n g t o t h e t r a n s p o r t ATPase from o u t e r m e d u l l a o f c a n i n e k i d n e y . J . B i o l . Chem. 348:7197-7200. Lane, L.K., Gupte, S.S., C o l l i n s , J.A., W a l l i c k , E.T., Joh n s o n , J.D. and S c h w a r t z , A. ( 1 9 7 9 ) . 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 lamb k i d n e y N a + ,K + -ATPase . i j i N a + , K + - A T P a s e : S t r u c t u r e and K i n e t i c s . Skou, J.C. and Norby, J.G. e d s . (Academic P r e s s , New Y o r k ) , pp. 33-44. Lo o m i s , W.F. ( 1 9 7 5 ) . D i c t y o s t e l i u m d i s c o i d e u m . A d e v e l o p m e n t a l s y s t e m . Academic P r e s s , New Y o r k . L oomis, W.F., K l e i n , C. and B r a c h e t , P. ( 1 9 7 8 ) . The e f f e c t o f d i v a l e n t c a t i o n s on a g g r e g a t i o n o f D i c t y o s t e l i u m d i s c o i d e u m . D i f f e r e n t i a t i o n . 122:83-89. L o t e r s z t a j n , S., Hanoune , J . and P e c k e r , F. (1 9 81) . A h i g h a f f i n i t y C a + 2 s t i m u l a t e d M g + 2 d e p e n d e n t ATPase i n r a t l i v e r p lasma membranes. Dependence on an endogenous 128 p r o t e i n a c t i v a t o r d i s t i n c t form c a l m o d u l i n . J . B i o l . Chem. 256:11209-11215. L y n c h , T. J . and Cheung, W.Y. ( 1 9 7 9 ) . Human e r y t h r o c y t e C a + 2 , M g + 2 - A T P a s e : Mechanisms o f s t i m u l a t i o n by Ca + 2 . A r c h . Biochem. B i o p h y s . 194:165-170-MacDonald, J . I . S . and Weeks, G. ( 1 9 8 4 ) . A p l a s m a membrane M g + 2 - A T P a s e i n t h e c e l l u l a r s l i m e mold D i c t y o s t e l i u m  d i s c o i d e u m . A r c h . Biochem. B i o p h y s . 23 5: 1-7. Malchow, D., N a n j u n d i a h , V. Wuster, B., E c k s t e i n , F. and G e r i s c h , G. ( 1 9 7 8 a ) . C y c l i c AMP i n d u c e d pH c h a n g e s i n D i c t y o s t e l i u m d i s c o i d e u m and t h e i r c o n t r o l by C a + 2 . B i o c h i m . B i o p h y s . A c t a . 538:473-480. Malchow, D., N a n j u n d i a h , V. and G e r i s c h , G. ( 1 9 7 8 b ) . pH o s c i l l a t i o n s i n c e l l s u s p e n s i o n s o f D i c t y o s t e l i u m  d i s c o i d e u m ; t h e i r r e l a t i o n t o cAMP s i g n a l s . J . C e l l S c i . 30:319-330. M a r i n , F.T. and Rothman , F.G. ( 1 9 8 0 ) . R e g u l a t i o n o f d e v e lopment i n D i c t y o s t e l i u m d i s c o i d e u m IV. E f f e c t s o f i o n s on t h e r a t e o f d i f f e r e n t i a t i o n and c e l l u l a r r e s p o n s e o f cAMP . J . C e l l B i o l . 87:823-827 . Mason, J.W., Rasmussen, H. and D i b e l l a , F. ( 1 9 7 1 ) . 3'5*-AMP and Ca i n s l i m e mold a g g r e g a t i o n . Exp. C e l l Res. 67:156-160. McLennan, D.H., B r a n d l , C., K o r e z a k , B. and Green, N.M. ( 1 9 8 5 ) . Amino a c i d s e q u e n c e o f a C a + 2 Mg + 2 d e p e n d e n t ATPase from r a b b i t muscle s a r c o p l a s m i c r e t i c u l u m , deduced from i t s c o m p l e m e n t a r y DNA s t r u c t u r e . N a t u r e 316:696-700. M o k r i n , S.C. and S p u d i c h , J.A. ( 1 9 7 6 ) . C a l c i u m c o n t r o l o f a c t i n a c t i v a t e d myosin ATPase from D i c t y o s t e l i u m  d i s c o i d e u m . P r o c . N a t l . A c a d . S c i . U.S.A. 73:2321-2325. M o r r i s s e y , J.H. (1981 ) . S i l v e r s t a i n f o r p r o t e i n s i n p o l y a c r y l a m i d e g e l s : A m o d i f i e d p r o c e d u r e w i t h enhanced s e n s i t i v i t y . A n a l . Biochem. 117:307-310. M u r r a y , B.A. ( 1 9 8 2 ) . Membranes, i j i The d e v e l o p m e n t o f D i c t y o s t e l i u m d i s c o i d e u m . L o o m i s , W.F., e d . (Academic P r e s s , New Y o r k ) , pp. 71-116. N i g g l i , V., Adunyah, E.S., P e n n i s t o n , J.T. and C a r a f o l i , E. ( 1 9 8 1 ) . P u r i f i e d ( C a + 2 , M g + 2 ) - A T P a s e o f t h e e r y t h r o c y t e membrane. R e c o n s t i t u t i o n and e f f e c t o f c a l m o d u l i n and p h o s p h o l i p i d s . J . B i o l . Chem. 256:395-401 . Ochs, D.L. and Reed, P.W. ( 1 9 8 4 ) . C a + 2 s t i m u l a t e d , M g + 2 dependent ATPase a c t i v i t y i n n e u t r o p h i l p l a s m a membrane 129 v e s i c l e s . C o u p l i n g 259 :102-1 06 . to Ca+d t r a n s p o r t . J . B i o l . Chem. O l i v a , J.M., d e H e i s , L. and I n e s i , G. ( 1 983 ). C a l m o d u l i n s t i m u l a t e s b o t h a d e n o s i n e 5 ' - t r i p h o s p h a t e h y d r o l y s i s and s y n t h e s i s c a t a l y z e d by a c a r d i a c C a + 2 i o n d e p e n d e n t ATPase. B i o c h e m i s t r y . 22: 5822-5825 . O ' N e i l , S.D. and S p a n s w i c k , R.M. ( 1 9 8 4 a ) . S o l u b i l i z a t i o n and r e c o n s t i t u t i o n o f a v a n a d a t e s e n s i t i v e H +-ATPase from the plasma membrane o f B e t a v u l g a r i s . J . Membrane B i o l . 79:231-244. O ' N e i l , S.D. and S p a n s w i c k , R.M. ( 1 9 8 4 b ) . C h a r a c t e r i z a t i o n o f n a t i v e and r e c o n s t i t u t e d p l a s m a membrane H +-ATPase from t h e plasma membrane o f B e t a v u l g a r i s . J . Membrane B i o l . 79_:245-256. Ono, K - I . , O c h i a i , H. and T o d a , K. ( 1 9 7 8 ) . " G h o s t s " f o r m a t i o n and t h e i r i s o l a t i o n from t h e c e l l u l a r s l i m e mold D i c t y o s t e l i u m d i s c o i d e u m . Exp. C e l l Res. 112:175-185 . P a r i s h , R.W. and M u l l e r , V. ( 1 9 7 6 ) . The i s o l a t i o n o f p l a s m a membranes from the c e l l u l a r s l i m e mold P i c t y o s t e l i u m  d i s c o i d e u m u s i n g ConA and T r i t o n X-100. FEBS L e t t s . 63_: 40-44. P a r i s h , R.W. and W e i b e l , M. ( 1 9 8 0 ) . E x t r a c e l l u l a r ATP, e c t o -ATPase and C a + 2 e f f l u x i n D i c t y o s t e l i u m d i s c o i d e u m c e l l s . FEBS L e t t s . 1 1 8: 263-266. P a r k i n s o n , D .K . and Radde, I.C. ( 1 9 7 1 ) . P r o p e r t i e s o f t h e M g + 2 and C a + 2 s t i m u l a t e d ATP h y d r o l y z i n g enzyme i n r a t k i d n e y c o r t e x . B i o c h i m . B i o p h y s . A c t a . 242:238-246. P e n n i s t o n , J . T . ( 1 9 8 3 ) . Plasma membrane C a + 2 - A T P a s e s as a c t i v e Ca pumps. ±n_ C a l c i u m and C e l l F u n c t i o n , v o l . IV. Cheung, W.Y. e d . (Academic P r e s s , New Y o r k ) , pp. 99-149. P e r l i n , D.S., Kasamo , K., B r o o k e r , R.T. and Slayman, C.W. ( 1 9 8 4 ) . E l e c t r o g e n i c H + t r a n s l o c a t i o n by the p l a s m a membrane ATPase o f N e u r o s p o r a . S t u d i e s on p l a s m a membrane v e s i c l e s and r e c o n s t i t u t e d enzyme. J . B i o l . Chem. 259:7884-7892. P e r r o n e , J.R., Hackney, J . F . , D i x o n , J . F . and H o k i n , L . E . ( 1 9 7 5 ) . M o l e c u l a r p r o p e r t i e s o f t h e p u r i f i e d N a + , K + -ATPase and t h e i r s u b u n i t s from the r e c t a l g l a n d o f S q u a l u s a c a n t h i a s and t h e e l e c t r i c o r g a n o f E l e c t r o p h o r u s e l e c t r i c u s . J . B i o l . Chem. 250: 4178-4184. Pogge-von Strandmann, R., Kay, R.R. and D u f o u r , J-P. ( 1 9 8 4 ) . An e l e c t r o g e n i c p r o t o n pump i n p l a s m a membranes from 1 3 0 t h e c e l l u l a r s l i m e mold D i c t y o s t e l i u m d i s c o i d e u m . L e t t s . 175:422-428. FEBS Q u i v i g e r , B., de C h a s t e l l i e r , C. and R y t e r , A. ( 1 9 7 8 ) . C y t o c h e m i c a l d e m o n s t r a t i o n o f a l k a l i n e p h o s p h a t a s e i n the c o n t r a c t i l e v a c u o l e o f D i c t y o s t e l i u m d i s c o i d e u m . J . U l t r a s t r u c . Res. 62: 228-236 . Q u i v i g e r , B., B e n i c h o u , J-C . and R y t e r , A. ( 1 9 8 0 ) . C o m p a r a t i v e c y t o c h e m i c a l l o c a l i z a t i o n o f a l k a l i n e and a c i d p h o s p h a t a s e s d u r i n g s t a r v a t i o n and d i f f e r e n t i a t i o n o f D i c t y o s t e l i u m d i s c o i d e u m . B i o l . C e l l u l a i r e . 3 7:241-250. R a n d a l l , S.K. and S z e , H. ( 1 9 8 6 ) . p r o p e r t i e s o f t h e p a r t i a l l y p u r i f i e d t o n o p l a s t H + pumping ATPase from o a t r o o t s . J . B i o l . Chem. 261:1364-1371. R a t n e r , D . I . ( 1 9 8 6 ) . E q u i v a l e n c e o f i n t r a c e l l u l a r pH o f d i f f e r e n t i a t i n g D i e t y o s t e l i u m c e l l t y p e s . N a t u r e ( L o n d o n ) . 321:180-182. Rea, P.A. and P o o l e , R . J . ( 1 985 ). P r o t o n t r a n s l o c a t i n g i n o r g a n i c p y r o p h o s p h a t a s e i n r e d b e e t ( B e t a v u l g a r i s ) t o n o p l a s t v e s i c l e s . P l a n t P h y s i o l . 77:46-52. Rea, P.A. and P o o l e , R . J . ( 1 9 8 6 ) . C h r o m a t o g r a p h i c r e s o l u t i o n o f H + t r a n s l o c a t i n g p y r o p h o s p h a t a s e from H + t r a n s l o c a t i n g ATPase o f h i g h e r p l a n t t o n o p l a s t . P l a n t P h y s i o l . 81 : 126-129. R o g e r s , T.B., L a p a l u s , P. and L a z d u n s k i , M. ( 1 9 7 9 ) . C o v a l e n t l a b e l i n g o f t h e o u a b a i n r e c e p t o r , i j i N a + K + - A T P a s e : S t r u c t u r e and K i n e t i c s . Skou , J.C. and Norby, J.G. e d s . (Academic P r e s s , New Y o r k ) , pp. 431-441. Rossomando, E . F . and C u t l e r , L.S. ( 1 9 7 5 ) . L o c a l i z a t i o n o f a d e n y l a t e c y c l a s e i n D i c t y o s t e l i u m d i s c o i d e u m . I . P r e p a r a t i o n and b i o c h e m i c a l c h a r a c t e r i z a t i o n s o f c e l l f r a c t i o n s and i s o l a t e d p l a s m a membrane v e s i c l e s . Exp. C e l l Res. 95_:67-78. Rossomando, E . F . and Hodge-Jahngen , J . ( 1983). S o l u b i l i z a t i o n and s u b s t r a t e s p e c i f i c i t y o f membrane bound n u c l e o t i d e p h o s p h o d i e s t e r a s e - p y r o p h o s p h o h y d r o l a s e a c t i v i t i e s from D i c t y o s t e l i u m d i s c o i d e u m . J . B i o l . Chem. 258:7653-7660. Rossomando, E . F . and Sussman , M. ( 1 9 7 3 ) . A 5 ' - a d e n o s i n e monophosphate d e p e n d e n t a d e n y l a t e c y c l a s e and an a d e n o s i n e 3 ' , 5 ' - c y c l i c monophosphate 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 e p y r o p h o s p h o h y d r o l a s e i n D i c t y o s t e l i u m d i s c o i d e u m . P r o c . N a t l . A c a d . S c i . U.S.A. 10:1254-1257. 1 3 1 R o u f a g a l i s , B.D. ( 1 9 7 9 ) . R e g u l a t i o n o f c a l c i u m t r a n s l o c a t i o n a c r o s s the r e d b l o o d c e l l membrane. Can. J . P h y s i o l . P h a r m a c o l . 57:1331-13^9-Ruoho, A. and K y t e , J . ( 1 9 7 4 ) . P h o t o a f f i n i t y l a b e l i n g o f the o u a b a i n b i n d i n g s i t e on Na+,K +-ATPase. P r o c . N a t l . A c ad. S c i . U.S.A. JJ.: 2352-2356 . S a i t o , M. ( 1 9 7 9 ) . E f f e c t o f e x t r a c e l l u l a r C a + 2 on t h e mo r p h o g e n e s i s o f D i c t y o s t e l i u m d i s c o i d e u m . Exp. C e l l Res. 123:79-86. Sanderman, H. and S t r o m i n g e r , J . L . ( 1 9 7 2 ) . B i o s y n t h e s i s o f p e p t i d o g l y c a n o f b a c t e r i a l c e l l w a l l , 27: 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 C ^ ^ _ i S O p r e n o i d a l c o h o l p h o s p h o k i n a s e from S t a p h y l o c o c c u s a u r e u s . J . B i o l . Chem. 247:5123-5131. S c a r b o r o u g h , G.A. ( 1 9 8 0 ) . P r o t o n t r a n s l o c a t i o n c a t a l y z e d by the e l e c t r o g e n i c ATPase i n t h e p l a s m a membrane o f N e u r o s p o r a . B i o c h e m i s t r y 19:2925-2931. S c a r b o r o u g h , G.A. ( 1 9 8 2 ) . C h e m o t a c t i c models f o r t h e mechanisms o f t h e c a t i o n - m o t i v e A T P a s e s . Ann. New York Acad. S c i . 402:99-115. S c a r b o r o u g h , G.A. and Dame, J.B. ( 1 9 8 4 ) . On 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 N e u r o s p o r a p l a s m a membrane ATPase o f N e u r o s p o r a c r a s s a . J . B i o l . Chem. 256: 12343-12349. S c h o n e r , W., K i r c h , V. and Halbwachs, C. ( 1 9 7 9 ) . A s t u d y on the i n t e r a c t i o n o f N a + and o f K + w i t h t h e o u a b a i n r e c e p t o r complex I and I I o f b e e f b r a i n , i i i N a + , K + -ATPase: S t r u c t u r e and K i n e t i c s . Skou , J.C. and Norby, J.G. e d s . (Academic P r e s s , New Y o r k ) , pp. 421-430. S e r r a n o , R., Cano , A . and P e r t a n a , A. ( 1985 ). The pla s m a membrane ATPase o f D i c t y o s t e l i u m d i s c o i d e u m . B i o c h i m . B i o p h y s . A c t a . 812:553-560. S e r r a n o , R., K i e l l a n d - B r a n d t , M.C. and F i n k , G.R. ( 1 9 8 6 ) . y e a s t plasma membrane ATPase i s e s s e n t i a l f o r g r o w t h and has homology w i t h ( N a + + K + ) , K + and C a + 2 - A T P a s e s . Natu r e ( L o n d o n ) . 319:689-693. Shami, Y. and Radde, I.C. ( 1 9 7 1 ) . C a l c i u m s t i m u l a t e d ATPase o f g u i n e a p i g p l a c e n t a . B i o c h i m . B i o p h y s . A c t a . 249:345-352. S h u l l , G.E., S c h w a r t z , A. and L i n g r e l , J.B. ( 1 9 8 5 ) . Amino a c i d s e quence o f t h e c a t a l y t i c s u b u n i t o f the N a + , K + -ATPase deduced from c o m p l e m e n t a r y DNA s t r u c t u r e . N a t u r e ( L o n d o n ) . 31 6 : 691 -695 . 132 S i u , C.H., L e r n e r , R.A. and Loomis, W.F. ( 1 977 ) . R a p i d a c c u m u l a t i o n and d i s a p p e a r a n c e o f plasma membrane p r o t e i n s d u r i n g d e v e l o p m e n t o f w i l d t y p e and mutant s t r a i n s o f P i c t y o s t e l i u m d i s c o i d e u m . J . M o l . B i o l . 116:469-488. S m a l l , N.V., E u r o p e - F i n n e r , G.N. and N e w e l l , P.C. ( 1 986 ). C a l c i u m i n d u c e s cGMP f o r m a t i o n i n D i c t y o s t e l i u m . FEBS L e t t s . 203:11-14. S o b o l e w s k i , A., Neave, N. and Weeks, G. ( 1 9 8 3 ) . The i n d u c t i o n o f s t a l k c e l l d i f f e r e n t i a t i o n i n submerged m o n o l a y e r s o f P i c t y o s t e l i u m d i s c o i d e u m . C h a r a c t e r i z a t i o n o f t h e t e m p o r a l s e q u e n c e f o r t h e m o l e c u l a r r e q u i r e m e n t s . P i f f e r e n t i a t i o n 25: 93-1 00 • S p u d i c h , J.A. and S p u d i c h , A. ( 1 9 8 2 ) . C e l l m o t i l i t y . _in The Development o f P i c t y o s t e l i u m d i s c o i d e u m . L o o m i s , W.F. ed . (Academic P r e s s , new Y o r k ) , pp. 1 69-1 94 . Sussman, M. ( 1 9 6 6 ) . B i o c h e m i c a l and g e n e t i c methods i n t h e s t u d y o f t h e c e l l u l a r s l i m e mold P i c t y o s t e l i u m  d i s c o i d e u m . Meth. C e l l P h y s i o l . 2_: 397-41 0 . S z e , H. ( 1983). P r o t o n pumping 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 membrane v e s i c l e s o f t o b a c c o c a l l u s . S e n s i t i v i t y t o v a n a d a t e and K + . B i o c h i m . B i o p h y s . A c t a . 732:586-594. S z e , H. ( 1 9 8 5 ) . H + t r a n s l o c a t i n g ATPase: a d v a n c e s u s i n g membrane v e s i c l e s . Ann. Rev. P l a n t P h y s i o l . 36:175-208. T e c h n i c a l B u l l e t i n #1089. The B i o - R a d S i l v e r S t a i n . ( 1 9 8 2 ) . Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a . Town, C.D. ( 1 9 8 4 ) . D i f f e r e n t i a t i o n o f D i c t y o s t e l i u m d i s c o i d e u m i n m o n o l a y e r c u l t u r e s and i t s m o d i f i c a t i o n by i o n i c c o n d i t i o n s . D i f f e r e n t i a t i o n 27:29-35. Tsukamoto, Y., S u k i , W.N., L i a n g , C.T. and S a c k t o r , B. ( 1 9 8 6 ) . C a + 2 d e p e n d e n t ATPases i n t h e b a s o l a t e r a l membrane o f r a t k i d n e y c o r t e x . J . B i o l . Chem. 261:2718-2724. U c h i d a , E . , Ohsumi, Y. and Anraku , Y. ( 1 9 8 5 ) . 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 H + t r a n s l o c a t i n g , M g + 2 ATPase from v a c u o l a r membranes o f S a c c h a r o m y c e s c e r i v i s i a e . J . B i o l . Chem. 260:1090-1095. V a r a , F. and S e r r a n o , R. ( 1 9 8 2 ) . P a r t i a l p u r i f i c a t i o n o f t h e p r o t o n t r a n s l o c a t i n g ATPase o f p l a n t membranes. J . B i o l . Chem. 257:12826-12830. Verma, A'.K . and P e n n i s t o n , J . T . (19 81 ) . A h i g h a f f i n i t y C a + s t i m u l a t e d M g + 2 dependent ATPase i n r a t c o r p u s l u t e u m 1 3 3 plasma membrane f r a c t i o n s . J . B i o l . Chem. 256:1269-1275. V i l l a l o b o , A. ( 1 9 8 2 ) . P o t a s s i u m t r a n s p o r t c o u p l e s to ATP h y d r o l y s i s i n r e c o n s t i t u t e d p r o t e o l i p o s o m e s o f y e a s t p lasma membrane ATPase. J . B i o l . Chem. 257:1824-1828. V i l l a l o b o , A. ( 1 9 8 4 ) . E n e r g y d e p e n d e n t H + and K + t r a n s l o c a t i o n by the r e c o n s t i t u t e d y e a s t p l a s m a membrane ATPase. Can. J . Biochem. C e l l B i o l . 62:865-877. V i l l a l o b o , A. B o u t r y , M. and G o f f e a u , A. ( 1 9 8 1 ) . E l e c t r o g e n i c p r o t o n t r a n s l o c a t i o n c o u p l e d t o ATP h y d r o l y s i s by the plasma membrane M g + 2 d e p e n d e n t ATPase o f y e a s t i n r e c o n s t i t u t e d p r o t e o l i p o s o m e s . J . B i o l . Chem. 256 : 12081-12087. Walderhaug, M.O., P o s t , R.L., S a c c o m a n i , G., L e o n a r d , R.T. and B r i s k e n , D.P. ( 1 9 8 5 ) . S t r u c t u r a l r e l a t e d n e s s o f t h r e e i o n t r a n s p o r t ATPases a r o u n d t h e i r a c t i v e s i t e o f p h o s p h o r y l a t i o n . J . B i o l . Chem. 260:3852-3859. W a l k e r , R.R. and L e i g h , R.A. ( 1 9 8 1 a ) . C h a r a c t e r i z a t i o n o f a s a l t s t i m u l a t e d ATPase a c t i v i t y a s s o c i a t e d w i t h v a c u o l e s i s o l a t e d from s t o r a g e r o o t s o f r e d b e e t s ( B e t a  v u l g a r u s L.. ) . P l a n t a . 153 : 1 40-1 49 . W a l k e r , R.R. and L e i g h , R.A. (19 81b ) . Mg + 2 d e p e n d e n t , c a t i o n s t i m u l a t e d i n o r g a n i c p y r o p h o s p h a t a s e a s s o c i a t e d w i t h v a c u o l e s i s o l a t e d from s t o r a g e r o o t s o f r e d b e e t s ( B e t a  v u l g a r u s L . ) . P l a n t a . 153:150-155. W a l l i c k , E.T., Anner, B.M., Ray, M.V. and S c h w a r t z , A. ( 1 9 7 8 ) . E f f e c t o f t e m p e r a t u r e on p h o s p h o r y l a t i o n and o u a b a i n b i n d i n g to N - e t h y l m a l e i m i d e t r e a t e d N a + , K + -ATPase. J . B i o l . Chem. 253:8778-8786. Wang, Y., L e i g h , R.A., K a e s t n e r , K.H. and S z e , H. ( 1 9 8 6 ) . E l e c t r o g e n i c , H +-pumping p y r o p h o s p h a t a s e i n t o n o p l a s t v e s i c l e s o f o a t r o o t s . P l a n t P h y s i o l . 81:497-502. Weeks, C. and Weeks, G. ( 1 9 7 5 ) . C e l l s u r f a c e c h a n g es d u r i n g t h e d i f f e r e n t i a t i o n o f D i c t y o s t e l i u m d i s c o i d e u m , Exp. C e l l Res. 92:372-382. Wick, V., Malchow, D. and G e r i s c h , G. ( 1 9 7 8 ) . C y c l i c AMP s t i m u l a t e d C a + 2 i n f l u x i n t o a g g r e g a t i n g c e l l s o f D i c t y o s t e l i u m d i s c o i d e u m . C e l l B i o l . I n t . R e p o r t s . 2:71-79. W i n t e r , C.G. and Moss J r . , A . J . ( 1 9 7 9 ) . U l t r a c e n t r i f u g a l a n a l y s i s o f t h e e n z y m a t i c a l l y a c t i v e f r a g m e n t s p r o d u c e d by d i g i t o n i n a c t i o n on N a + , K + - A T P a s e . in_ N a + , K + - A T P a s e : 134 S t r u c t u r e and K i n e t i c s . Skou, J.C. and Norby, J.G. eds. (Academic P r e s s , New Y o r k ) , pp. 25-32. 135 

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