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Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium.. 1987

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GUANINE NUCLEOTIDE BINDING PROPERTIES AND ATTEMPTED IMMUNOPURIFICATION OF RAS PROTEIN FROM DICTYOSTELIUM DISCOIDEUM By BHARYL ELIZABETH BRAMBLE B . S c , The U n i v e r s i t y of Saskatchewan, 1984 A THESIS SUBMITTED!IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 © S h a r y l E l i z a b e t h Bramble, 1987 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 written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i A B S T R A C T O n e p u r p o s e o f t h i s s t u d y w a s t o d e t e r m i n e w h e t h e r t h e r a s p r o t e i n 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 ( p 2 3 ) b i n d s g u a n i n e n u c l e - o t i d e s l i k e t h e r a s p r o t e i n s f r o m m a m m a l s ( p 2 1 ) a n d y e a s t . T h e o t h e r p u r p o s e o f t h i s i n v e s t i g a t i o n w a s t o p u r i f y o r e n r i c h f o r p 2 3 r a s f r o m D . d i s c o i d e u m b y i m m u n o a f f i n i t y c h r o m a t o g r a p h y . A n u m b e r o f d i f f e r e n t a p p r o a c h e s w e r e u s e d t o d e t e r m i n e g u a n i n e n u c l e o t i d e b i n d i n g b y p 2 3 . A s i m p l e f i l t e r b i n d i n g a s s a y , b i n d i n g t o W e s t e r n b l o t s , a n d p h o t o a f f i n i t y l a b e l i n g a l l f a i l e d t o d e m o n s t r a t e s p e c i f i c b i n d i n g w i t h l y s a t e s o f D . d i s - ITcl S c o i d e u m c e l l s . I n c o n t r a s t p 2 1 f r o m t r a n s f o r m e d N I H - 3 T 3 c e l l l y s a t e w a s s u c c e s s f u l l y p h o t o a f f i n i t y l a b e l e d i n t h e p r e - s e n c e o f ^ 2 ^ - c x - g u a n o s i n e 5 ' - t r i p h o s p h a t e ( G T P ) s u g g e s t i n g t h a t t h e t e c h n i q u e h a d b e e n p e r f o r m e d c o r r e c t l y . I t w a s c o n c l u d e d t h a t e i t h e r p 2 3 h a s a v e r y l o w a f f i n i t y f o r g u a n i n e n u c l e o - t i d e s s u c h t h a t G T P b i n d i n g w a s n o t d e t e c t a b l e i n t h e s e e x p e r i - m e n t s o r t h a t t h e r a s p r o t e i n f r o m D . d i s c o i d e u m s i m p l y d o e s n o t b i n d g u a n i n e n u c l e o t i d e s . T h e p u r i f i c a t i o n o f p 2 3 f r o m D . d i s c o i d e u m c e l l s w a s a t t e m p t e d i n o r d e r t o p r o v i d e a p u r i f i e d p r o t e i n p r e p a r a t i o n f o r g u a n i n e n u c l e o t i d e b i n d i n g a n d f o r r e c o n s t i t u t i o n s t u d i e s . A n a n t i ^ r a s m o n o c l o n a l a n t i b o d y ( Y 1 3 - 2 5 9 ) w a s u s e d a s t h e l i g a n d f o r t h e i m m u n o a f f i n i t y c h r o m a t o g r a p h y . T h i s a p p r o a c h w a s n o t s u c c e s s f u l i n t h a t t h e r a s p r o t e i n c o u l d n o t b e e n - r i c h e d r e l a t i v e t o o t h e r p r o t e i n s b e c a u s e t h e i m m u n o a f f i n i t y c o l u m n s d i d n o t b i n d p 2 3 i i i TABLE.OF CONTENTS ABSTRACT i i L I S T OF TABLES V L I S T OF FIGURES v i ACKNOWLEDGEMENT v i i INTRODUCTION 1 MATERIALS AND METHODS I . MATERIALS 7 I I . METHODS 8 A . Organi sms and C u l t u r e C o n d i t i o n s 8 B . R a d i o l a b e l i n g o f D. d i s c o i d e u m c e l l s 8 C . P r e p a r a t i o n o f C e l l - F r e e E x t r a c t s 8 D. I m m u n o p r e c i p i t a t i o n 10 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 10 F . P r o t e i n D e t e r m i n a t i o n 11 G . F i l t e r A s s a y f o r Guanine N u c l e o t i d e B i n d i n g . . 11 1. Guanos ine 5 1 - D i p h o s p h a t e (GDP) B i n d i n g . . . . 11 2 . G u a n o s i n e 5 1 - T r i p h o s p h a t e (GTP) B i n d i n g . . . 11 H . W e s t e r n B l o t A s s a y f o r N u c l e o t i d e B i n d i n g . . . . 12 I . W e s t e r n Immunoblot A s s a y f o r p23 13 J . P h o t o a f f i n i t y L a b e l i n g 14 K . E n z y m e - L i n k e d Immunoadsorbent A s s a y (ELISA) . . 16 L . P r e p a r a t i o n o f A n t i b o d i e s 17 M . Removal o f T r i t o n X-100 18 N . I m m u n o a f f i n i t y Chromatography 18 0 . Dot B l o t . . : 20 i v RESULTS SECTION I. GUANINE NUCLEOTIDE BINDING A. F i l t e r Assay 21 B. Western B l o t Probed with GTP 23 C. Pho t o a f f i n i t y L a b e l i n g with GTP 23 SECTION I I . ATTEMPTED PURIFICATION OF p2 3 r a s A. S o l u b i l i z a t i o n C o n d i t i o n s 30 B. D e t e c t i o n by EL ISA 34 C. D e t e c t i o n by Immunoprecipitation 39 DISCUSSION 43 REFERENCES 50 V LIST OF TABLES 1. Guanine N u c l e o t i d e B i n d i n g F i l t e r Assay 22 2. ELISA Using V e g e t a t i v e Membranes as A n t i g e n 35 3. Two-Step ELISA with Goat A n t i - R a t Immunoglobulin G (Garat) Conjugated to A l k a l i n e Phosphatase 36 4. ELISA with V e g e t a t i v e Membranes and A c e t o n e - P r e c i - p i t a t e d 0.3% T r i t o n X-100 Membrane E x t r a c t 38 5. Immunoaffinity Chromatography wi t h Y13-259 Column E q u i l i b r a t e d i n T r i s - S a l t s Plus 1% T r i t o n X-100 and 0.5% Sodium Dodecyl Sulphate (SDS) 41 6. Immunoaffinity Chromatography wi t h Y13-259 Column E q u i l i b r a t e d i n T r i s - S a l t s 4 2 v i L I S T O F F I G U R E S 1. G T P B i n d i n g t o D . d i s c o i d e u m P r o t e i n s 2 4 2 . D i r e c t P h o t o a f f i n i t y L a b e l i n g w i t h G T P 2 6 3 . P h o t o a c t i v a t i o n a n d I m m u n o p r e c i p i t a t i o n 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 1 mM G T P 2 7 4 . G T P B i n d i n g t o p 2 1 f r o m H a r v e y M u r i n e S a r c o m a V i r u s ( H a M S V ) T r a n s f o r m e d N I H - 3 T 3 C e l l s 2 9 5 . S o l u b i l i z a t i o n o f p 2 3 w i t h T r i t o n X - 1 0 0 3 1 6 . E f f e c t o f L o w C o n c e n t r a t i o n s o f T r i t o n X - 1 0 0 o n t h e S o l u b i l i z a t i o n o f p 2 3 3 2 7 . E f f e c t o f H i g h S a l t , 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 D T A ) , o r H i g h p H o n S o l u b i l i z a t i o n o f p 2 3 . . 3 3 v i i A C K N O W L E D G E M E N T I w o u l d l i k e t o t h a n k t h r e e p e o p l e f o r t h e i r a s s i s t a n c e i n t h e c o m p l e t i o n o f t h i s t h e s i s : D r . G . W e e k s f o r h i s h e l p - f u l s u g g e s t i o n s a n d f o r t h e u s e o f h i s o f f i c e , S t e v e R o b b i n s f o r t h e u s e o f h i s c o m p u t e r , a n d A d a m B . L o s s i n g f o r h i s e f f i - c i e n t p h o t o g r a p h i c w o r k . 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 c e l l u l a r s l i m e m o l d t h a t p r o v i d e s a s i m p l e m o d e l s y s t e m f o r t h e s t u d y o f d e v e l o p m e n t ( L o o m i s , 1 9 8 2 ) . I t i s u n i q u e a m o n g e u k a r y o t e s b e c a u s e t h e p r o c e s s e s o f p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n a r e s e p a r a t e d . T h e v e g e t a t i v e a m o e b a e p r o l i f e r a t e i n t h e p r e s e n c e o f a n u - t r i e n t s o u r c e a n d u p o n s t a r v a t i o n , g r o w t h s t o p s a n d d i f f e r - e n t i a t i o n t h e n o c c u r s o v e r a 2 4 h o u r p e r i o d i n t h e l a b o r a t o r y t o p r o d u c e t w o c e l l t y p e s . C e l l - f r e e e x t r a c t s o f D . d i s c o i d e u m c o n t a i n a p r o t e i n o f m o l e c u l a r w e i g h t ( M r ) 2 3 , 0 0 0 ( p 2 3 ) " ( P a w s o n e t a l . , 1 9 8 5 ) t h a t i s s p e c i f i c a l l y i m m u n o p r e c i p i t a t e d b y a n a n t i b o d y o r i g i n a l l y r a i s e d a g a i n s t t h e t r a n s f o r m i n g p r o t e i n ( p 2 1 ) e n c o d e d b y t h e v i r a l r a s g e n e o f H a r v e y m u r i n e s a r c o m a v i r u s ( F u r t h , 1 9 8 2 ) . T h e r a s p r o t e i n i s d e v e l o p m e n t - a l l y r e g u l a t e d i n D . d i s c o i d e u m , p r o v i d i n g a n o p p o r t u n i t y t o s t u d y t h e n o r m a l f u n c t i o n o f t h e r a s p r o t e i n d u r i n g c e l l g r o w t h a n d d i f f e r e n t i a t i o n i n a n o r g a n i s m c o n s i d e r a b l y l e s s c o m p l e x t h a n m a m m a l s . T h e h u m a n r a s o n c o g e n e f a m i l y c o n s i s t s o f t h r e e m e m b e r s : H a r v e y , K i r s t e n , a n d N - r a s . T h e f i r s t t w o w e r e i d e n t i f i e d a s h o m o l o g u e s o f t h e t r a n s f o r m i n g r a s g e n e s o f H a r v e y a n d K i r s t e n m u r i n e s a r c o m a v i r u s e s ( C h a n g e t a l . , 1 9 8 2 ) w h e r e a s t h e N - r a s g e n e w a s o r i g i n a l l y i s o l a t e d f r o m a n e u r o b l a s t o m a c e l l l i n e ( H a l l e t a l . , 1 9 8 3 ) . S i n c e t h e n , c e l l u l a r h o m o l o g u e s o f t h e r a s g e n e h a v e b e e n i d e n t i f i e d i n o r g a n i s m s a s d i v e r s e a s D i c - t y o s t e l i u m d i s c o i d e u m , A p l y s i a , D r o s o p h i l i a m e l a n o g a s t e r , a n d S a c c h a r o m y c e s c e r e v i s i a e ( L e v i n s o n , 1 9 8 6 ) . 2 T h e p r e s e n c e o f t h e r a s g e n e i n n o r m a l c e l l s a n d i t s e v o l u t i o n a r y c o n s e r v a t i o n s u g g e s t t h a t i t h a s a n i m p o r t a n t r o l e i n c e l l u l a r p h y s i o l o g y . T r a n s f o r m a t i o n i s l i k e l y a r e s u l t o f t h e l o s s o f c o n t r o l o f t h i s n o r m a l f u n c t i o n ( B i s h o p , 1 9 8 3 ) . T h e m a m m a l i a n r a s p r o t o - o n c o g e n e c a n b e a c t i v a t e d t o a t r a n s - f o r m i n g o n c o g e n e b y a m p l i f i c a t i o n o f t h e g e n e ' s e x p r e s s i o n i n t h e c e l l ( P u l c i a n i e t a l . , 1 9 8 5 ) o r b y a c q u i r i n g a p o i n t m u t a - t i o n t h a t r e s u l t s i n a s i n g l e a m i n o a c i d c h a n g e ( B o s e t a l . , 1 9 8 7 ; F o r r e s t e r e t a l . , 1 9 8 7 ; Q u a i f e e t a l . , 1 9 8 7 ) a t o n e o f f i v e s p e c i f i c r e s i d u e s i n t h e e n c o d e d p r o t e i n ( F a s a n o e t a l . , 1 9 8 4 ; M a r s h a l l , 1 9 8 6 ) . A l l t h r e e m a m m a l i a n r a s g e n e s e n c o d e p r o t e i n s o f 2 1 , 0 0 0 M r t h a t a r e c l o s e l y h o m o l o g o u s i n t h e N - t e r m i n a l r e g i o n a n d h a v e a v a r i a b l e C - t e r m i n a l d o m a i n ( G i b b s e t a l . , 1 9 8 5 ) . A l - t h o u g h l i t t l e i s k n o w n a s t o t h e p h y s i o l o g i c a l f u n c t i o n o f t h e m a m m a l i a n r a s p r o t e i n s , t h e y b i n d g u a n i n e n u c l e o t i d e s s p e c i - f i c a l l y ( S c o l n i c k e t a l . , 1 9 7 9 ; S h i h e t a l . , 1 9 8 0 ; M a n n e e t a l . , 1 9 8 4 ; T r a h e y e t a l . , 1 9 8 7 ) a n d p o s s e s s a G T P a s e a c t i v i t y ( M a r - s h a l l , 1 9 8 6 ) . P 2 1 i s l o c a t e d a t t h e c y t o p l a s m i c f a c e o f t h e p l a s m a m e m b r a n e ( P a p a g e o r g e e t a l . , 1 9 8 2 ) , a n c h o r e d b y a f a t t y a c i d c o v a l e n t l y l i n k e d t o t h e C - t e r m i n u s o f t h e p r o t e i n ( S e f t o n e t a l . , 1 9 8 2 ) . T h e a b i l i t y t o b i n d G T P , t h e G T P a s e a c t i v i t y , a n d t h e m e m b r a n e l o c a l i z a t i o n a r e a l l c h a r a c t e r i s t i c s t h a t a r e v e r y s i m i l a r t o t h o s e o f a c l a s s o f r e g u l a t o r y G p r o t e i n s t h a t f u n c t i o n i n s i g n a l t r a n s d u c t i o n a c r o s s t h e p l a s m a m e m b r a n e ( N e w b o l d , 1 9 8 4 ) . W h e n G T P i s b o u n d , t h e p r o t e i n i s i n a n a c - t i v e s t a t e a n d h a s a n e f f e c t o n a t a r g e t e n z y m e . T h e h y d r o l y s i s 3 of GTP t o GDP i s thought t o e l i m i n a t e t h i s s i g n a l . T r a n s f o r m - i n g r a s genes have a reduced GTPase a c t i v i t y compared to p r o t o - oncogenes (McGrath e t a l . , 1984; Gibbs e t a l . , 1984) which r e - s u l t i n an i n c o r r e c t l y r e g u l a t e d s i g n a l . A l s o , membrane l o c a l - i z a t i o n of p21 i s r e q u i r e d f o r t r a n s f o r m a t i o n (Willumsen & C h r i s t e n s e n , 1984) making t h i s G p r o t e i n ana logy an a t t r a c t i v e h y p o t h e s i s . Yea s t RASl and RAS2 genes encode p r o t e i n s t h a t a l s o pos - sess the b i o c h e m i c a l c h a r a c t e r i s t i c s o f G p r o t e i n s ( T a t c h e l l , 1986). The RAS p r o t e i n s are f a t t y a c y l a t e d and membrane a s so- c i a t e d (Fuj iyama & Tamanoi , 1986) but these p r o p e r t i e s are not e s s e n t i a l f o r RAS f u n c t i o n (Deschenes & B r o a c h , 1987) i n Sac- char omyces c e r e v i s i a e . A c t i v a t e d RASl (Temeles e t a l . , 1985) and RAS2 (Tamanoi e t a l . , 1985) genes , w i t h muta t ions a n a l o g - ous t o those o f oncogenic mammalian r a s genes , encode p r o t e i n s w i t h reduced GTPase a c t i v i t y as compared t o w i l d - t y p e gene p r o d u c t s . The c o n s e r v e d b i o c h e m i c a l p r o p e r t i e s between ra s p r o t e i n s from y e a s t and mammals suggest t h a t these c h a r a c t e r - i s t i c s may be e s s e n t i a l to the r a s gene p r o d u c t ' s c e l l u l a r f u n c t i o n . In y e a s t , membrane r e c o n s t i t u t i o n exper iments have shown t h a t the RAS p r o t e i n i s i n v o l v e d i n the a c t i v a t i o n of a d e n y l a t e c y c l a s e (Broek e t a l . , 1985) . However, p u r i f i e d £* cl S p21 does not s t i m u l a t e or i n h i b i t the a d e n y l a t e c y c l a s e of mammalian membranes (Beckner e t a l . , 1985) . JL7 cl S R e c e n t l y , mammalian p21 has been i m p l i c a t e d i n the p h o s p h o i n o s i t i d e pathway o f i n t r a c e l l u l a r s i g n a l t r a n s d u c t i o n . I n o s i t o l l i p i d s are i n v o l v e d i n s i g n a l t r a n s d u c t i o n from 4 c e r t a i n growth f a c t o r r e c e p t o r s on mammalian c e l l membranes to i n t r a c e l l u l a r second messengers , i n i t i a t i n g responses l e a d i n g t o c e l l g rowth . In response to growth f a c t e r o c c u p a t i o n of i t s s p e c i f i c r e c e p t o r , there i s an a c t i v a t i o n of a p h o s p h o l i p a s e C t h a t i s s p e c i f i c f o r the h y d r o l y s i s of p h o s p h a t i d y l i n o s i t o l 4 , 5 - b i s p h o s p h a t e (PIP 2 ) t o 1 , 2 - d i a c y l g l y c e r o l and i n o s i t o l 1, 4 , 5 - t r i p h o s p h a t e ( 1 ,4 ,5 - IP . j ) . These p r o d u c t s s t i m u l a t e p r o - t e i n k ina se C and m o b i l i z e i n t r a c e l l u l a r c a l c i u m , r e s p e c t i v e l y ( M i c h e l l & K i r k , 1986; Majerus e t a l . , 1985). The P I P 2 i s genera ted by s e q u e n t i a l p h o s p h o r y l a t i o n o f p h o s p h a t i d y l i n o s i - t o l , by two d i f f e r e n t k i n a s e s ( B e r r i d g e , 1986) . There i s e v i - dence t h a t a G T P - b i n d i n g p r o t e i n i s i n v o l v e d i n the r e g u l a t i o n o f p h o s p h o l i p a s e C (Cockcro f t & Gomper i s , 1985; L a p e t i n a & Reep, 1987) but tehe p r o t e i n has not as y e t been c h a r a c t e r i z e d . I" 3. S Mammalian c e l l s t r ans formed w i t h p21 pos ses s e l e v a t e d l e v e l s o f 1 , 2 - d i a c y l g l y c e r o l and i n c r e a s e d p r o t e i n k inase C a c t i v i t y compared to untrans formed c e l l s (Wolfman & Macara , 1987) and the a n t i - r a s a n t i b o d y , Y13-259, i n h i b i t s the i n s u l i n - d e p e n d e n t i n d u c t i o n o f Xenopus l a e v i s oocyte m a t u r a t i o n (Deshpande & Kung, 1987) s u g g e s t i n g t h a t p 2 1 r a s i s i n v o l v e d i n the i n o s i t o l s i g n a l l i n g pathway i n response to i n s u l i n . The GDP-bound form o f p21 has been shown to a t t enua te the a u t o p h o s p h o r y l a t i o n o f the i n s u l i n r e c e p t o r ( O ' B r i e n e t a l . , 1987) but i t s p h y s i o - l o g i c a l s i g n i f i c a n c e remains u n c l e a r . A l t h o u g h i t i s t empt ing ITcl S t o p o s t u l a t e t h a t p21 encodes the G p r o t e i n t h a t r e g u l a t e s p h o s p h o l i p a s e C and i n t e r a c t s w i t h the i n s u l i n r e c e p t o r t o 5 t ransduce a s i g n a l t o t h i s enzyme, t h e r e i s no d i r e c t ev idence i n support of t h i s h y p o t h e s i s . D. d i s c o i d e u m a l s o has an i n t r a c e l l u l a r s i g n a l l i n g p a t h - way i n v o l v i n g p h o s p h o i n o s i t i d e l i p i d s a l t h o u g h , i n compari son t o mammalian c e l l s , l e s s i s known about t h i s pathway. The s i g - n a l l i n g system of D. d i s c o i d e u m does not i n v o l v e hormones but r a t h e r c e l l s communicate w i t h c y c l i c adenos ine 5 1 -monophosphate (cAMP) and f o l a t e (Devreotes , 1983). C e l l s are a t t r a c t e d t o f o l a t e d u r i n g v e g e t a t i v e growth and as c e l l s s t a r t t o d i f f e r e n - t i a t e , they l o se t h e i r s e n s i t i v i t y t o f o l a t e and g a i n s e n s i t i v - i t y t o cAMP, the c h e m o a t t r a c t a n t r e s p o n s i b l e f o r a g g r e g a t i o n . The b i n d i n g o f f o l a t e or cAMP t o t h e i r r e s p e c t i v e c e l l sur face r e c e p t o r s induces a t r a n s i e n t accumula t ion o f c y c l i c guanosine 5 1 -monophosphate (cGMP), t h a t peaks a t 8 to 10 seconds a f t e r b i n d i n g o f the a t t r a c t a n t (Mato e t a l . , 1977; Wurster e t a l . , 1977) , presumably due to a t r a n s i e n t a c t i v a t i o n o f guanyla te c y c l a s e . C a l c i u m (Small e t a l . , 1986) and 1 , 4 , 5-IP^ (Europe- F i n n e r & N e w e l l , 1985), when added to p e r m e a b i l i z e d amoebae, mimic the a c t i o n o f the c h e m o a t t r a c t a n t s and induce cGMP f o r - m a t i o n . More d i r e c t ev idence f o r p h o s p h o i n o s i t i d e involvement i n the c h e m o s t a t i c s i g n a l l i n g pathway was o b t a i n e d when 1 , 4 , 5 ,-IP^ was shown to accumulate r a p i d l y i n response to s t i m u l a t i o n by cAMP (Europe-F inner & N e w e l l , 1987) . A model f o r chemosensory t r a n s d u c t i o n has been proposed i n which the a c t i v a t e d cAMP and f o l a t e r e c e p t o r s i n t e r a c t w i t h a membrane p h o s p h o l i p a s e t h a t h y d r o l y s e s P I P 2 t o 1 / 2 - d i a c y l g l y c e r o l and 1 , 4 , 5-IPo ( s i m i l a r t o the response i n mammalian c e l l s to growth 6 hormones) . The i n t r a c e l l u l a r messenger 1 , 4 , 5 - I P g then mobi- l i z e s i n t r a c e l l u l a r c a l c i u m s t o r e s and t h i s i n t u r n s t i m u l a t e s guany la te c y c l a s e a c t i v i t y t o produce cGMP. R e c e n t l y , i t has been shown t h a t a D i c t y o s t e l i u m r a s gene, mutated i n an ana- logous p o s i t i o n t o oncogenic mammalian ras gene, caused a r e - d u c t i o n i n the l e v e l o f cGMP i n D. d i s c o i d e u m c e l l s (Van H a a s t e r t e t a l . , 1987) a f t e r c7\MP or f o l a t e s t i m u l a t i o n . T h i s 3i S suggests a r o l e f o r p23 i n the c h e m o t a c t i c s i g n a l l i n g p a t h - way o f D. d i s c o i d e u m . One purpose of t h i s s tudy was to i n v e s t i g a t e the suppo- IT cl S s i t i o n t h a t p23 b i n d s guanine n u c l e o t i d e s . The o t h e r p u r - pose o f t h i s i n v e s t i g a t i o n was t o immunopurify the r a s p r o t e i n from D. d i s c o i d e u m because a p u r i f i e d p r e p a r a t i n of p2 3 c o u l d be used i n membrane r e c o n s t i t u t i o n exper iments to d i r e c t l y s tudy i t s f u n c t i o n . 7 MATERIALS AND METHODS I . MATERIALS B a c t e r i o l o g i c a l peptone and y e a s t e x t r a c t were from Oxoid and Bac to -Agar was from D i f c o . D u l b e c c o ' s modif ied. Eag le me- dium and f e t a l bov ine serum were from G i b c o . T r i t o n X-100 was from Amersham. N - t e t r a d e c y l - N , N - d i m e t h y l - 3 - a m m o n i o - l - p r o p a n e - s u l f o n a t e (Z-14) was from C a l b i o c h e m . 5.0 ym pore s i z e f i l t e r s were from N u c l e o p o r e . N i t r o c e l l u l o s e (0.45 ym pore s i ze ) was from S c h l e i c h e r and S c h u e l l . A l l r a d i o i s o t o p e s were from New England N u c l e a r . F o r m a l i n f i x e d S t a p h y l o c o c c u s aureus (Staph) was from The Enzyme C e n t r e . R a b b i t a n t i - r a t immunoglobul in G (RARIG) was from Cooper B i o m e d i c a l . Goat a n t i - r a b b i t immuno- g l o b u l i n G (GARIG) and GARAT a l k a l i n e phosphatase con juga te s were from K i r k e g a a r d and P e r r y L a b o r a t o r i e s , r e s p e c t i v e l y . The r a t myeloma c e l l l i n e Y 3 - A G 1 . 2 . 2 (Y3) was from the Amer- i c a n Type C u l t u r e C o l l e c t i o n . Y13-259 p u r i f i e d by h i g h p r e s - sure l i q u i d chromatography (HPLC) was from Oncogene S c i e n c e . D i e t h y l a m i n o e t h y l (DEAE)-Sephace1 was purchased as p r e - s w o l l e n beads from Pharmac ia . SDS ( s p e c i a l l y pure) was from BDH. B io-Beads SM-2, A f f i - G e l 10, a c r y l a m i d e , and t e t r a m e t h y l e t h y 1 - enediamine were from B i o - R a d . B i s - a c r y l a m i d e and XAR-5 f i l m were from KODAK. A l l o t h e r c h e m i c a l s were of reagent grade from 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 . 8 I I . METHODS A) Organisms and C u l t u r e C o n d i t i o n s D i c t y o s t e l i u m d i s co ideum w i l d - t y p e s t r a i n V12-M2 was used, throughout t h i s s tudy . S t r a i n V12-M2 was 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 aerogenes on n u t r i e n t agar p l a t e s u n t i l c l e a r i n g o f the b a c t e r i a l lawn was d i s c e r n i b l e (Sussman, 1966). The c e l l s were h a r v e s t e d from growth p l a t e s u s i n g s t e r i l e phosphate b u f f e r (20 mM K H 2 P 0 4 , K 2 H P 0 4 , pH 6.0) and then wash- ed f r ee o f b a c t e r i a w i t h four 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 s (700 x g f o r 2 m i n ) . Harvey murine sarcoma v i r u s (HaMSV) t r a n s f o r m e d , nonpro- ducer NIH-3T3 mouse f i b r o b l a s t s were grown i n Dulbecco modified Eag le medium c o n t a i n i n g 10% f e t a l bov ine serum at 3 7 ° C . B) R a d i o l a b e l i n g o f D. d i s c o i d e u m c e l l s F o r the p r e p a r a t i o n o f r a d i o l a b e l e d c e l l e x t r a c t s , c e l l s 35 were l a b e l e d w i t h S-methionine a t the o u t s e t o f d i f f e r e n t i a - t i o n as d e s c r i b e d p r e v i o u s l y (Weeks e t a l . , 1987). C) P r e p a r a t i o n o f C e l l - F r e e E x t r a c t s Crude c y t o s o l and membrane f r a c t i o n s were p repared by g re su spend ing the washed D. d i s c o i d e u m c e l l s to 1 x 10 c e l l s per ml o f T r i s - s a l t s (lOmM T r i s - H C l , pH 7 .5 , 100 mM N a C l , 5 mM M g C l 2 ) , c o n t a i n i n g 40 yg/ml l e u p e p t i n and 40 ug/ml a n t i p a i n . The c e l l s were broken by p a s s i n g them through a 5.0 'um pore s i z e p o l y c a r b o n a t e f i l t e r 2 to 3 t i m e s , as d e s c r i b e d by Das and Henderson (1983), u n t i l over 99% of the c e l l s had been broken as a s se s sed by m i c r o s c o p y . The l y s a t e was c e n t r i f u g e d at 9 100,000 x g f o r 1 h , a t 4 ° C t o separa te a crude c y t o s o l ( super- nate) and a crude membrane ( p e l l e t ) f r a c t i o n . The 0.3% T r i t o n X-100 membrane e x t r a c t s were r o u t i n e l y prepared by re suspend ing the crude membrane f r a c t i o n i n an r -equal volume o f T r i s - s a l t s p l u s 40 yg/ml: l e u p e p t i n , 40 yg/ml a n t i p a i n , and 0.3% (v/v) T r i t o n X-100. T h i s suspens ion was incuba ted f o r 15 min on i c e , and then centr id luged at 100,000 x g f o r 1 h , a t 4 ° C . Membrane e x t r a c t s c o n t a i n i n g 1% T r i t o n and 0.5% SDS were p repared f o r i m m u n o a f f i n i t y chromatography u s i n g the same procedure except t h a t the crude membrane f r a c t i o n was resuspended i n T r i s - s a l t s c o n t a i n i n g 40 yg /ml l e u p e p t i n , 40 yg /ml a n t i p a i n , 1% (v/v) T r i - ton X-100, and 0.5% (w/v) SDS. Whole c e l l l y s a t e s were prepared f o r i m m u n o p r e c i p i t a t i o n as d e s c r i b e d p r e v i o u s l y (Pawson e t a l . , 1985) except the l y s i s b u f f e r c o n t a i n e d T r i s - s a l t s , 40 yg/ml l e u p e p t i n , 40 yg/ml a n t i - p a i n , and 1% (v/v) T r i t o n X-100. The e x t r a c t s were passed three t imes through an 18 gauge need le and p a r t i c u l a t e matter was removed by c e n t r i f u g a t i o n at 100,000 x g f o r l h , a t 4 ° C . These l y s a t e s w i l l be r e f e r r e d t o as T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e throughout the remainder o f t h i s t h e s i s . HaMSV-transformed NIH-3T3 c e l l s were h a r v e s t e d and l y s e d as d e s c r i b e d by S c o l n i c k e t a l . , (1979) except the c e l l s were p l a c e d on i c e a f t e r removal o f the medium and washed w i t h T r i s - b u f f e r e d s a l i n e . C e l l s were l y s e d by s c r a p i n g them i n t o 1 ml o f T r i s - s a l t s , 40 yg/ml l e u p e p t i n , 40 yg/ml a n t i p a i n , and 1% T r i t o n X-100. The e x t r a c t s were passed three t imes through a 23 gauge needle and c l a r i f i e d a t 100,000 x g f o r 1 h , a t 4 ° C . 10 D) I m m u n o p r e c i p i t a t i o n A l l s teps i n t h i s procedure were per formed at 4 ° C . C e l l l y s a t e s were a d j u s t e d to 1% T r i t o n X-100 and 0.5% SDS. For the s o l u b i l i z a t i o n exper iments (F igure s 5, 6, and 7) p a r t i c u l a t e matter was removed by c e n t r i f u g a t i o n at 15,000 x g f o r 1 h . A l i q u o t s (0.5 ml) were p r e c l e a r e d by an i n i t i a l i n c u b a t i o n f o r 15 min w i t h 50 u l o f a 1:10 suspens ion of S taph , f o l l o w e d by a second i n c u b a t i o n w i t h 50 u l o f a 1:10 suspens ion of RARIG- c o a t e d Staph (RARIG-Staph) f o r 15 min . The Staph was p e l l e t e d by c e n t r i f u g a t i o n at 13,000 x g f o r 2 min and the supernate was t r a n s f e r r e d to a new tube . Samples were i n c u b a t e d w i t h 50 t o 100 u l of ammonium s u l p h a t e - p r e c i p i t a t e d Y13-259 f o r 1 h . C o n t r o l i m m u n o p r e c i p i t a t i o n s c o n t a i n e d an e q u a l volume of n o r - mal r a t serum or ammonium s u l p h a t e - p r e c i p i t a t e d supernate (Jonak, 1980) from the Y13-259 p a r e n t a l myeloma, Y3 . A l l sam- p l e s were i n c u b a t e d w i t h 50 yfl' o f a 1:10 suspens ion o f RARIG- Staph fo r 1 h . The immune complexes were p e l l e t e d by c e n t r i f li- g a t i o n at 13,000 x g f o r 1 min and the supernates were removed by a s p i r a t i o n . The immune complexes were resuspended by p i p e t - t i n g i n 0.5 ml o f T r i s - s a l t s c o n t a i n i n g 1% T r i t o n X-100 and 0.5% SDS and washed by r e p e a t e d c e n t r i f u g a t i o n (3 to 5 t i m e s ) . Where i n d i c a t e d , SDS a d d i t i o n or p r e c l e a r i n g were o m i t t e 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 A l l immunoprec ip i t a t e s and l y s a t e s were heated a t 1 0 0 ° C i n SDS sample b u f f e r fo r 3 min u n l e s s o therwise i n d i c a t e d . A l l samples were e l e c t r o p h o r e s e d through 11.25% or 12.5% SDS-poly- a c r y l a m i d e g e l s (Pawson e t a l . , 1985). 11 F) P r o t e i n D e t e r m i n a t i o n P r o t e i n was de termined by the F o l i n method, as m o d i f i e d by Sandermann and S t rominger (1972), o r w i t h the amido b l a c k p r o t e i n assay (Schaf fner & Weissman, 1973) i f the p r o t e i n c o n - c e n t r a t i o n was l e s s than 1 mg/ml. G) F i l t e r Assay f o r Guanine N u c l e o t i d e B i n d i n g 1) Guanosine 5 ' - D i p h o s p h a t e (GDP) B i n d i n g . A 500 y l a l i - quot o f T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e (2.36 mg) was i n - -5 3 cubated w i t h 10 M H-GDP (10 Ci/mmol) f o r 1 h a t 4 ° C . The samples were i m m u n o p r e c i p i t a t e d d i r e c t l y w i t h o u t the a d d i t i o n of SDS or p r e c l e a r i n g , u s i n g e i t h e r normal r a t serum o f Y13-2 59 f o l l o w e d by i n c u b a t i o n w i t h RARIG-coated agarose beads (100 y l of a 1:10 suspension) f o r 1 h a t 4 ° C . A f t e r wash ing , the immune complexes were c o l l e c t e d on 0.45 ym pore s i z e f i l t e r s and wash- ed t h r e e more t imes w i t h 5 ml o f i c e - c o l d T r i s - s a l t s c o n t a i n i n g 3 1% T r i t o n X-100. The amount o f H-GDP r e t a i n e d by the f i l t e r s was determined 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 . The p u r i t y 3 o f the H-GDP was a s ses sed by t h i n l a y e r chromatography on poly- ( e t h y l e n e i m i n e ) - c e l l u l o s e p l a t e s u s i n g 1.6 M L i C l i n d e i o n i z e d H2O as s o l v e n t (Randerath & Randera th , 1964). R a d i o a c t i v e spots were d e t e c t e d by expos ing the chromatographed t h i n l a y e r to XAR-5 f i l m and q u a n t i t a t e d by<:cutting the spots out and d i s s o l v - i n g them i n s c i n t i l l a t i o n f l u i d f o r c o u n t i n g . 2) Guanosine 5 ' - T r i p h o s p h a t e (GTP) B i n d i n g . A l i q u o t s (200) y l of T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e (0.79 mg) were p r e c l e a r e d and then 10 y l of 40 yM S-Y-GTP and 2 y C i (2 .7 ' yl) 35 S—Y-GTP were added to each sample. The samples were i n c u b a t e d 12 f o r l h a t 4 ° C and then d i r e c t l y i m m u n o p r e c i p i t a t e d w i t h e i t h e r normal r a t serum o r Y13-259 w i t h o u t a d d i t i o n o f SDS. The im- mune complexes were washed and c o l l e c t e d as d e s c r i b e d above. A l t e r n a t i v e l y 600 u l a l i q u o t s o f T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e (0.96 mg) were p r e c l e a r e d and i m m u n o p r e c i p i t a t e d ( in the absence of 0.5% SDS) w i t h e i t h e r normal r a t serum or Y13-259. The immune complexes were washed and then resuspended i n 200 "uiL of T r i s - s a l t s p l u s 1% T r i t o n X-100. Two uM S-Y'-GTP (10 u l o f 40 yM stock) and 2.7 u l 3 5 - S - y-GTP (2 yCi) were added to each a l i q u o t and i n c u b a t e d f o r l h a t 4 ° C . The immune com- p l e x e s were then c o l l e c t e d on f i l t e r s , washed, and counted as d e s c r i b e d above. H) Western B l o t Assay f o r N u c l e o t i d e B i n d i n g P r o t e i n s were e l e c t r o b l o t t e d from SDS-po lyacry l amide g e l s to n i t r o c e l l u l o s e as d e s c r i b e d by Towbin e t a l . (1979) . T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e was i m m u n o p r e c i p i t a t e d i n the ab- sence o f 0.5% SDS. The T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e samples and the immunoprec ip i t a ted samples were not heated be- fore e l e c t r o p h o r e s i s . The e l e c t r o p h o r e s e d p r o t e i n was e l e c t r o - b l o t t e d and probed as d e s c r i b e d p r e v i o u s l y f o r p21 r a s (McGrath 35 e t a l . , 1984) except 50 y C i S-y-GTP was used i n p l a c e of 32 P-a-GTP i n 4 ml of b i n d i n g b u f f e r (50 mM T r i s - H C l , pH 7 .5 , 5 mM M g C l 2 , 0.1% (v/v) NP40, 1 mg/ml bovine serum a l b u m i n ) . A f t e r i n c u b a t i o n f o r l h a t 3 7 ° C i n b i n d i n g b u f f e r , the e l e c t r o - b l o t t e d p r o t e i n was washed f o r 10 min three t imes i n 4 ml o f b i n d i n g b u f f e r l a c k i n g bov ine serum albumin (BSA) and GTP. 1 3 3 The d r i e d f i l t e r was sprayed w i t h En hance and was then s u b j e c t - ed t o f l u o r o g r a p h y at - 7 0 ° C . I) Western Immunoblot Assay f o r p2 3 A l i q u o t s o f 1% T r i t o n X-100 membrane e x t r a c t s (1.67 mg) were e i t h e r d i r e c t l y e l e c t r o p h o r e s e d , or immunoprec ip i t a t ed w i t h e i t h e r Y13-259 or normal r a t serum and then e l e c t r o p h o r e s e d , through 12.5% S D S - p o l y a c r y l a m i d e . The p r o t e i n was e l e c t r o b l o t - t ed o v e r n i g h t a t 30V i n p r e c o o l e d t r a n s f e r b u f f e r (20 mM NaPO^, pH 8 . 0 ) . The Western b l o t s were i n c u b a t e d i n 15 ml o f b l o c k i n g b u f f e r (3.0% (w/v) BSA, 50 mM T r i s - H C l , pH 7 .4 , 150 mM NaCl) f o r 1 h at 3 7 ° C , on a g y r a t o r y i n c u b a t o r . The b l o t was then i n c u b a t e d i n 15 ml o f Y13-259 d i l u t e d 1/200 i n b l o c k i n g b u f f e r , a t 4 ° C , o v e r n i g h t . The f i l t e r was washed t h r e e t imes f o r 15 m i n , a t room temperature w i t h washing b u f f e r (0.6% BSA, 50 mM T r i s - H C l , pH 7 .4 , 150 mM N a C l ) . RARIG (preadsorbed w i t h metha- n o l - f i x e d v e g e t a t i v e c e l l s ) was d i l u t e d 1/400 i n 15 ml o f b l o c k - i n g b u f f e r and t h i s was i n c u b a t e d w i t h the f i l t e r f o r l h a t 3 7 ° C / : s h a k i n g . The b l o t was then washed as d e s c r i b e d above and then i n c u b a t e d f o r l h a t 3 7 ° C on a g y r a t o r y shaker w i t h GARIG c o n j u - gated t o a l k a l i n e phosphatase d i l u t e d 1/3000 i n 15 ml of b l o c k - i n g b u f f e r . The b l o t was washed twice as be fore and once f o r 15 min i n washing b u f f e r w i t h o u t BSA. The b l o t was then i n c u - ba ted fo r 30 min at 3 7 ° C w i t h 15 ml o f d e v e l o p i n g s o l u t i o n (0.01 mg/ml 5 - b r o m o - 4 - c h l o r o - 3 - i n d o l y l phosphate (BCIP) and 0.1 mg/ml n i t r o b l u e t e t r a z o l i u m (NBT) i n 1 M T r i s - H C l , pH 8.8 c o n t a i n i n g 4 mM M g C l 2 ) , r i n s e d twice w i t h d e i o n i z e d H 2 0 and a i r - d r i e d . 14 F o r the Western immunoblots and the Western b l o t s used f o r guanine n u c l e o t i d e b i n d i n g , l anes c o n t a i n i n g s i z e markers o f known m o l e c u l a r weight were c u t from the f i l t e r a f t e r e l e c t r o - b l o t t i n g f o r s t a i n i n g w i t h I n d i a Ink (Hancock & Tsang , 1983) . J) P h o t o a f f i n i t y L a b e l i n g P h o t o a f f i n i t y l a b e l i n g w i t h crude c y t o s o l and membrane f r a c t i o n s was conducted as d e s c r i b e d p r e v i o u s l y ( S c h l e i c h e r e t a l . , 1986) , w i t h a few m o d i f i c a t i o n s . The membrane p e l l e t was resuspended i n T r i s - s a l t s c o n t a i n i n g 40 yg/ml l e u p e p t i n and 40 yg/ml a n t i p a i n . The p a r t i c u l a t e mat ter was removed by c e n t r i f u - g a t i o n a t 100,000 x g f o r l h a t 4 ° C b e f o r e p r o t e i n d e t e r m i n a t i o n . C e l l l y s a t e s were a d j u s t e d to 2 mg/ml p r o t e i n and 50 y l a l i quo t s 32 were p r e i n c u b a t e d on i c e w i t h 5 y C i P-a-GTP (750-800 Cifaimol) i n m i c r o t i t e r w e l l s f o r 30 m i n . I r r a d i a t i o n w i t h short-wave u l t r a v i o l e t (UV) l i g h t (maximal e m i s s i o n , 254 nm) was f o r 60 m i n , a t 4 ° C . A f t e r i r r a d i a t i o n , samples were e i t h e r mixed w i t h SDS sample b u f f e r and e l e c t r o p h o r e s e d d i r e c t l y or immunopreci- p i t a t e d w i t h e i t h e r Y3 o r Y13-259 be fore e l e c t r o p h o r e s i s through a 12.5% SDS-po lyacry l amide g e l . S i ze markers of known m o l e c u l a r weight were c o e l e c t r o p h o r e s e d and t h e i r m o b i l i t i e s were determined by Coomassie b lue s t a i n i n g . G e l s were d r i e d and a u t o r a d i o g r a p h e d w i t h a sc reen a t - 7 0 ° C o r - 2 0 ° C . Til o t h e r exper iment s , T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e was i m m u n o p r e c i p i t a t e d be fore p h o t o a f f i n i t y l a b e l i n g w i t h GTP. T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e was p r e p a r e d i n T r i s - s a l t s c o n t a i n i n g 40 yg/ml l e u p e p t i n , 40 yg/ml a n t i p a i n , 1% T r i t o n X- 100, and 0.5% (w/v) SDS and 500 y l a l i q u o t s (3.34 mg p r o t e i n ) 15 were i m m u n o p r e c i p i t a t e d w i t h e i t h e r Y3 or Y13-259. The immune complexes were washed and then resuspended i n T r i s - s a l t s . The i m m u n o p r e c i p i t a t e d samples were p l a c e d i n m i c r o t i t e r w e l l s f o r 32 p h o t o a f f i n i t y l a b e l i n g w i t h P-a-GTP as d e s c r i b e d above. A f t e r i r r a d i a t i o n , samples were mixed w i t h SDS sample b u f f e r and e l e c - t r o p h o r e s e d through a 12.5% p o l y a c r y l a m i d e g e l as d e s c r i b e d above. To study the p o s s i b l e d e l e t e r i o u s e f f e c t of p h o t o a f f i n i t y 35 l a b e l i n g on i m m u n o p r e c i p i t a t i o n , S-methionine r a d i o l a b e l e d T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e was a d j u s t e d t o 4 mg/ml p r o t e i n and 0.5% T r i t o n X-100. A l i q u o t s (100 yg) were i n c u - bated w i t h or wi thout 1 mM GTP on i c e f o r 30 min and i r r a d i a t e d , i m m u n o p r e c i p i t a t e d and e l e c t r o p h o r e s e d as d e s c r i b e d above. N u c l e o t i d e p h o t o a f f i n i t y - l a b e l i n g of HaMSV-transformed NIH-3T3 c e l l e x t r a c t was performed as d e s c r i b e d by S t e i n e t a l . (1984) except f o r a few m o d i f i c a t i o n s . A l i q u o t s (1 ml) were c o n c e n t r a t e d 1 0 - f o l d w i t h a M i n i c o n B15 f i l t e r system at 4 ° C (about 2 h ) . A f t e r c o n c e n t r a t i o n , 40 yg l e u p e p t i n and 40 yg a n t i p a i n were added and the sample was s t o r e d at - 7 0 ° G . Each 32 sample was p r e i n c u b a t e d w i t h 1.2 yM P-a-GTP (750-800 Ci/mmol) i n m i c r o t i t e r w e l l s f o r l h i n the d a r k , on i c e . I r r a d i a t i o n w i t h short-wave UV l i g h t was f o r 30 min a t 4 ° C . A f t e r i r r a d i a - t i o n , samples were i m m u n o p r e c i p i t a t e d w i t h e i t h e r Y3 or HPLC- p u r i f i e d Y13-259 and e l e c t r o p h o r e s e d as d e s c r i b e d above. A 0.3% T r i t o n X-100 membrane e x t r a c t was p r e p a r e d from D. d i s - coideum v e g e t a t i v e c e l l s and t r e a t e d i n the same manner as the t rans formed mammalian c e l l e x t r a c t . 16 K) Enzyme-Linked Immunoadsorbent Assay (ELISA) The s t andard ELISA was performed u s i n g a crude membrane f r a c t i o n from v e g e t a t i v e c e l l s as a n t i g e n . The p r o t e i n was d i l u t e d to 0.1 mg/ml i n carbonate b u f f e r (14 mM Na 2 CC" 3 , 35 mM NaHC0 3 , 0.02% (w/v) NaN^, 5 mM M g C l 2 , pH 9.6) and 100 y l was added t o each w e l l of a 9 6 - w e l l m i c r o t i t e r p l a t e (except the s u b s t r a t e b l ank w e l l s i n the f i r s t row) f o r a d s o r p t i o n a t 4 ° C , o v e r n i g h t . The w e l l s were then washed three t imes w i t h 0.3% BSA i n p h o s p h a t e - b u f f e r e d s a l i n e (137 mM N a C l , 1.2 mM K H 2 P 0 4 , 8 mM N a 2 H P 0 4 , 2.7 mM K C l , 0.02% (w/v) NaN^, pH 7.2) p l u s 0.05% (v/v) Tween 20 (PBS-Tween). Y13-259 ammonium-sulphate p r e c i p i t a t e d and c o n t r o l a n t i b o d y (normal r a t serum or Y3) were d i l u t e d 1/200 i n PBS-Tween p l u s 0.3% BSA and 100 y l was added to the a p p r o p r i a t e w e l l s f o r i n c u b a t i o n at 3 7 ° C f o r 1 h . N o - a n t i b o d y c o n t r o l w e l l s c o n t a i n e d PBS-Tween p l u s 0.3% BSA. Subs t r a t e b lank c o n t r o l s t h a t c o n t a i n e d o n l y s u b s t r a t e s o l u t i o n ( p - n i t r o - p h e n y l phosphate) were a l s o i n c l u d e d i n each a s say . A f t e r the i n c u b a t i o n , the w e l l s were washed three t imes w i t h PBS-Tween. RARIG (preadsorbed w i t h v e g e t a t i v e c e l l s ) was d i l u t e d 1/2000 i n PBS-Tween and 100 y l o f t h i s s o l u t i o n was added to each w e l l (except s u b s t r a t e b l a n k ) . The p l a t e s were i n c u b a t e d at 3 7 ° C f o r 1 h . The w e l l s were washed three t imes w i t h PBS-Tweenoand 100 y l o f G A R I G - a l k a l i n e phosphatase c o n j u - gate d i l u t e d 1/2000 i n PBS-Tween was added to each w e l l except the s u b s t r a t e b l a n k . The ELISA p l a t e s were i n c u b a t e d f o r 1 h a t 3 7 ° C and then washed three t imes w i t h PBS-Tween. F i n a l l y , 17 100 y l o f a 1 mg/ml s o l u t i o n of p_-nitropheny 1 phosphate d i s - s o l v e d i n d i e t h a n o l a m i n e b u f f e r (1 M d i e t h a n o l a m i n e , 0.0 2% NaN-j/ 0.5 mM MgCl^) was added to each w e l l . A f t e r i n c u b a t i n g w i t h s u b s t r a t e a t 3 7 ° C f o r 30 to 90 m i n , the O . D . ^ Q ^ of each w e l l was taken u s i n g a T i t e r t e k . The s u b s t r a t e b l ank w e l l s gave an average O . D . ^ Q J - o f 0.002 o r l e s s . The n o - a n t i b o d y c o n t r o l w e l l s had an average O . D . ^ Q ^ t h a t was 0.0 5 t o 1.0 u n i t lower than the average O . D . ^ Q ^ o b t a i n e d from the w e l l s i n c u - bated w i t h c o n t r o l an t ibody (Y3 or normal r a t serum). Any changes from the s t andard ELISA are noted i n the t e x t or the f o o t n o t e s to the t a b l e s . V e g e t a t i v e c e l l s f o r the p r e a d s o r p t i o n of RARIG were p r e - pared by re su spend ing washed and p e l l e t e d V12-M2 c e l l s i n an- 7 hydrous a b s o l u t e methanol a t 4 ° C t o 10 c e l l s / m l . C e l l s were f i x e d by end- to -end r o t a t i o n f o r 30 min a t 4 ° C . The methanol was washed from the c e l l s by repea ted c e n t r i f u g a t i o n (three t imes) at 700 x g f o r 2 min i n PBS. The p e l l e t e d c e l l s were then resuspended i n RARIG ( r e c o n s t i t u t e d a c c o r d i n g to manufac- g t u r e r ' s i n s t r u c t i o n s ) to 10 c e l l s / m l and the se ra was adsorbed on the end- to -end r o t a t o r o v e r n i g h t a t 4 ° C . The c e l l s were p e l l e t e d at 700 x g f o r 5 min and the RARIG was removed to an- o t h e r tube f o r s torage a t - 2 0 ° C . L) P r e p a r a t i o n o f A n t i b o d i e s Ammonium s u l p h a t e - p r e c i p i t a t e d Y13-259 or Y3 was p u r i f i e d by i o n exchange chromatography on a DEAE-Sephace l column as d e s c r i b e d p r e v i o u s l y (Fahey & T e r r y , 1973) . Immunoglobulin G 18 (IgG) was d e t e c t e d i n f r a c t i o n s e l u t e d from the column u s i n g RARIG i n an O u c h t e r l o n y t e s t . M) Removal of T r i t o n X-100 T r i t o n X-100 was removed from samples p r i o r to the a d s o r p t i o n o f the p r o t e i n to the ELISA p l a t e u s i n g B io-Beads SM-2 u s i n g the b a t c h procedure d e s c r i b e d by Hol loway (1973). Removal o f T r i t o n X-100 by acetone p r e c i p i t a t i o n was done by add ing an e q u a l volume of i c e - c o l d acetone to the membrane ex- t r a c t and i n c u b a t i n g on i c e f o r 30 m i n . The samples were c e n - t r i f u g e d at 13,000 x g f o r 5 min and the supernates were poured o f f . The p r e c i p i t a t e s were p l a c e d under vacuum i n a c e n t r i f u g e (Speed-Vac) f o r 30 min t o remove a l l the a c e t o n e . P e l l e t s were resuspended i n T r i s - s a l t s c o n t a i n i n g 40 yg/ml l e u p e p t i n and 40 yg/ml a n t i p a i n . N) Immunoaf f in i ty Chromatography A f f i - G e l 10 was c o u p l e d to D E A E - p u r i f i e d Y13-259 or Y3 i n 0 .1 M MOPS, pH 7 . 5 , f o r 4 h a t 4 ° C . Between 5 and 12 mg a n t i b o d y was c o u p l e d per ml g e l . F o r the exper iments u t i l i z i n g d e t e c t i o n o f p23 by ELISA, 5 ml immunoa f f in i ty columns were u s e d . Columns were e q u i l i - b r a t e d w i t h T r i s - s a l t s . The membrane e x t r a c t was a p p l i e d to the column and the f l o w - t h r o u g h was r e c y c l e d 3 t o 5 t i m e s . The column was washed and e l u t e d as d e s c r i b e d i n the R e s u l t s . One ml f r a c t i o n s were e l u t e d i n t o tubes c o n t a i n i n g 40 yg l e u p e p t i n and 40 yg a n t i p a i n . The f r a c t i o n s were d i a l y s e d a g a i n s t 1 1 of T r i s - s a l t s p l u s 100 yg/ml p_-aminobenzamidine o v e r n i g h t at 4 ° C 19 and the p r o t e i n conten t was d e t e r m i n e d . E i t h e r 10 yg o f p r o t e i n or 100 y l o f each f r a c t i o n was used as a n t i g e n f o r the ELISA and was adsorbed i n b u f f e r a d j u s t e d to pH of approx imate ly 10 u n i t s . F o r exper iments u t i l i z i n g the d e t e c t i o n o f p23 by immuno- p r e c i p i t a t i n of r a d i o l a b e l e d membrane e x t r a c t s , columns o f 1 ml bed volume were u s e d . The columns were e q u i l i b r a t e d w i t h the b u f f e r s d e s c r i b e d i n the R e s u l t s . Membrane e x t r a c t s were 8 3 5 p r e p a r e d from 5 x 10 S-methionine l a b e l e d c e l l s and a p p l i e d t o the columns i n a volume of 1 m l . The f l o w - t h r o u g h was r e - c y c l e d 3 to 5 t i m e s . T o t a l p r o t e i n c o n t e n t was e s t i m a t e d by t r i c h l o r o a c e t i c a c i d (TCA) p r e c i p i t a t i o n o f 5 y l a l i q u o t s t h a t had been d r i e d on g l a s s f i b e r f i l t e r s . The f i l t e r s were washed w i t h 20% TCA, 5% TCA and methano l , r e s p e c t i v e l y , and then the r a d i o a c t i v i t y was de termined by l i q u i d s c i n t i l l a t i o n i n a T r i - ton X-100 based s c i n t i l l a t i o n f l u i d . The columns were washed w i t h d i f f e r e n t d e t e r g e n t - c o n t a i n i n g T r i s - s a l t s b u f f e r s u n t i l a l l the T C A - p r e c i p i t a b l e r a d i o a c t i v i t y had been e l u t e d . Each wash b u f f e r was c o n c e n t r a t e d to 1 m l . The e l u t i o n c o n d i t i o n s t h a t were used depended on the exper iment and are d e s c r i b e d i n the f o o t n o t e s t o t a b l e s . E l u t e d f r a c t i o n s were d i a l y s e d : a g a i n s t 2 1 o f T r i s - s a l t s p l u s 100 yg /ml p_-aminobenzamidine o v e r n i g h t a t 4 ° C u n l e s s they c o n t a i n e d T r i s - s a l t s a l r e a d y . The samples were s p l i t i n t o two 0.5 ml a l i q u o t s and were immunoprec ip i t a t ed w i t h e i t h e r Y13-259 or Y3 . The i m m u n o p r e c i p i t a t e d p r o t e i n s were e l e c t r o p h o r e s e d through a 12.5% SDS-po lyacry lamide g e l . 20 3 F o l l o w i n g t reatment w i t h En hance , the d r i e d g e l was exposed a g a i n s t XAR-5 f i l m f o r one to s i x weeks. The p2 3 bands were q u a n t i t a t e d by d e n s i t o m e t r y and these va lue s were used t o e v a l u a t e r e c o v e r y of p23 from the columns . O) Dot B l o t The amount o f p2 3 i n v e g e t a t i v e membrane p r e p a r a t i o n s was a l s o de termined by the dot b l o t method d e s c r i b e d by Jahn e t a l . (1984) except the b l o t s were i n c u b a t e d ini . e i t h e r Y13-259 or normal r a t serum d i l u t e d 1/100 or 1/200 f o r 1 h a t 3 7 ° C o r o v e r n i g h t at 4 ° C . A f t e r washing and r e b l o c k i n g the n i t r o c e l l u - lo se s h e e t s , they were i n c u b a t e d w i t h RARIG (preadsorbed w i t h v e g e t a t i v e c e l l s ) d i l u t e d 1/400 f o r 1 h . A f t e r washing f o r 5 min f i v e t i m e s , the b l o t s were i n c u b a t e d w i t h G A R I G - a l k a l i n e phosphatase con jugate t h a t had been d i l u t e d 1/3000 i n b l o c k i n g s o l u t i o n c o n t a i n i n g 0.1% (v/v) T r i t o n X-100 f o r 1 h . The b l o t s were washed four t imes f o r 5 min i n T r i s / N a C l / 0 . 1 % T r i - ton X-100 and then four t imes f o r 25 min i n T r i s / N a C l / 0 . 1 % T r i - ton X-100. The b l o t s were i n c u b a t e d f o r 30 min at 3 7 ° C i n 10 ml o f D'.M T r i s - H C l , pH 8.8 c o n t a i n i n g 4 mg/ml M g C l 2 , 0.01 mg/ml BCIP and 0.1 mg/ml NBT. The b l o t s were r i n s e d w i t h d e i o n i z e d H^O and a i r - d r i e d . D E A E - p u r i f i e d normal r a t serum and d i f f e r e n t d i l u t i o n s of RARIG were used u n s u c c e s s f u l l y to t r y to lower the background . 21 RESULTS SECTION I . GUANINE NUCLEOTIDE BINDING 1" cl S The p u r i f i e d ra s p r o t e i n from mammalian c e l l s (p21 ) -9 -9 has a b i n d i n g c o n s t a n t o f 8 x 10 M f o r GTP and 7 x 10 M f o r GDP (Shih e t a l . , 1980). U s i n g at l e a s t t en t imes h i g h e r c o n c e n t r a t i o n s o f guanine n u c l e o t i d e , a number of d i f f e r e n t approaches were used to t r y t o d e t e c t guanine n u c l e o t i d e b i n d i n g by p 2 3 r a S . A) F i l t e r assay A p r e v i o u s study had shown t h a t 100 ug o f a c e l l - f r e e l y s a t e o f K i r s t e n murine sarcoma v i r u s (KiMSV) t rans formed NIH- 3 3T3 c e l l s w i l l b i n d 1.10 pmoles of H-GDP when immunoprec ip i - 3 t a t e d w i t h a n t i - p 2 1 a n t i b o d y whereas o n l y 0.05 pmoles o f H-GDP are bound u s i n g c o n t r o l serum ( S c o l n i c k e t a l . , 1979) . In a f i l t e r assay u s i n g s i m i l a r c o n d i t i o n s , p23 d i d not b i n d GDP (Table 1 ) . The l e v e l of bound GDP i n c e l l l y s a t e s immunoprec i- p i t a t e d w i t h the a n t i - r a s a n t i b o d y was i d e n t i c a l to t h a t i n c e l l l y s a t e s i m m u n o p r e c i p i t a t e d by normal r a t serum. U s i n g RARIG-Staph i n s t e a d o f RARIG-coated agarose f o r immunoprec ip i - t a t i o n d i d not change the r e s u l t and add ing SDS to the immuno- p r e c i p i t a t i o n i n c r e a s e d the background l e v e l s o f GDP bound w i t h both s p e c i f i c an t ibody and normal r a t serum (data not shown). 3 The H-GDP used i n these exper iments was determined t o be 93 t o 96 p e r c e n t pure by t h i n l a y e r chromatography t h e r e f o r e the low amount o f GDP b i n d i n g was not due t o degraded or contaminated GDP. 22 Table 1. Guanine N u c l e o t i d e B i n d i n g F i l t e r Assay 3 5S-7~GTP bound (pmol/mg) b H-GDP bound Incubate Immunoprec ip i ta te A n t i b o d y (fmol/mg) F i r s t F i r s t Normal r a t 21. .8 ± 9.2 1. .93 ± 0. . 32 12. .45 ± 4. . 47 serum Y-13-259 16. ,8 ± 13.2 1. .92 ± 0, .29 13. .21 ± 6, .69 a . The means and s t andard d e v i a t i o n s o f two d e t e r m i n a t i o n s are g i v e n . b . The numbers are the means o f four d e t e r m i n a t i o n s ± the s t andard d e v i a t i o n . " Incubate F i r s t " i n d i c a t e s GTP b i n d - i n g f o l l o w e d by i m m u n o p r e c i p i t a t i o n and " Immunoprec ip i - t a t e F i r s t " i n d i c a t e s i m m u n o p r e c i p i t a t i o n f o l l o w e d by GTP b i n d i n g . 23 F i l t e r assays u s i n g the n o n h y d r o l y z a b l e GTP-ana logue , S - y - G T P , r e v e a l e d h i g h e r l e v e l s of b i n d i n g than those o b t a i n e d w i t h GDP but t h e r e was no i n d i c a t i o n of any s p e c i f i c b i n d i n g t o p23 r a s (Table 1) . When e x t r a c t s were e n r i c h e d f o r p23 by immu- n o p r e c i p i t a t i n g f i r s t and then i n c u b a t i n g w i t h GTP, the o v e r a l l amount of GTP bound was i n c r e a s e d but there was no i n d i c a t i o n o f s p e c i f i c b i n d i n g t o the p r o t e i n i m m u n o p r e c i p i t a t e d w i t h Y13- 259 (Table 1 ) . B) Western B l o t Probed w i t h GTP When..'proteins were separa ted by SDS e l e c t r o p h o r e s i s and then e l e c t r o b l o t t e d onto n i t r o c e l l u l o s e , s e v e r a l o f the b l o t t e d 35 p r o t e i n s bound S-y-GTP. However, there was no l a b e l i n g i n IT cl S the r e g i o n t h a t cor re sponded t o p2 3 . The number o f GTP- b i n d i n g p r o t e i n s was decrea sed i f the l y s a t e s were f i r s t immu- n o p r e c i p i t a t e d p r i o r to e l e c t r o p h o r e s i s , but the same G T P - b i n d - i n g p r o t e i n s were i m m u n o p r e c i p i t a t e d by a n t i - r a s a n t i b o d y and by normal r a t serum ( F i g . 1 ) . A g a i n , there was no i n d i c a t i o n of GTP b i n d i n g to a p r o t e i n o f the m o l e c u l a r weight o f p23 C) P h o t o a f f i n i t y L a b e l l i n g w i t h GTP N u c l e o t i d e s can be c o v a l e n t l y c r o s s - l i n k e d to n u c l e c t i d e - b i n d i n g p r o t e i n s u s i n g p h o t o c h e m i c a l methods. Under the d i r e c t a c t i o n o f u l t r a v i o l e t l i g h t , the b i n d i n g of the l i g a n d r e q u i r e s a c t i v e enzyme and o n l y o c c u r s a t the l i g a n d - b i n d i n g s i t e on the p r o t e i n (Yue & Schimmel, 1977). T h i s t echn ique has been used t o d e t e c t s e v e r a l G T P - b i n d i n g p r o t e i n s i n D. d i s c o i d e u m , i n - c l u d i n g a s t r o n g l y b i n d i n g p r o t e i n of M of 24,000 ( S c h l e i c h e r 24 66K- l i .« 3.6K- 29K- t 20 K- 14 K- A B F i g u r e 1. GTP B i n d i n g t o D. d i s c o i d e u m P r o t e i n s . Samples were e l e c t r o p h o r e s e d t h r o u g h an 11.25% p o l y a c r y l a m i d e g e l and the p r o t e i n was transferred t o n i t r o c e l l u l o s e . The b l o t was probed w i t h 50 y C i S-y-GTP as d e s c r i b e d i n Methods . Two samples o f T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e (1.2 mg p r o t e i n ) were t r e a t e d with normal r a t serum (Lane A) o r Y13-259 (Lane B) and the i m m u n o p r e c i p i t a t e d m a t e r i a l was 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 . Lane C : 120 yg t o t a l p r o t e i n . M o l e c u l a r we ight markers were v i s u a l i z e d by the I n d i a Ink s t a i n i n g method. 25 e t a l . , 1986) . In o r d e r t o determine whether t h i s G T P - b i n d i n g 10 3, S p r o t e i n was a c t u a l l y the p23 , e x t r a c t s were p h o t o a f f i n i t y l a b e l e d w i t h GTP and then e i t h e r s u b j e c t e d t o SDS e l e c t r o p h o r e - s i s d i r e c t l y o r i m m u n o p r e c i p i t a t e d and then s u b j e c t e d to SDS e l e c t r o p h o r e s i s . The r e s u l t i n g g e l s were d r i e d and a u t o r a d i o - graphed . A number o f p r o t e i n s i n crude c y t o s o l and membrane f r a c t i o n s bound GTP, i n c l u d i n g a p r o t e i n o f M r about 24,000 ( F i g . 2) but none of these p r o t e i n s were i m m u n o p r e c i p i t a t e d w i t h e i t h e r the s p e c i f i c Y13-259) or the c o n t r o l a n t i b o d y (Y3). 10 Si s These r e s u l t s i n d i c a t e t h a t the p23 p r o t e i n does not b i n d GTP under these c o n d i t i o n s and t h a t the p r e v i o u s l y i d e n t i f i e d 24,000 M r G T P - b i n d i n g p r o t e i n i s not the r a s p r o t e i n . To e n - 10 ci S r i c h f o r p23 i n t h i s a s say , samples o f T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e (3.34 mg p r o t e i n ) were i m m u n o p r e c i p i t a t e d w i t h e i t h e r c o n t r o l or Y13-259 f i r s t and then the immunopreci- p i t a t e d p r o t e i n s were p h o t o a f f i n i t y l a b e l e d and e l e c t r o p h o r e s e d (see Methods ) . The r e s u l t s o b t a i n e d were the same as those TJ* Si S when p23 was p h o t o a f f i n i t y l a b e l e d and then immunoprec ip i - t a t e d . 10 Si s A l t h o u g h Y13-259 n e u t r a l i z e s p21 a c t i v i t y , i t does not i n t e r f e r e w i t h the b i n d i n g o f GDP ( H a t t o r i e t a l . , 1987) or GTP ( L a c a l & A a r o n s o n , 1986). However, i t i s p o s s i b l e t h a t GTP 10 cl S b i n d i n g p r e v e n t e d the i m m u n o p r e c i p i t a t i o n of p23 i n these 35 e x p e r i m e n t s . To address t h i s q u e s t i o n , S-methionine l a b e l e d T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e was p h o t o a f f i n i t y l a b e l e d i n the presence and absence o f 1 mM GTP and then immunoprec ip i - t a t e d w i t h c o n t r o l o r a n t i - r a s an t ibody ( F i g . 3) . In both 2 6 F i g u r e 2. D i r e c t P h o t o a f f i n i t y L a b e l i n g w i t h GTP. Three a l i - quots each of 100 u g p r o t e i n from crude c y t o s o l and membrane., I n a c t i o n s w e r e i n c u b a t e d i n t h e d a r k , o n i c e , w i t h 1.4 x 10 M P-a-GTP f o r 30 m i n . The samples were then i r r a d i a t e d w i t h s h o r t wave UV l i g h t f o r 60 m i n . One c y t o s o l (Lane A) and one membrane (Lane B) sample was mixed w i t h SDS sample b u f f e r and s t o r e d a t - 2 0 ° C w h i l e two samples each o f c y t o s o l (Lanes C and D) and membrane (Lanes E and F) p r o t e i n s were i m m u n o p r e c i p i - t a t e d w i t h e i t h e r c o n t r o l a n t i b o d y (Lanes C and E) o r Y13-259 (Lanes D and F ) . A l l samples were then s u b j e c t e d to e l e c t r o - p h o r e s i s through a 12.5% p o l y a c r y l a m i d e g e l . The g e l was c u t to separa te Lanes A and B from Lanes C t h r o u g h F so t h a t the d r i e d g e l s c o u l d be' exposed to f i l m (with a s c reen and at - 7 0 ° C ) f o r d i f f e r e n t p e r i o d s o f t ime . Lanes A and B : exposed f o r 23 h . Lanes C through F : exposed f o r one week. 27 20.1K 14.2K 1 2 3 4 F i g u r e 3. P h o t o a c t i v a t i o n and I m m u n o p r e c i p i t a t i o n i n the P r e - sence and Absence o f 1 mM GTP. Equa l amounts (100 yg p r o t e i n ) of S-methionine l a b e l e d T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e were i n c u b a t e d i n the presence (1) and absence (2) o f 1 mM GTP i n the da rk , on i c e , f o r 30 min. The samples were then i r r a - d i a t e d w i t h s h o r t wave UV l i g h t fo r 60 m i n . Samples were then i m m u n o p r e c i p i t a t e d w i t h e i t h e r c o n t r o l (Lanes A and C) or a n t i - ras (Lanes B and D) a n t i b o d y and e l e c t r o p h o r e s e d through a 12.5% p o l y a c r y l a m i d e g e l as d e s c r i b e d i n Methods . The d r i e d g e l was exposed t o XAR-5 f i l m f o r 10 d a y s . 2 8 cases the p23 p r o t e i n was i i tununoprecipi ta ted by the Y13-259 a n t i b o d y sugge s t ing t h a t GTP b i n d i n g does not i n t e r f e r e w i t h i m m u n o p r e c i p i t a t i o n . A p o s i t i v e c o n t r o l f o r GTP b i n d i n g was p r o v i d e d by p h o t o - a f f i n i t y l a b e l i n g a l y s a t e from HaMSV-transformed r a t c e l l s and then i m m u n o p r e c i p i t a t i n g w i t h e i t h e r a n t i - r a s (Y13-259) or c o n t r o l a n t i b o d y (Y3) . The i m m u n o p r e c i p i t a t e s were then e l e c - 2Tcl S t r o p h o r e s e d . The p21 p r o t e i n d i d b i n d GTP under these c o n - d i t i o n s ( F i g . 4 ) . A 0.3% T r i t o n X-100 D. d i s c o i d e u m membrane e x t r a c t was p h o t o a f f i n i t y l a b e l e d under the same c o n d i t i o n s and then i m m u n o p r e c i p i t a t e d w i t h e i t h e r Y13-259 or Y3 ant ibody. The i m m u n o p r e c i p i t a t e d p r o t e i n s were then e l e c t r o p h o r e s e d and a u t o r a d i o g r a p h e d . There i s no i n d i c a t i o n o f GTP b i n d i n g by 3tr SL S p23 under these c o n d i t i o n s . 29 66K -36K •29K •(24K) A B C D F i g u r e 4. GTP B i n d i n g t o p21 from Harvey Mur ine Sarcoma V i r u s (HaMSV) Trans formed NIH-3T3 C e l l s . E q u a l amounts o f p r o t e i n (2.5 mg i n about 1 ml volume) from e i t h e r a 0.3% T r i - ton X-100 D. d i s c o i d e u m membrane e x t r a c t (Lanes A and B) or a l y s a t e of HaMSV-transformed NIH-3T3 c e l l s (Lanes C and D) were c o n c e n t r a t e d t o 100 y l a t 4 ° C . Two 50 y l a l i q u o t s o f ^ | c h sample were i n c u b a t e d i n m i c r o t i t e r w e l l s w i t h 1.2o.pM P-crGTP i n the dark f o r 60 m i n , on i c e . The samples were then i r r a - d i a t e d w i t h short-wave UV l i g h t f o r 30 m i n . The p h o t o a f f i n i t y - l a b e l e d samples were then i m m u n o p r e c i p i t a t e d w i t h e i t h e r 5 y l o f Y3 (Lanes A and C) or 5 y l of H P L C - p u r i f i e d Y13-259 (Lanes B and D ) . The i m m u n o p r e c i p i t a t e d p r o t e i n s were e l e c t r o p h o r e s e d through a 12.5% S D S - p o l y a c r y l a m i d e g e l . The d r i e d g e l was exposed to f i l m a t - 2 0 ° C w i t h a screen f o r 4 d a y s . 30 SECTION I I . ATTEMPTED PURIFICATION OF p 2 3 r a S Attempts were made to p u r i f y p23 s i n c e a p a r t i a l l y p u r i f i e d p r e p a r a t i o n s h o u l d p r o v i d e more d e f i n i t i v e r e s u l t s i n a G T P - b i n d i n g assay i n t h a t o ther G T P - b i n d i n g p r o t e i n s should be removed and there shou ld be r e l a t i v e l y more p23 p r o t e i n . 3T cl S U l t i m a t e l y p u r i f i e d p23 c o u l d be used i n membrane r e c o n s t i - t u t i o n exper iments to s tudy the f u n c t i o n of the p r o t e i n . Immu- n o a f f i n i t y chromatography was chosen because i t p o t e n t i a l l y p r o v i d e s a one-s tep p u r i f i c a t i o n p r o c e d u r e , the immunopreci- p i t a t i o n wi th Y13-259 works w e l l , and t h e r e i s an u n l i m i t e d supply o f the monoc lona l a n t i b o d y . A) S o l u b i l i z a t i o n C o n d i t i o n s 2T cl S The p2 3 i s e x c l u s i v e l y found i n the crude membrane f r a c t i o n r a t h e r than the c y t o s o l i c f r a c t i o n a f t e r h i g h - s p e e d c e n t r i f u g a t i o n i n the presence of the pro tea se i n h i b i t o r s l e u - p e p t i n and a n t i p a i n (Weeks & Pawson, 1987). The p23 p r o t e i n was removed from the membrane by T r i t o n X-100 ( F i g . 5 ) , a c o n - c e n t r a t i o n o f 0.3% (w/v) b e i n g r e q u i r e d f o r t o t a l s o l u b i l i z a - t i o n ( F i g . 6 ) . T r i t o n X-100 s o l u b i l i z a t i o n r e s u l t e d i n an enr ichment of p2 3 of a p p r o x i m a t e l y f o u r - f o l d over T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e s . The p 2 3 r a S was not s o l u b i l i z e d from the membrane by h i g h pH , h i g h s a l t , or the presence of EDTA ( F i g . 7 ) , sugges t ing t h a t p2 3 i s t i g h t l y bound to the membrane b i l a y e r . In f a c t , p23 i s a c y l a t e d (Weeks e t a l . , 1987) l i k e the mammalian ras p r o t e i n , and the a c y l group may anchor the p r o t e i n to the membrane. 31 1 2 3 4 5 6 7 8 9 10 11 12 — 66K — 4 5 K 36K 2 9 K p23 — 20.1 K 14.2K F i g u r e 5. S o l u b i l i z a t i o n of p23 w i t h T r i t o n X-100 . E q u a l amounts of S - m e t h i o n i n e - l a b e l e d crude membranes were r e s u s - pended i n 1 ml o f T r i s - s a l t s c o n t a i n i n g 40 yg l e u p e p t i n , 40 yg a n t i p a i n , and 0% (Lanes 1 t o 4) , 0.3% (Lanes 5 t o 8 ) , o r 1.0% (Lanes 9 to 12) T r i t o n X-100 and mixed w e l l by v o r t e x i n g . A f t e r a 15 min i n c u b a t i o n a t 4 ° C , the samples were r e c e n t r i - fuged t o separate u n s o l u b i l i z e d p e l l e t (Lanes 1, 2, 5, 6, 9, 10) and s o l u b i l i z e d superna tan t (Lanes 3, 4, 7, 8, 11, 12) . The p e l l e t s were then resuspended i n 1 ml o f b u f f e r and each sample was s p l i t i n t o two 0.5 ml a l i q u o t s f o r i m m u n o p r e c i p i - t a t i o n w i t h 75 y l o f e i t h e r normal r a t serum (Lanes 1, 3, 5, 1, 9, 11) or Y13-259 Lanes 2, 4, 6, 8, 10, 12 ) . The immuno- p r e c i p i t a t e d p r o t e i n was e l e c t r o p h o r e s e d as d e s c r i b e d i n Methods and f l u o r o g r a p h e d f o r 9 days . 32 — 66K —45K —36K 29K p23 —20.1 K — 14.2K 1 2 3 4 5 6 7 8 9 10 11 12 F i g u r e 6. E f f e c t o f Low C o n c e n t r a t i o n s o f T r i t o n X-100 on the S o l u b i l i z a t i o n o f p23 . E q u a l amounts o f r a d i o l a b e l e d crude membrane f r a c t i o n s were resuspended i n 1 ml o f T r i s - s a l t s c o n t a i n i n g 40 yg l e u p e p t i n , 40 yg a n t i p a i n , and 0.01% (Lanes 1 t o 4 ) , 0.03% (Lanes 5 t o 8 ) , or 0.1% (Lanes 9 t o 12) T r i t o n X-100. A f t e r a 15 min i n c u b a t i o n , the samples were r e c e n t r i - fuged t o separate u n s o l u b i l i z e d p e l l e t (Lanes 1, 2, 5, 6, 9, 10) and s o l u b i l i z e d superna tan t (Lanes 3, 4, 7, 8, 11, 12) . The p e l l e t s were resuspended i n 1 ml o f b u f f e r and each sample was s p l i t i n t o two 0.5 ml a l i q u o t s f o r i m m u n o p r e c i p i t a t i o n w i t h e i t h e r normal r a t serum (Lanes 1, 3, 5, 7, 9, 11) or Y13- 259 (Lanes 2, 4, 6, 8, 10, 12) . The i m m u n o p r e c i p i t a t e d p r o t e i n was e l e c t r o p h o r e s e d and f l u o r o g r a p h e d f o r 4 d a y s . 3 3 — 66K — 45K — 3 6 K — - 2 9 K < - p 2 3 — 2 W K — 14.2K 3 4 6 7 8 9 10 11 12 F i g u r e 7. E f f e c t o f High S a l t , 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 (EDTA), o r H i g h pH on S o l u b i l i z a t i o n o f p23. E q u a l amounts of r a d i o l a b e l e d crude membranes were re suspended i n 1 ml o f T r i s - s a l t s c o n t a i n i n g 40 yg l e u p e p t i n , 40 yg a n t i - p a i n , and 0.5 M N a C l (Lanes 1 t o 4 ) , 10 mM EDTA (Lanes 5 t o 8 ) , or 100 mM Na^COg (Lanes 9 to 12 ) . A f t e r a 15 min i n c u - b a t i o n , the samples were r e c e n t r i f u g e d t o s epara te u n s o l u - b i l i z e d p e l l e t (Lanes 1, 2, 5, 6, 9, 10) and s o l u b i l i z e d v>. <•:•• supernatant (Lanes 3, 4, 7, 8, 11, 12 ) . The s u p e r n a t a n t s were d i a l y s e d a g a i n s t T r i s - s a l t s o v e r n i g h t a t 4 ° C and the p e l l e t s were resuspended i n 1 ml of b u f f e r and s t o r e d o v e r - n i g h t a t - 7 0 ° C . Each sample was s p l i t i n t o two 0.5 ml a l i - quots and i m m u n o p r e c i p i t a t e d w i t h e i t h e r normal r a t serum (Lanes 1, 3, 5, 7, 9, 11) or Y13-259 (Lanes 2, 4, 6, 8, 10, 12) . The i m m u n o p r e c i p i t a t e d p r o t e i n was e l e c t r o p h o r e s e d and f l u o r o g r a p h e d f o r 4 d a y s . 34 B) D e t e c t i o n by ELISA The ELISA Is a r a p i d and s imple method t h a t can be used to m o n i t o r p r o t e i n p u r i f i c a t i o n . An assay was deve loped u s i n g v e g e t a t i v e membranes as the a n t i g e n , as d e s c r i b e d under Methods. In an at tempt t o lower backgrounds , a v a r i e t y o f m o d i f i c a t i o n s were t r i e d : BSA, sheep IgG (Table 2 ) , or 1% SDS (data not shown) as b l o c k i n g agent s , ther-use o f D E A E - p u r i f i e d Y13-259, and the use of enzyme-ant ibody con juga te t h a t had been f i r s t adsorbed w i t h v e g e t a t i v e c e l l s (data no t shown). The b e s t r e s u l t s were o b t a i n e d w i t h 0.3% BSA as b l o c k i n g agent w i t h Y3 as c o n t r o l a n t i b o d y (Table 2 ) . In a d d i t i o n , v a r y i n g the i n c u - b a t i o n t imes d i d not improve the a c t i v i t y over background. A two-step ELISA, u s i n g goat a n t i - r a t IgG (GARAT) c o n j u - gated to a l k a l i n e phosphatase as the second a n t i b o d y , gave h i g h a c t i v i t y to c o n t r o l r a t i o s but o n l y a t very h i g h c o n c e n t r a t i o n s of GARAT (Table 3 ) . F o r example, u s i n g Y3 as the c o n t r o l a n t i - body, a 1/100 d i l u t i o n o f GARAT gave an a c t i v i t y t o c o n t r o l r a t i o o f 3.3 whereas the s t andard assay u s i n g the a l k a l i n e phosphatase con juga te a t a d i l u t i o n o f 1/2000 gave an a c t i v i t y to c o n t r o l r a t i o o f 1.7 (Table 2 ) . However:, the two-step ELISA proved to be c o s t - p r o h i b i t i v e due to the h i g h c o n c e n t r a t i o n s cf GARAT r e q u i r e d f o r a h i g h a c t i v i t y t o c o n t r o l r a t i o . In the s t andard ELISA, c o n c e n t r a t i o n s o f T r i t o n X-100 i n excess of 0.00 3% reduce the Y13-259 a c t i v i t y t o the same l e v e l as the c o n t r o l a c t i v i t y (data not shown). Other d e t e r g e n t s (po lyoxye thy lene (20) s o r b i t a n monolaurate (Tween 20) , 3- (3- cho lamidopropy l )d imethy l -ammonio (CHAPS), Z-14, p o l y o x e t h y l e n e Table 2. ELISA U s i n g V e g e t a t i v e Membranes as A n t i g e n ° ' D - 4 0 5 C o n d i t i o n Normal Rat Serum Y3 Y13 -259 A c t i v i t y - C o n t r o l A / C R a t i o 2 0.3% BSA 0.495 N / D b 0 . 710 0.215 1.4 ± 0.028 + 0 .024 ± 0.037 0.3% BSA N/D 0.426 0 . 710 0 .284 1.7 ± 0.037 + 0 .024 ± 0.044 0.001% 0 .374 N/D 0 .535 0 .161 1.4 Sheep IgG ± 0.021 + 0 .023 ± 0.031 0.003% 0.417 N/D 0 .510 0.093 1.2 Sheep IgG 1 0.033 + 0 .020 ± 0 . 0 . 3 9 a . A c t i v i t y to c o n t r o l r a t i o . b . Not d e t e r m i n e d . The means and s t a n d a r d d e v i a t i o n s of e i g h t d e t e r m i n a t i o n s are g i v e n . These r e s u l t s are from a s i n g l e exper iment but are r e p r e s e n t a t i v e o f s e v e r a l exper iment s . Sheep IgG was added i n p l a c e of BSA. The i n c u b a t i o n w i t h s u b s t r a t e was f o r 60 min . 36 Tab le 3. Two-Step ELISA wi th Goat A n t i - R a t Immuno- g l o b u l i n G (GARAT) Conjugated t o A l k a l i n e Phosphatase O . D . 4 Q 5 D i l u t i o n Normal A c t i v i t y A / C of GARAT Rat Serum Y3 Y13-259 - C o n t r o l R a t i o 1/20' 0.086 ± 0.014 N/D 0.736 0.074 0 0 6 50 075 8.6 1/100 N/D 0.097 0.003 0.319 ± 0.007 0.222 ± 0.008 3.3 a . The means and s t andard d e v i a t i o n s o f seven d e t e r m i n a t i o n s are g i v e n . The i n c u b a t i o n w i t h s u b s t r a t e was f o r 30 min . b . The means and s t andard d e v i a t i o n s o f two d e t e r m i n a t i o n s are g i v e n . The i n c u b a t i o n w i t h s u b s t r a t e was f o r 90 min . 37 2 3 - l a u r y l e ther (Br i j -35 ) have the same d e l e t e r i o u s e f f e c t and n - o c t y l - g l u c o s i d e g i v e s a f a l s e p o s i t i v e r e s u l t (data not shown). Removal o f T r i t o n X-100 w i t h B io-Beads SM-2 does not remove s u f f i c i e n t d e t e r g e n t to a l l o w a c t i v i t y above c o n t r o l l e v e l s (data not shown). Acetone p r e c i p i t a t i o n o f samples d i d remove the T r i t o n X-100 but r e s u l t e d i n o n l y a very weak s i g n a l (Table 4 ) . X" cl S D e s p i t e these prob lems , I t r i e d to d e t e c t p23 i n f r a c - t i o n s s epara ted by i m m u n o a f f i n i t y chromatography w i t h the ELISA because I thought the p u r i f i e d p r o t e i n would g i v e a s t r o n g enough s i g n a l t o be d e t e c t e d w i t h t h i s a s say . Samples c o n t a i n - ing T r i t o n X-100 were a c e t o n e - p r e c i p i t a t e d b e f o r e b e i n g used fo r the ELISA. A 0.3% T r i t o n X-100 membrane e x t r a c t was a p p l i e d t o a column t h a t had been p r e - e q u i l i b r a t e d i n T r i s - s a l t s (10 mM T r i s - H C l , pH 7 . 5 , 5 mM M g C l 2 , 100 mM N a C l ) . In o r d e r to remove unbound p r o t e i n and d e t e r g e n t the column was washed w i t h T r i s - s a l t s u n t i l the O . D . _ o r i r eached z e r o . S e v e r a l c o n d i t i o n s were used t o t r y to e l u t e p 2 3 r a S : 0 .5 M N a C l ; 0 .1 N HC1; 0 .1 M g l y c i n e - H C l , pH 2.5 c o n t a i n i n g 50% e t h y l e n e g l y c o l ; 1 M and 4 M g u a n i d i n e - H C l ; and 3 M and 6 M NaSCN. In a l l exper iment s , ]C cl S p23 was bound to the column but was not e l u t e d by any of the e l u t i o n c o n d i t i o n s . A p p r o x i m a t e l y 25% of the t o t a l p r o t e i n a p p l i e d to the column was never r e c o v e r e d . T h i s suggested t h a t T r i t o n X-100 was needed d u r i n g chromatography to keep the mem- brane p r o t e i n s s o l u b i l i z e d . An assay was t h e r e f o r e r e q u i r e d 38; Table 4. ELISA wi th V e g e t a t i v e Membranes and A c e t o n e - P r e c i p i t a t e d 0.3% T r i t o n X-100 Membrane E x t r a c t O - D . 4 0 5 Normal A c t i v i t y C o n d i t i o n Rat Serum Y13-259 - C o n t r o l A / C R a t i o V e g e t a t i v e 0.464 0.872 0 .408 1.9 Membranes ± 0.070 ± 0.055 ± 0.089 A c e t o n e - 0 .110 0 .192 0 .082 1.7 P r e c i p i t a t e d ± 0.014 ± 0.113 ± 0.114 The means and s t andard d e v i a t i o n s of four d e t e r m i n a t i o n s are g i v e n . The RARIG was d i l u t e d 1/1000 f o r t h i s exper iment and the i n c u b a t i o n w i t h s u b s t r a t e was f o r 30 m i n . 39 t h a t c o u l d be used i n the presence o f d e t e r g e n t , s i n c e the ELISA had poor s e n s i t i v i t y on a c e t o n e - t r e a t e d f r a c t i o n s . Two immunoassays t h a t can be performed w i t h the a n t i g e n i n the presence o f d e t e r g e n t are the dot b l o t and the Western b l o t . The dot b l o t assay was i n s u f f i c i e n t l y s p e c i f i c to be of v a l u e i n t h a t there was an e q u a l l y s t r o n g a c t i v i t y when the a n t i g e n was i n c u b a t e d w i t h Y13-2 59 a n t i b o d y as when i t was i n - cubated wi th normal r a t serum. The Western immunoblot t ing Z? ci S t echn ique would o n l y d e t e c t p23 i f the p r o t e i n was immuno- p r e c i p i t a t e d p r i o r to e l e c t r o p h o r e s i s and then e l e c t r o b l o t t e d to n i t r o c e l l u l o s e f o r d e t e c t i o n , and t h i s method proved to be i n s u f f i c i e n t l y s e n s i t i v e (data no t shown). The most s e n s i t i v e assay t h a t c o u l d be performed w i t h the a n t i g e n i n the presence of d e t e r g e n t was i m m u n o p r e c i p i t a t i o n o f r a d i o a c t i v e l y - l a b e l e d p r o t e i n s f o l l o w e d by r a d i o a u t o g r a p h y . T h i s assay was t h e r e - fo re used to moni to r the f r a c t i o n a t i o n of membrane e x t r a c t s by immunoa f f in i ty chromatography. C) D e t e c t i o n by I m m u n o p r e c i p i t a t i o n 35 For these exper iment s , S - m e t h i o n i n e - l a b e l e d membrane e x t r a c t s were a p p l i e d to immunoa f f in i ty columns t h a t had been p r e - e q u i l i b r a t e d under v a r i o u s d e t e r g e n t c o n d i t i o n s . S ince the i m m u n o p r e c i p i t a t i o n o f p23 w i t h Y13-259 i s done i n the presence o f 1% T r i t o n and 0.5% SDS, an immunoa f f in i ty column e q u i l i - b r a t e d w i t h the same d e t e r g e n t s was i n i t i a l l y used t o t r y to p u r i f y p2 3. The membrane e x t r a c t a p p l i e d to t h i s column was a l s o s o l u b i l i z e d w i t h 1% T r i t o n X-100 and 0.5% SDS. About 30% 40 o f the t o t a l p r o t e i n bound n o n s p e c i f i c a l l y t o t h i s column but IT ci. S p23 d i d not b i n d under these c o n d i t i o n s (Table 5) so o ther b u f f e r c o n d i t i o n s were t r i e d . A membrane e x t r a c t s o l u b i l i z e d with 0.3% T r i t o n X-100 was a p p l i e d to a Y13-259 column t h a t had been e q u i l i b r a t e d i n T r i s - s a l t s b u f f e r (Table 6 ) . Under these c o n d i t i o n s there was l i t - t l e n o n s p e c i f i c b i n d i n g by t o t a l p r o t e i n but aga in none o f the p 2 3 r a s was bound. 4 1 T a b l e 5 . I m m u n o a f f i n i t y C h r o m a t o g r a p h y w i t h Y 1 3 - 2 5 9 C o l u m n E q u i l i b r a t e d i n T r i s - S a l t s P l u s 1% T r i t o n X - 1 0 0 a n d . 0 . 5 % S o d i u m D o d e c y l S u l p h a t e ( S D S ) . F l 6 w - N o t T h r o u g h E l u t i o n 1 E l u t i o n 2 R e c o v e r e d P r o t e i n (%) 6 8 0 . 8 0 . 2 3 1 P 2 3 r a s (%) 1 0 0 0 0 0 a . T h e c o l u m n w a s e l u t e d s e q u e n t i a l l y w i t h 0 . 1 M d i e t h y l a m i n e , 1 M g u a n i d i n e , a n d 1 M N a S C N , e a c h c o n t a i n i n g 1% T r i t o n X - 1 0 0 . b . T h e c o l u m n w a s t h e n e l u t e d w i t h t h e h a r s h e r c o n d i t i o n o f 6 M N a S C N + 1% T r i t o n X - 1 0 0 . 42 Table 6. Immunoaf f in i ty Chromatography w i t h Y13-259 Column E q u i l i b r a t e d i n T r i s - S a l t s F low- b Not Through E l u t i o n 1 E l u t i o n 2 Recovered P r o t e i n (%) 98 1.7 0 .3 0 p 2 3 r a S (%) 100 0 0 0 a . The column was e l u t e d s e q u e n t i a l l y w i t h T r i s - S a l t s + 0.5% SDS, 0.1 N H C l , and 0.1 M d i e t h y l a m i n e each c o n t a i n i n g 1% T r i t o n X-100 . b . The column was then s e q u e n t i a l l y e l u t e d w i t h 4M g u a n i d i n e and 6 M NaSCN, each c o n t a i n i n g 1% T r i t o n X-100. 43 DISCUSSION In t h i s s t u d y , I a t tempted to demonstrate GTP b i n d i n g by 3T cl S p23 u s i n g t h r e e d i f f e r e n t approaches : a f i l t e r a s say , a Western b l o t a s say , and p h o t o a f f i n i t y l a b e l i n g . The f i l t e r assay i n v o l v e d i n c u b a t i o n o f c e l l e x t r a c t s w i t h r a d i o l a b e l e d GDP or GTP f o l l o w e d by i m m u n o p r e c i p i t a t i o n wi th a n t i - r a s a n t i - body. The i m m u n o p r e c i p i t a t e d complexes were then c o l l e c t e d on n i t r o c e l l u l o s e f i l t e r s and counted d i r e c t l y . T h i s approach has been used s u c c e s s f u l l y t o demonstrate GDP and GTP b i n d i n g by KiMSV t rans formed c e l l l y s a t e s i m m u n o p r e c i p i t a t e d w i t h anti-p21 a n t i b o d y ( S c o l n i c k e t a l . , 1979) . Exper iments where the immune complexes were b o i l e d to r e l e a s e the p r o t e i n i n s t e a d of be ing c o l l e c t e d on n i t r o c e l l u l o s e f i l t e r s showed guanine n u c l e o t i d e b i n d i n g by y e a s t c e l l e x t r a c t s (Tamanoi e t a l . , 1984) . S ince the same monoclona l a n t i b o d y (Y13-259) s p e c i f i c a l l y immunopre- X* ci S c i p i t a t e s p21 and p23 (Pawson e t a l . , 1985), t h i s assay shou ld d e t e c t guanine n u c l e o t i d e b i n d i n g wi th D. d i s co ideum c e l l e x t r a c t s i f p23 does b i n d guanine n u c l e o t i d e s . One pos - i" cl S s i b l e reason f o r the absence o f s p e c i f i c GTP b i n d i n g by p23 i n the f i l t e r as says d e s c r i b e d i n t h i s i n v e s t i g a t i o n i s the h y d r o l y s i s o f bound GTP to GDP and i t s subsequent r e l e a s e by i" ci S the enzyme. However, s i n c e p23 d i d not b i n d a nonhydro- l y z a b l e GTP-ana logue , t h i s e x p l a n a t i o n was r e j e c t e d . F o r the Western b l o t assay p r o t e i n s from c e l l l y s a t e s and immunoprec ip i t a t ed p r o t e i n were separa ted on p o l y a c r y l a - mide g e l s and then e l e c t r o - b l o t t e d to n i t r o c e l l u l o s e . The 44 n i t r o - c e l l u l o s e was then i n c u b a t e d w i t h r a d i o l a b e l e d GTP. T h i s ^ Si S method had been used to show GTP b i n d i n g by p21 t h a t had been expres sed i n E s c h e r i c h i a c o l i under c o n d i t i o n s where t h i s p r o t e i n r e p r e s e n t e d 5 to 10 per c e n t o f the t o t a l i n t r a c e l l u l a r b a c t e r i a l p r o t e i n (McGrath e t a l . , 1984) . In v e g e t a t i v e c e l l s o f D. d i s c o i d e u m , p23 r e p r e s e n t s o n l y 0.02 t o 0.03 per c e n t of t o t a l p r o t e i n (Pawson e t a l . , 1985) . The low c e l l u l a r l e v e l 10 Si S of p23 may account f o r the f a i l u r e o f the Western b l o t assay to d e t e c t GTP b i n d i n g to the p r o t e i n . In the t h i r d approach , p . d i s co ideum c e l l l y s a t e s were p h o t o a f f i n i t y l a b e l e d w i t h GTP and the l a b e l e d p r o t e i n s were e i t h e r e l e c t r o p h o r e s e d d i r e c t l y o r i m m u n o p r e c i p i t a t e d and then e l e c t r o p h o r e s e d . In a d d i t i o n , c e l l l y s a t e s were immunoprec ip i - t a t e d and then p h o t o a f f i n i t y l a b e l e d w i t h GTP p r i o r t o e l e c t r o - p h o r e s i s . T h i s approach l e d to d e t e c t a b l e l a b e l i n g of the 2T cl S p21 p r o t e i n from HaMSV-transformed NIH-3T3 c e l l s i n the 10 cl S presence of 1.2 yM GTP ( F i g . 4 ) , but p23 e x h i b i t e d no d e t e c - t a b l e GTP b i n d i n g u s i n g the same e x p e r i m e n t a l c o n d i t i o n s . One p o s s i b l e reason f o r a l a c k of GTP b i n d i n g by c e l l l y - sa te s p h o t o a f f i n i t y l a b e l e d and then immunoprec ip i t a t ed i s t h a t the bound GTP i n t e r f e r e s w i t h i m m u n o p r e c i p i t a t i o n of p23 by Y13-259. However, i n c u b a t i o n of T r i t o n - s o l u b i l i z e d whole c e l l l y s a t e w i t h 1 mM GTP under s i m i l a r c o n d i t i o n s t o those used i n the p h o t o a f f i n i t y l a b e l i n g exper iments d i d not i n t e r f e r e w i t h i m m u n o p r e c i p i t a t i o n o f p23 by Yl3<+259 ( F i g . 3 ) . There are two p o s s i b l e e x p l a n a t i o n s f o r the l a c k o f 10 Si s GDP/GTP b i n d i n g by p23 : the p r o t e i n from D. d i s co ideum may 45 have a very low a f f i n i t y f o r guanine n u c l e o t i d e s and b i n d i n g i s t h e r e f o r e u n d e t e c t a b l e u s i n g the e x p e r i m e n t a l p rocedure s de- cl S s c r i b e d i n t h i s t h e s i s or p23 may n o t b i n d guanine n u c l e o - t i d e s a t a l l . The f i r s t p o s s i b i l i t y c o u l d t h e o r e t i c a l l y be t e s t e d by u s i n g h i g h c o n c e n t r a t i o n s o f GTP but t h i s exper iment i s i m p r a c t i c a l i n terms o f the l a r g e amounts o f r a d i o l a b e l t h a t would be r e q u i r e d . The second p o s s i b i l i t y seems u n l i k e l y i n view of the f a c t t h a t the RASl and RAS2 p r o t e i n s of Saccharo- myces c e r e v i s i a e b i n d GTP and GDP s p e c i f i c a l l y (Temeles e t a l . , 1985; Tamanoi e t a l . , 1984) l i k e the mammalian r a s p r o t e i n s ( S c o l n i c k e t a l . , 1979; S h i h e t a l . , 1980; Trahey e t a l . , 1987) a l t h o u g h they a p p a r e n t l y pe r fo rm d i f f e r e n t p h y s i o l o g i c a l func- t i o n s (Beckner e t a l . , 1985; Toda e t a l . , 1985; Uno e t a l . , 1985) . A number of d i f f e r e n t enzyme a c t i v i t i e s i n D. d i s co ideum c e l l s t h a t had been t r a n s f e c t e d w i t h the missense muta t ion Dd- 12 r a s - T h r have been examined (Van Haas te r e t a l . , 1987). T h i s m u t a t i o n , analogous t o t h a t found i n oncogenic mammalian ra s genes (Newbold, 1984), r e s u l t s i n abberant morphogenesis of D. 12 d i s c o i d e u m c e l l s (Reymond e t a l . , 1986). The D d - r a s - T h r t r a n s f o r m a n t s e x h i b i t normal f o l a t e and cAMP-induced a c t i v a t i o n and d e s e n s i t i z a t i o n of a d e n y l a t e c y c l a s e but the t r a n s i e n t i n - c rea se i n cGMP i s t e r m i n a t e d e a r l i e r i n these c e l l s than i n untrans formed c e l l s . T h i s l a t t e r phenomenon i s not due to a lower a c t i v i t y o f guany la te c y c l a s e or to a h i g h e r a c t i v i t y of c G M P - s t i m u l a t e d p h o s p h o d i e s t e r a s e , but r a t h e r to an a l t e r e d 17 cl S d e s e n s i t i z a t i o n o f guany la te c y c l a s e . Thus p23 does not 46 appear to be i n v o l v e d i n the r e g u l a t i o n o f a d e n y l a t e c y c l a s e but t o be d i r e c t l y or i n d i r e c t l y i n v o l v e d i n the enhanced de- s e n s i t i z a t i o n of guany la te c y c l a s e . The Dd-ras gene used i n the s t u d i e s d e s c r i b e d above was o r i g i n a l l y d e t e c t e d as a homologue of the mammalian r a s gene (Reymond e t a l . , 1984). I t encodes a p r o t e i n of M r of 24 ,000 . X" cl S There are d i s t i n c t d i f f e r e n c e s i n the e x p r e s s i o n of p23 and the messenger r i b o n u c l e i c a c i d (mRNA) t h a t i s h y b r i d i z e d by the r a s gene d u r i n g development . S y n t h e s i s o f p2 3 i s most r a - p i d i n v e g e t a t i v e c e l l s and then drops d u r i n g d i f f e r e n t i a t i o n u n t i l the p s e u d o p l a s m o d i a l stage where there i s a s l i g h t i n - crease i n the r a t e o f s y n t h e s i s be fore the r a t e c o n t i n u e s to drop t o about 10% of the o r i g i n a l ameoboid l e v e l a t the end of development (Pawson e t a l . , 1985) . In c o n t r a s t r a s mRNA i s p r e s e n t i n low l e v e l s i n v e g e t a t i v e c e l l s , but i n h i g h l e v e l s i n pseudoplasmodia (Reymond e t a l . , 1984) . A t the p s e u d o p l a s - m o d i a l s tage , newly s y n t h e s i z e d p23 i s l o c a l i z e d i n pre spore c e l l s (Weeks & Pawson, 1987) whereas p24 mRNA i s l o c a l i z e d i n p r e s t a l k c e l l s (Reymond e t a l . , 1984). I t i s p o s s i b l e t h a t X" cl S p23 and the p r o d u c t o f the c l o n e d r a s gene are n o t i d e n t i c a l and t h a t each p r o t e i n has a s p e c i f i c f u n c t i o n a t p a r t i c u l a r X* cl S p o i n t s of development . Thus p2 3 may not be i n v o l v e d i n the d e s e n s i t i z a t i o n o f guany la te c y c l a s e l i k e the p r o d u c t o f the c l o n e d r a s gene and i t may not a c t as a GTP b i n d i n g r e g u l a t o r y p r o t e i n s i n c e there i s ev idence from t h i s study sugge s t ing cl S t h a t i t does not b i n d guanine n u c l e o t i d e s . However p23 and 47 the c l o n e d ra s gene p r o d u c t may be i d e n t i c a l i n t h a t the p r o - t e i n i m m u n o p r e c i p i t a t e d by Y13-259 i s r e c o g n i z e d by a p o l y c l o - n a l an t ibody r a i s e d a g a i n s t the r a s gene p r o d u c t . A l s o Y13- 259 thus f a r has been shown to immunoprec ip i t a te a l l known ras gene p r o d u c t s . I t i s i n t e r e s t i n g t o note t h a t F i r t e l ' s group have not r e p o r t e d guanine n u c l e o t i d e b i n d i n g by the Dd-ras gene p r o d u c t and i t i s p o s s i b l e t h a t they have at tempted to demonstrate GTP b i n d i n g , but have a l s o been u n s u c c e s s f u l . R a s - r e l a t e d genes have been found i n D r o s o p h o l i a melanogaster (Weinberg & S h i l o , 1981), i n the m o l l u s c A p l y s i a (Madaule & A x e l , 1985) , and Schizosaccharomyces pombe (Fukui & K a z i r o , 1985) , but there have been no r e p o r t s t h a t t h e i r gene p r o d u c t s b i n d GTP or GDP. The d e f i n i t i v e d e t e r m i n a t i o n of n u c l e o t i d e b i n d i n g by p23 awai t s the p u r i f i c a t i o n of t h i s p r o t e i n . In a d d i t i o n , func- t i o n a l s t u d i e s on the ra s p r o t e i n i n D. d i s co ideum w i l l r e q u i r e p u r i f i e d p r o t e i n . My at tempts a t p u r i f i c a t i o n o f p23 by immu- n o a f f i n i t y chromatography have not been s u c c e s s f u l but I be- l i e v e t h a t t h i s p r o t e i n can at l e a s t be e n r i c h e d by t h i s method because o ther membranous p r o t e i n s have been p u r i f i e d o r g r e a t - l y e n r i c h e d u s i n g immunoa f f in i ty chromatography, i n c l u d i n g the s rc oncogene p r o d u c t ( E r i k s o n e t a l . , 1979). There seems to be one main prob lem w i t h the immunoaf f i - cl S n i t y p u r i f i c a t i o n o f p23 : p23 does not b i n d to the column (Tables 5 and 6 ) . The D. d i s co ideum p r o t e i n may be a g g r e g a t i n g and t h i s may p r e v e n t b i n d i n g of p23 to the a n t i b o d y on the 48 co lumn. A l t e r i n g the e q u i l i b r a t i o n and sample b u f f e r s may a l l o w the d i s a g g r e g a t i o n o f the p r o t e i n and a l l o w the b i n d i n g 3T cl S o f p23 t o the co lumn. Membrane-assoc iated p r o t e i n s from o t h e r organisms have been p u r i f i e d by i m m u n o a f f i n i t y chromatography i n combina t ion w i t h o ther chromatographic methods (Aubry e t a l . , 1987; Shen & T a i , 1986). T h i s approach i s d i f f i c u l t i n the case o f D. d i s c o i d e u m membrane p r o t e i n s s ince they n o n s p e c i f i c a l l y b i n d and aggregate very e a s i l y and do not f r a c t i o n a t e e a s i l y on g e l f i l t r a t i o n and ion-exchange columns (MacDonald, 1986). Other membrane p r o t e i n s have been p u r i f i e d by p a s s i n g the p r e p a r a t i o n over a column of the m a t r i x i t s e l f (Hugues & Augus t , 1982) or a column made w i t h a n o n s p e c i f i c a n t i b o d y (Suzuki e t a l . , 1987) to remove n o n s p e c i f i c a l l y b i n d i n g p r o t e i n be fore a p p l i c a t i o n to the i m m u n o a f f i n i t y co lumn. T h i s approach was not neces sary f o r t h i s study because there was no problem w i t h n o n s p e c i f i c b i n d i n g i f the column was e q u i l i b r a t e d i n the absence of d e t e r g e n t (Table 6) . A l t h o u g h i t would be worthwhi le to s y s t e m a t i c a l l y vary the c o n d i t i o n s o f e q u i l i b r a t i o n and b i n d i n g i n an attempt to o b t a i n an enr ichment of p2 3 by i m m u n o a f f i n i t y chromatography, because the p r o t e i n i s found at such low l e v e l s i n the c e l l , i t may be s i m p l e r to i s o l a t e the genomic ra s c l o n e and express p23 i n a s u i t a b l e organism (such as E . c o l i ) so t h a t t h e r e i s an enr ichment f o r p23 be fore , a p p l i c a t i o n t o an immunoa f f in i ty co lumn. T h i s would a i d i n the d e t e c t i o n o f the p r o t e i n d u r i n g 49 chromatography and s h o u l d e l i m i n a t e the problems o f n o n s p e c i f i c a g g r e g a t i o n by D. d i s c o i d e u m p r o t e i n s . 50 REFERENCES A u b r y , M . , B e r t e l o o t , A . , Beaumont, A . , Roques, B . P . , & C r i n e , P. (1987). 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