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UBC Theses and Dissertations

Fujinami sarcoma virus P140 proteolysis and peptide purification Brose, Michael C. 1985

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FUJINAMI SARCOMA VIRUS P140 PROTEOLYSIS AND PEPTIDE PURIFICATION By MICHAEL CARL BROSE B.S.; S e a t t l e P a c i f i c U n i v e r s i t y , 1978 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF June © M i c h a e l C a r l BRITISH COLUMBIA 1985 B r o s e , 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of r_r-& I io fgcj y  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date t t P ABSTRACT F u j i n a m i sarcoma v i r u s e n c o d e s a 140/000 m.w. p o l y p e p t i d e (P140) w h i c h has been c o r r e l a t e d a s the a g e n t o f t r a n s f o r m a t i o n i n h o s t c h i c k e n f i b r o b l a s t s and mammalian f i b r o b l a s t s . To c o n c l u s i v e l y i d e n t i f y the r o l e of P140 i n the t r a n s f o r m a t i o n p r o c e s s i t w i l l be n e c e s s a r y t o o b t a i n i n t a c t / p u r i f i e d P140. The a v a i l a b i l i t y o f an a n t i b o d y m o n o c l o n a l l y s p e c i f i c t o the N - t e r m i n a l gag enco d e d p o r t i o n o f P140 s u g g e s t e d a o n e - s t e p i m m u n o a f f i n i t y p u r i f i c a t i o n o f P140. A f t e r p u r i f i c a t i o n o f the a n t i b o d y o u t o f mouse a s c i t e s f l u i d , by 50% ammonium s u l f a t e f r a c t i o n a t i o n and i o n exchange c h r o m a t o g r a p h y , a n t i b o d y was l i n k e d to a S e p h a r o s e 4-B m a t r i x a c t i v a t e d w i t h c yanogen b r o m i d e . The a n t i - p l 9 a f f i n i t y m a t r i x bound i n t a c t P140 a s a d o u b l e t r e l a t i v e t o a p o l y c l o n a l a n t i - p l 9 . C h a o t r o p i c a g e n t s , h i g h pH and low pH t r e a t m e n t s a l l f a i l e d t o e l u t e the bound P140 from the a f f i n i t y m a t r i x . F a i l i n g the p u r i f i c a t i o n o f i n t a c t P140 a method of p a r t i a l p r o t e o l y s i s was u s e d t o p r o d u c e v a r y i n g s i z e d f r a g m e n t s o f P140/ w i t h the g o a l o f p u r i f y i n g t h e s e f r a g m e n t s f o r f u r t h e r work on the r o l e o f P140. T r y p s i n a l o n e i n a l i m i t e d p r o t e o l y s i s p r o d u c e d s m a l l , u n s t a b l e p e p t i d e s t o o c l o s e i n s i z e d i s t r i b u t i o n t o be e f f e c t i v e l y p u r i f i e d . C h y m o t r y p s i n a l o n e p r o d u c e d a b r o a d r a n g e o f more s t a b l e p e p t i d e s , w i t h a p r e d o m i n a n c e o f a 45,000 m.w. p e p t i d e . i i i C h y m o t r y p s i n - t r y p s i n c o n s e c u t i v e p r o t e o l y s i s p r o d u c e d a v e r y s t a b l e 35,000 m.w. p e p t i d e . G e l f i l t r a t i o n o f the c h y m o t r y p t i c p e p t i d e s was i n e f f e c t i v e a s the p e p t i d e s coraplexed and were n o t f r a c t i o n a t e d . Ion exchange c h r o m a t o g r a p h y f r a c t i o n a t e d the c o m p l e x i n g c h y m o t r y p t i c p e p t i d e s , making p o s s i b l e the p u r i f i c a t i o n o f t h e s e p e p t i d e s . The s t a b l e 45,000 m.w. p e p t i d e r e t a i n e d some k i n a s e a c t i v i t y , a s i t p h o s p h o r y l a t e d the s u b s t r a t e e n o l a s e , s i m i l a r to b u t l e s s i n t e n s e t h a n i n t a c t P140. A 30,000 m.w. p e p t i d e o n l y p h o s p h o r y l a t i n g a f t e r i o n exchange d i d n o t p h o s p h o r y l a t e e n o l a s e . i v TABLE OF CONTENTS Page 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 i L i s t o f F i g u r e s v i i i A b b r e v i a t i o n s i x Acknowledgements x i I n t r o d u c t i o n 1 M a t e r i a l s , and Methods 7 I C e l l s and P a r e n t a l V i r u s e s 7 I I I m m u n o p r e c i p i t a t i o n 7 I I I Iri V i t r o K i n a s e R e a c t i o n 8 1 Immune Complex A s s a y 8 2. Column F r a c t i o n A s s a y 8 3. A f f i n i t y M a t r i x A s s a y 8 IV 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 ' 9 V A n t i b o d y P r e p a r a t i o n 9 1. Ammonium S u l f a t e f r a c t i o n a t i o n 9 2. D i a l y s i s and p r o t e i n c o n c e n t r a t i o n 10 3. Ion exchange 10 VI Enzyme L i n k e d Immunosorbent A s s a y 10 V I I Cyanogen Bromide A c t i v a t i o n 11 V I I I A f f i n i t y M a t r i x E l u t i o n 12 1. I n t e r n a l probe 12 2. B a t c h e l u t i o n 12 IX P r o t e o l y s i s o f P140 12 1. P r e - p h o s p h o r y l a t e d P140 p r o t e o l y s i s 13 2. N o n - p h o s p h o r y l a t e d P140 p r o t e o l y s i s 13 A. T r y p s i n p r o t e o l y s i s 13 B. C h y m o t r y p s i n p r o t e o l y s i s 14 C. C h y m o t r y p s i n - T r y p s i n p r o t e o l y s i s 14 X P u r i f i c a t i o n of P r o t e o l y t i c P e p t i d e s 14 1. G e l f i l t r a t i o n 14 2. Ion exchange 15 XI Exogenous S u b s t r a t e P h o s p h o r y l a t i o n 16 C h a p t e r I . I m m u n o a f f i n i t y P u r i f i c a t i o n o f P140 17 I n t r o d u c t i o n 17 R e s u l t s 18 I A n t i - p l 9 m o n o c l o n a l a n t i b o d y 18 I I 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 19 A. P r e p a r a t i o n o f m a t r i x 19 B. E l u t i o n c o n d i t i o n s o f the a f f i n i t y m a t r i x 22 1. KSCN e l u t i o n 22 2. H i g h and low pH e l u t i o n 23 D i s c u s s i o n 26 C h a p t e r I I . L i m i t e d P r o t e o l y s i s o f P140 30 I n t r o d u c t i o n 30 I L i m i t e d p r o t e o l y s i s 31 v i R e s u l t s 33 I T r y p s i n p r o t e o l y s i s • 33 I I C h y m o t r y p s i n p r o t e o l y s i s 36 A. P r o t e o l y s i s o f p r e - p h o s p h o r y l a t e d P140 36 B. P r o t e o l y s i s o f n o n - p h o s p h o r y l a t e d P140 39 I I I C h y m o t r y p s i n - t r y p s i n c o n s e c u t i v e p r o t e o l y s i s 42 A. P r o t e o l y s i s o f n o n - p h o s p h o r y l a t e d P140 42 B. P r o t e o l y s i s of p r e - p h o s p h o r y l a t e d P140 43 D i s c u s s i o n 46 C h a p t e r I I I P u r i f i c a t i o n o f P r o t e o l y t i c P e p t i d e s 51 I n t r o d u c t i o n 51 I G e l f i l t r a t i o n 52 A. Column p r e p a r a t i o n 52 B. Sample p r e p a r a t i o n 53 R e s u l t s 53 I C h y m o t r y p s i n p e p t i d e s 53 A. G e l f i l t r a t i o n 53 I I C h y m o t r y p s i n - t r y p s i n p e p t i d e s 54 A. G e l f i l t r a t i o n 54 B. Ion exchange 57 1. P r e - p h o s p h o r y l a t e d c h y m o t r y p t i c p e p t i d e s 60 2. N o n - p h o s p h o r y l a t e d c h y m o t r y p t i c p e p t i d e s 63 I I I Exogenous s u b s t r a t e p h o s p h o r y l a t i o n 64 D i s c u s s i o n 71 B i b l i o g r a p h y 74 v i i L I S T OF TABLES Page T a b l e I . P140 P r o t e o l y t i c P e p t i d e s 50 v i i i LI S T OF FIGURES Page F i g u r e 1. A b i l i t y o f A n t i - p l 9 A f f i n i t y M a t r i x t o B i n d P140 21 F i g u r e 2. E l u t i o n of P140 From A n t i - p l 9 A f f i n i t y M a t r i x 24 F i g u r e 3. T r y p s i n P r o t e o l y s i s o f P140 35 F i g u r e 4. C h y m o t r y p s i n P r o t e o l y s i s o f P h o s p h o r y l a t e d P140 38 F i g u r e 5. C h y m o t r y p s i n , C h y m o t r y p s i n - T r y p s i n C o n s e c u t i v e P r o t e o l y s i s o f N o n - P h o s p h o r y l a t e d P140 41 F i g u r e 6. C h y m o t r y p s i n , C h y m o t r y p s i n - T r y p s i n C o n s e c u t i v e P r o t e o l y s i s o f P h o s p h o r y l a t e d P140 45 F i g u r e 7. G e l F i l t r a t i o n o f P140 C h y m o t r y p t i c P e p t i d e s .56 F i g u r e 8. G e l F i l t r a t i o n o f P140 C h y m o t r y p s i n - T r y p s i n P r o t e o l y t i c P e p t i d e s 59 F i g u r e 9. Ion Exchange o f P140 C h y m o t r y p t i c P e p t i d e s 62 F i g u r e 10. Ion Exchange o f P140 C h y m o t r y p t i c P e p t i d e s 66,67 F i g u r e 11. E n o l a s e P h o s p h o r y l a t i o n by P140 C h y m o t r y p t i c P e p t i d e s 70 ABBREVIATIONS ATP - A d e n o s i n e t r i p h o s p h a t e CNBr - Cyanogen bromide DEAE - D i e t h y l a m i n o e t h y l a n i o n e x c h a n g e r EDTA - E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ELISA - Enzyme l i n k e d immunosorbent a s s a y env - v i r a l gene c o d i n g f o r e n v e l o p e g l y c o p r o t e i n s FSV - F u j i n a m i sarcoma v i r u s f p s - u n i q u e F u j i n a m i sarcoma v i r u s t r a n s f o r m i n g gene gag - v i r a l gene c o d i n g f o r g r o u p s p e c i f i c s t r u c t u r a l p r o t e i n s HCl - H y d r o c h l o r i c a c i d K - 1,000 KSCN - P o t a s s i u m i s o t h i o c y a n a t e m.w. - M o l e c u l a r w e i g h t M - M o l a r i t y mM - M i l l i m o l a r c o n c e n t r a t i o n N a C l - Sodium c h l o r i d e NaSCN - Sodium i s o t h i o c y a n a t e O.D. - O p t i c a l d e n s i t y peg - P o l y e t h y l e n e g l y c o l p o l - v i r a l gene e n c o d i n g r e v e r s e t r a n s c r i p t a s e p l 9 - 19/000 m o l e c u l a r w e i g h t gag e n c o d e d v i r a l p r o t e i n P140 - F u j i n a m i sarcoma v i r u s e n c o d e d 140,000 m.w. p h o s p h o p r o t e i n p p 6 0 s r c _ Rous sarcoma v i r u s e n c o d e d 60/000 m.w. p h o s p h o p r o t e i n RNA - Ribonucleic acid RSV - Rous sarcoma virus S. aureus - Staphylococcus aureus SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis "Sepharose p e l l e t " - Sepharose deposit after centrifugation src - Rous sarcoma virus transforming gene TPCK - T o l y l s u l f o n y l phenylalanyl chloromethyl ketone ACKNOWLEDGEMENTS x i I g r a t e f u l l y a c k n o w l e d g e the h e l p o f Dr. Tony Pawson i n s e e i n g me t h r o u g h t h i s work/ and the examples from whom I have l e a r n e d much a b o u t s c i e n c e i n the Department o f M i c r o b i o l o g y . I thank my p a r e n t s f o r t h e i r p a t i e n c e and h e l p i n p r e p a r a t i o n o f the m a n u s c r i p t . F i n a l l y , I am i n d e b t e d t o my w i f e , Dawn, w i t h o u t whom I would n o t have been a b l e t o p e r s e v e r e . E c c l e s i a s t e s 2:11, 24. / 1 INTRODUCTION Fo r some t i m e , r e t r o v i r u s e s have been known a s u s e f u l t o o l s i n the s t u d y o f o n c o g e n e s i s , owing t o t h e i r a b i l i t y t o p r o d u c e tumors i n a n i m a l s ( D o u g h e r t y and Rasmussen, 1964; Ha n a f u s a and H a n a f u s a , 1966) and t h e i r e f f i c i e n c y a t i n d u c i n g n e o p l a s t i c t r a n s f o r m a t i o n i n c e l l c u l t u r e s (Temin and R u b i n , 1 9 5 8 ) . R e c e n t l y g e n e t i c ( T o y o s h i m a and V o g t , 1969; Vogt/ 1971; M a r t i n , 1970) and b i o c h e m i c a l e v i d e n c e (Brugge and E r i k s o n , 1977) has been g a t h e r e d t h a t has i d e n t i f i e d the v i r a l components r e s p o n s i b l e f o r t h e s e b i o l o g i c a l e f f e c t s . A c u t e l y o n c o g e n i c r e t r o v i r u s e s c a r r y a t r a n s f o r m i n g gene/ w h i c h has no e s s e n t i a l v i r a l g r o w t h o r r e p r o d u c t i v e f u n c t i o n , b u t whose se q u e n c e i s c l o s e l y r e l a t e d to s e q u e n c e s i n n o r m a l h o s t c e l l s . These t r a n s f o r m i n g genes seem t o have o r i g i n a t e d by a r e t r o v i r u s a c q u i r i n g a n o r m a l c e l l u l a r s e q uence v i a a t r a n s d u c t i o n e v e n t , t h u s g e n e r a t i n g a v i r a l oncogene (one) ( B a l t i m o r e , 1975; D u e s b e r g , 1 9 8 3 ) . R e t r o v i r u s e s can be d i v i d e d i n t o two c l a s s e s ; t h e r a r e , h i g h l y o n c o g e n i c c l a s s w h i c h has an one gene, and a much more common c l a s s w i t h o u t one genes ( D u e s b e r g , 1 9 8 0 ) . These v i r u s e s can be f u r t h e r c l a s s i f i e d by the p r e s e n c e o r a b s e n c e o f one o r more o f the t h r e e v i r i o n genes e s s e n t i a l f o r r e p l i c a t i o n : the 2 gag gene, w h i c h c o d e s f o r the s t r u c t u r a l p r o t e i n s o f the v i r u s , termed the g r o u p - s p e c i f i c a n t i g e n s ; the p o l gene, w h i c h c o d e s f o r the DNA p o l y m e r a s e r e v e r s e t r a n s c r i p t a s e , and the env gene, w h i c h c o d e s f o r the v i r a l e n v e l o p e g l y c o p r o t e i n s ( T o o z e , 1973; B a l t i m o r e , 1 9 7 5 ) . In e a c h g r o u p many t y p e s o f v i r u s e s can be i d e n t i f i e d b a s e d on the t y p e o f c a n c e r w i t h w h i c h t h e y a r e a s s o c i a t e d . These g r o u p s a r e ; the sarcoma v i r u s e s , the a c u t e l e u k e m i a v i r u s e s t h a t t r a n s f o r m f i b r o b l a s t s , the a c u t e l e u k e m i a v i r u s e s t h a t do n o t t r a n s f o r m f i b r o b l a s t s and the n o n - d e f e c t i v e l y m p h a t i c l e u k e m i a v i r u s e s ( D e u s b e r g , 1 9 8 0 ) . The sarcoma v i r u s e s c a u s e s o l i d tumors i n the a n i m a l w i t h i n one t o two weeks, and e f f i c i e n t l y t r a n s f o r m f i b r o b l a s t s i n c e l l c u l t u r e . Sarcoma v i r u s e s v a r y i n the number o f e s s e n t i a l v i r i o n genes t h e y c a r r y , and t h i s work w i l l be f o c u s e d on the g r o u p o f a v i a n tumor v i r u s e s d e f e c t i v e i n r e p l i c a t i o n . Rous sarcoma v i r u s (RSV) i s the p r o t o t y p e o f the a v i a n sarcoma v i r u s e s , and much i s known a l r e a d y from work i n t h i s s y s t e m . RSV t r a n s f o r m s f i b r o b l a s t s i n c u l t u r e , i s r e p l i c a t i o n c o m p e t e n t , and c a r r i e s a gene termed s r c whose p r o d u c t i s the a g e n t o f t r a n s f o r m a t i o n by RSV. R e p l i c a t i o n d e f e c t i v e s t r a i n s o f RSV have been d i s c o v e r e d w h i c h have l o s t the s r c gene. The s r c gene was t h o u g h t to be the o n l y t r a n s f o r m i n g gene i n the a v i a n sarcoma v i r u s e s u n t i l work w i t h a sarcoma v i r u s i s o l a t e d some time ago from c h i c k e n tumors ' (-Fujinami and Inamoto, 1914). The F u j i n a m i sarcoma v i r u s (FSV) d e m o n s t r a t e d a t r a n s f o r m i n g gene i n a r e p l i c a t i o n - d e f e c t i v e v i r u s w h i c h i s d i s t i n c t from s r c and any o f the e s s e n t i a l v i r i o n genes (Lee e_t a_l, 1980; H a n a f u s a e t a l , 1 9 8 0 ) . T h i s RNA tumor v i r u s p o s s e s s e s a 4.5 kb 3 genome w h i c h c o n t a i n s an i n t e r n a l / v i r u s s p e c i f i c s e quence/ termed f p s / s e e m i n g l y a r i s i n g from a r e c o m b i n a t i o n e v e n t between a n o n t r a n s f o r m i n g / r e p l i c a t i o n - c o m p e t e n t r e t r o v i r u s and the c e l l u l a r homologue o f the v i r a l f p s gene/ termed c e l l u l a r f p s . T h i s i n t e r n a l sequence i s bounded on the 5' and 3 1 ends by s e q u e n c e s s h a r e d w i t h i t s n o n t r a n s f o r m i n g h e l p e r v i r u s ; the 5' l k b c o n t a i n i n g d e f e c t i v e gag gene s e q u e n c e s and the 3' 0.5kb c o n t a i n i n g n o n c o d i n g s e q u e n c e s (Lee e_t a_l, 1980; H a n a f u s a e_t a l , 1980; S h i b u y a e t a l , 1 9 8 0 ) . The FSV RNA i s t r a n s l a t e d i n t o a 140/000 m o l e c u l a r w e i g h t p o l y p e p t i d e (P140) (Lee e t a ^ , 1980; Hana f u s a e t a_l, 1 980). P140 i s f o u n d i n FSV t r a n s f o r m e d c e l l s , and i s the o n l y p r o d u c t o f an i n v i t r o t r a n s l a t i o n o f f u l l l e n g t h FSV genome (Lee e_t a l _ , 1 9 8 0 ) . S i n c e no subgenomic FSV mRNAs have been d e t e c t e d , i t a p p e a r s t h a t FSV e n c o d e s o n l y t h i s s i n g l e p r o t e i n . P140 has been i m m u n o p r e c i p i t a t e d from c e l l s t r a n s f o r m e d by FSV u s i n g a n t i s e r u m t o the v i r a l gag 19/000 m.w. p r o t e i n p l 9 ( B i s t e r e_t a l _ , 1 9 8 0 ) . When i m m u n o p r e c i p i t a t e d from u n l a b l e d FSV i n f e c t e d c e l l s and i n c u b a t e d i n the i j i v i t r o k i n a s e r e a c t i o n , P140 i s p h o s p h o r y l a t e d a t t y r o s i n e r e s i d u e s ( B i s t e r e_t a_l, 1 9 8 0 ) . In a d d i t i o n , c e l l s i n f e c t e d w i t h FSV/ as w i t h o t h e r a v i a n sarcoma v i r u s e s / y i e l d a b n o r m a l l y h i g h l e v e l s o f p h o s p h o t y r o s i n e (Beemon, 1981)/ an amino a c i d n o t p h o s p h o r y l a t e d t o a g r e a t e x t e n t i n known no r m a l c e l l u l a r p r o c e s s e s ( K r e b s and Beavo, 1 9 7 9 ) . The p h o s p h o r y l a t i o n o f c e r t a i n c e l l u l a r " t a r g e t " p r o t e i n s i s r e s p o n s i b l e f o r the i n c r e a s e d l e v e l s o f p h o s p h o t y r o s i n e i n the i n f e c t e d c e l l s ( C o o p e r e t a_l, 1982)/ seems t o c o r r e l a t e w i t h m o r p h o l o g i c a l and gro w t h c h a r a c t e r i s t i c s o f c e l l u l a r t r a n s f o r m a t i o n and seems to 4 be a f u n c t i o n o f a t y r o s i n e - s p e c i f i c p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h P140 and o t h e r ASV t r a n s f o r m i n g p r o t e i n s . R e c e n t l y d i s c o v e r e d m utants o f FSV t e m p e r a t u r e s e n s i t i v e f o r t r a n s f o r m a t i o n have g i v e n i n s i g h t i n t o the r e l a t i o n s h i p between o n c o g e n e s i s and t y r o s i n e s p e c i f i c p h o s p h o r y l a t i o n (Pawson e_t a l _ , 1980; H a n a f u s a e t a_l, 1 9 8 1 ) . C e l l s i n f e c t e d by the v a r i a n t FSV s t r a i n w i l l t r a n s f o r m a t the p e r m i s s i v e t e m p e r a t u r e 38 °c, b u t w i l l r e g a i n o r r e m a i n p h e n o t y p i c a l l y n o r m a l upon s h i f t back t o o r c o n s t a n t g r o w t h a t the n o n - p e r m i s s i v e t e m p e r a t u r e 41.5 °c. At 38 °C P140 i s h i g h l y p h o s p h o r y l a t e d i n the c e l l , c o n t a i n i n g p h o s p h o t y r o s i n e and p h o s p h o s e r i n e . T h i s P140 i s p h o s p h o r y l a t e d a t s e v e r a l t y r o s i n e r e s i d u e s upon i m m u n o p r e c i p i t a t i o n and i n c u b a t i o n i n the i n  v i t r o k i n a s e r e a c t i o n . A t 41.5 °c P140 i s w e a k l y p h o s p h o r y l a t e d a t s e r i n e r e s i d u e s , i s n o t p h o s p h o r y l a t e d a t t y r o s i n e r e s i d u e s and does n o t p h o s p h o r y l a t e t y r o s i n e r e s i d u e s i n the i j i v i t r o k i n a s e a s s a y . In f a c t , c e l l s i n f e c t e d w i t h FSV grown a t 38 °c c o n t a i n 3-4 t i m e s the l e v e l o f p h o s p h o t y r o s i n e as do u n i n f e c t e d c e l l s i n s i m i l a r c o n d i t i o n s . Lee e t a l ^ (1982) f o u n d a c o r r e l a t i o n between t r a n s f o r m a t i o n o f mammalian c e l l s and p h o s p h o r y l a t i o n o f P140, i n d i c a t i n g p h o s p h o r y l a t i o n o f P140 to be the n e c e s s a r y s t e p i n mammalian c e l l t r a n s f o r m a t i o n . T h i s i s the same P140 i n v o l v e d i n a v i a n c e l l t r a n s f o r m a t i o n . O t h e r ASV t r a n s f o r m e d c e l l s a l s o have i n c r e a s e d l e v e l s o f p h o s p h o t y r o s i n e , i n c e r t a i n c e l l u l a r t a r g e t p r o t e i n s . P h o s p h o r y l a t i o n o f t y r o s i n e r e s i d u e s on t h e s e c e l l u l a r p r o t e i n s u b s t r a t e s c o r r e l a t e s w i t h the t r a n s f o r m a t i o n p r o c e s s and seems to be a f u n c t i o n of the p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h 5 the v i r a l t r a n s f o r m i n g p r o t e i n . To c l a r i f y the r o l e o f P140 i n t y r o s i n e - s p e c i f i c p r o t e i n k i n a s e a c t i v i t y , a more c a r e f u l d e f i n i t i o n and c h a r a c t e r i z a t i o n o f the r e g i o n s on P140 r e q u i r e d f o r t h i s e n z y m a t i c a c t i v i t y has been done (Weinmaster e_t a_l, 1982; Weinmaster e_t a l , 1 9 8 3 ) . P a r t i a l d i g e s t i o n o f P140 w i t h two p r o t e o l y t i c enzymes has mapped the s i t e s o f t y r o s i n e p h o s p h o r y l a t i o n on P140. The major s i t e l i e s i n the C - t e r m i n a l end o f the f p s e n c o d e d sequence o f P140, w i t h a n o t h e r s i t e i n the m i d - f p s s e q u e n c e . A m i n o r s i t e i s l o c a t e d i n t h e N - t e r m i n a l gag e n c o d e d r e g i o n . These r e s u l t s l o c a l i z e the t y r o s i n e - s p e c i f i c p r o t e i n k i n a s e d o m a i n ( s ) t o the C - t e r t n i n a l r e g i o n of f p s e n c o d e d P140. F u r t h e r e v i d e n c e t h a t the f p s e n c o d e d s e q u e n c e o f P140 i s n e c e s s a r y i n o n c o g e n e s i s i s r e c e n t work showing t h a t the v i r a l f p s s e q u e n c e w i t h o u t the N - t e r m i n a l gag sequence w i l l i n d u c e t r a n s f o r m a t i o n o f c e l l s i n c u l t u r e ( F o s t e r and H a n a f u s a , 1 9 8 3 ) . T h i s i n d i c a t e d the g a g - e n c o d e d s e q u e n c e s o f P140 a r e n o t n e c e s s a r y f o r t r a n s f o r m a t i o n . F u r t h e r d e f i n i t i o n and c o n f i r m a t i o n o f the a c t u a l e n z y m a t i c a c t i v i t y , i f any, r e s i d i n g on FSV P140, the p h y s i c a l l o c a l i z a t i o n o f t h i s a c t i v i t y on the p r o t e i n and any d e t e r m i n a t i o n o f the r e l a t i o n s h i p between the a l r e a d y o b s e r v e d t y r o s i n e - s p e c i f i c p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h P140 and i t s r o l e i n the p r o c e s s e s o f o n c o g e n e s i s a w a i t s p u r i f i c a t i o n and i s o l a t i o n o f the P140 p r o t e i n and; s u b s e q u e n t l y , i t s e n z y m a t i c a l l y a c t i v e components. U t i l i z i n g the above f a c t s and t e c h n i c a l a p p r o a c h e s , t h i s work i s f o c u s e d on the p u r i f i c a t i o n o f e n z y m a t i c a l l y a c t i v e P140 a n d / o r the 6 components or "domains" of P140 and t h e i r a n a l y s e s i n the hope of d e s c r i b i n g and c o n f i r m i n g the p h y s i c a l l o c a l i z a t i o n of t h a t a r ea of P140, i f any, which i s a c t i v e i n and e s s e n t i a l f o r mechanisms of o n c o g e n e s i s . 7 MATERIALS AND METHODS C e l l s and P a r e n t a l V i r u s e s The p a r e n t a l r a t - 1 f i b r o b l a s t c e l l l i n e and i t s d e r i v a t i v e t r a n s f o r m e d by the t e m p e r a t u r e - r e s i s t a n t d e r i v a t i v e L5 FSV were a g i f t from J . Ston e and have been d e s c r i b e d (Topp, 1981; Ingman-Baker e t a_l, 1 9 8 4 ) . C e l l s were grown as d e s c r i b e d by Ingman-Baker e_t a_l (1984) and m a i n t a i n e d a t 37 °c u n l e s s s p e c i f i e d . L y s a t e s were p r o d u c e d a s d e s c r i b e d by Weinmaster e_t al_ (1983) e x c e p t t h a t the c e l l l y s i s b u f f e r d i d n o t c o n t a i n 2mM ATP. L y s a t e s were s t o r e d a t -196 °c u n t i l u s e d . I m m u n o p r e c i p i t a t i o n P r e s p u n c e l l l y s a t e s were i m m u n o p r e c i p i t a t e d a s d e s c r i b e d by Weinmaster e t a_l (1983) e x c e p t t h a t 3 ^ul a n t i s e r u m was us e d p e r 500 pi c e l l l y s a t e . In the c a s e s where a mouse d e r i v e d m o n o c l o n a l a n t i b o d y was u s e d , S. a u r e u s was p r e - c o a t e d w i t h r a b b i t a n t i mouse Ig (RAMIG:Cappel l a b s ) by p e l l e t i n g 1.0 ml o f 10% v o l / v o l S. a u r e u s s u s p e n s i o n and r e s u s p e n d i n g i n 0.25 ml RAMIG and 0.65 ml 8 l y s i s b u f f e r a s d e s c r i b e d by Ingman-Baker e_t a_l ( 1 9 8 4 ) . When i m m u n o p r e c i p i t a t i n g w i t h the a n t i - p l 9 S e p h a r o s e , 1 gram S e p h a r o s e was s u s p e n d e d i n 5 ml l y s i s b u f f e r , 100 pi o f t h i s s u s p e n s i o n i n c u b a t e d w i t h 500 _ul c e l l l y s a t e 60 m i n u t e s a t 4 °c, w i t h o c c a s i o n a l v o r t e x i n g . The s u s p e n s i o n was t h e n p e l l e t e d i n a m i c r o f u g e ( E p p e n d o r f ) , s u p e r n a t a n t drawn o f f and S e p h a r o s e p e l l e t washed w i t h i m m U n o p r e c i p i t a t i o n b u f f e r s ( d e s c r i b e d by Weinmaster e_t a_l 1 9 8 3 ) . In V i t r o K i n a s e R e a c t i o n (1) Immune co m p l e x e s were washed w i t h c e l l l y s i s b u f f e r and l a b e l e d a s d e s c r i b e d by Pawson e_t a_l (1980) e x c e p t t h a t 0.5 t o 1.0 u C i o f [ } ( - 3 2 p ] ( A m e r s h a m ) was use d and the r e a c t i o n done a t 4 °c. (2) Column f r a c t i o n a s s a y . A 30 >il sample was removed t o a s m a l l m i c r o f u g e t u b e . 1.0 M MnCl^ was added to 10 mM p e r sample, 0.5 ^aCi [ t f - 3 2 p ] A T P i n 2 u l T r i s - H C l pH 7.5 were added to sample and i n c u b a t e d 15 m i n u t e s a t 0 °c. SDS g e l sample b u f f e r ( d e s c r i b e d below) was added t o t e r m i n a t e the r e a c t i o n . (3) A f f i n i t y m a t r i x complexed P140 a s s a y . The S e p h a r o s e p e l l e t was washed t w i c e w i t h 10 mM T r i s - H C l (pH 7.5)-10 mM MnCl2 / r e s u s p e n d e d i n 35 j a l o f same and 0.1 p C i [}(- 3 2 p - j A T p a d d e d . The r e a c t i o n was i n c u b a t e d 15 m i n u t e s a t 4 o C / t e r m i n a t e d by a d d i t i o n o f SDS g e l sample b u f f e r o r w a s h i n g S e p h a r o s e w i t h c e l l l y s i s b u f f e r and u s i n g p e r e x p e r i m e n t . 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 (SDS-PAGE) 9 IX SDS g e l sample b u f f e r was p r e p a r e d a s d e s c r i b e d by Weinmaster e_t al_ ( 1 9 8 3 ) , 2X b u f f e r f o r aqueous s a m p l e s b e i n g a 2X c o n c e n t r a t i o n o f IX b u f f e r . Samples were p r e p a r e d f o r SDS-PAGE as d e s c r i b e d by Weinmaster e_t a_l ( 1 9 8 3 ) , the b u f f e r d e s c r i b e d h e r e a f t e r a s SDS g e l sample b u f f e r . E l e c t r o p h o r e s i s was done as d e s c r i b e d by Weinmaster e_t a l (1983) e x c e p t t h a t s e p a r a t i n g g e l s c o n t a i n e d 10% a c r y l a m i d e c r o s s l i n k e d w i t h 0.2% b i s - a c r y l a m i d e . E l e c t r o p h o r e s i s was done a t 2 w a t t s t h r o u g h the s t a c k i n g g e l and 3 t o 5 w a t t s t h r o u g h the s e p a r a t i n g g e l . G e l s t a i n i n g , d r y i n g and a u t o r a d i o g r a p h y were done a s d e s c r i b e d by Weinmaster e_t a_l ( 1 9 8 3 ) . A n t i b o d y P r e p a r a t i o n A s c i t e s f l u i d c o n t a i n i n g mouse m o n o c l o n a l a n t i - p l 9 a n t i s e r u m ( d e s c r i b e d by Ingman-Baker e_t a_l 1984) was a g i f t o f J . Ingman-Baker. A n t i b o d y was p u r i f i e d o u t o f the a s c i t e s f l u i d by a t h r e e s t e p p r o c e d u r e ; ammonium s u l f a t e f r a c t i o n a t i o n , d i a l y s i s and p r o t e i n c o n c e n t r a t i o n , i o n exchange c h r o m a t o g r a p h y . ( 1 ) . The a s c i t e s f l u i d was p r e c i p i t a t e d w i t h 50% ammonium s u l f a t e , e i t h e r by a d d i t i o n o f 2 volumes o f 100% s a t u r a t e d s o l u t i o n ammonium s u l f a t e to 1 volume a s c i t e s p l u s 1 volume o f p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , o r a d d i t i o n o f s o l i d ammonium 10 s u l f a t e t o 50% s a t u r a t i o n i n 1 volume a s c i t e s p l u s 1 volume PBS. T h i s was s t i r r e d o v e r n i g h t a t 4 o c a n c j p e l l e t e d a t 15,000 x g f o r 30 m i n u t e s . The s u p e r n a t a n t was s a v e d t o a s s a y f o r a n t i b o d y . (2) . The p e l l e t was d i s s o l v e d i n an a p p r o p r i a t e volume o f 10 mM T r i s - H C l (pH 7 . 5 ) - 0.1 M sodium a z i d e ( h e r e a f t e r r e f e r r e d t o as T r i s b u f f e r ) . D i a l y s i s was p e r f o r m e d 3 t i m e s a g a i n s t the T r i s b u f f e r i n d i a l y s i s t u b i n g ( p r e v i o u s l y b o i l e d 10 m i n u t e s i n d e i o n i z e d H^O and EDTA) and p r o t e i n c o n c e n t r a t e d by c o v e r i n g the d i a l y s i s t u b i n g w i t h s o l i d p o l y e t h y l e n e g l y c o l (PEG) carbowax 20,000 a t 4 °c. P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d by use o f a c o m m e r c i a l l y a v a i l a b l e a s s a y k i t ( B i o - R a d ) and u s e d as d i r e c t e d by t h e m a n u f a c t u r e r . , (3) . The d i a l y s e d , c o n c e n t r a t e d a s c i t e s a n t i b o d y p r e p a r a t i o n was l o a d e d on t o a 50 ml DEAE S e p h a c e l ( P h a r m a c i a ) column, p r e v i o u s l y d e - f i n e d as d i r e c t e d by the m a n u f a c t u r e r and e q u i l i b r a t e d i n T r i s b u f f e r . The a n t i b o d y s o l u t i o n was washed t h r o u g h the column w i t h T r i s b u f f e r and the e l u a n t c o l l e c t e d i n 0.5 ml f r a c t i o n s . F r a c t i o n s were a s s a y e d f o r p r o t e i n by m e a s u r i n g a b s o r b a n c e a t 280 nm r e l a t i v e t o T r i s b u f f e r , b e f o r e and d u r i n g e l u t i o n . Washing was c o n t i n u e d u n t i l p r o t e i n was a t b a c k g r o u n d l e v e l s i n the e l u a n t . The column was e l u t e d w i t h a l i n e a r N aCl g r a d i e n t . Enzyme L i n k e d Immunosorbent A s s a y ( E L I S A ) The ELISA was u s e d as a r o u t i n u e a s s a y f o r p r e s e n c e o f the a n t i - p l 9 a n t i b o d y i n t h i s work. The i n d i r e c t m i c r o p l a t e ELISA 11 method was use d as d e s c r i b e d by V o l l e r e_t a_l (1980) w i t h the f o l l o w i n g a d a p t a t i o n s : N P 4 0 - d i s r u p t e d RSV v i r i o n s a t 0.5 ug/ml i n the d e s c r i b e d c a r b o n a t e b u f f e r were us e d a s i n i t i a l a n t i g e n base on the p l a t e , 0.1 ml volumes o f t e s t s a m p l e s , d e v e l o p i n g a n t i b o d y ( r a b b i t a n t i - m o u s e Ig) and enzyme s u b s t r a t e were u s e d , and a b s o r b a n c e p e r m i c r o p l a t e w e l l was measured u s i n g a M u l t i s k a n T i t e r t e k a t 405 nm. Cyanogen Bromide A c t i v a t i o n o f S e p h a r o s e 4-B. F u n c t i o n a l g r o u p s on S e p h a r o s e 4-B were a c t i v a t e d by cyanogen bromide (CNBr) a c t i v a t i o n e s s e n t i a l l y a s d e s c r i b e d by Cooper (1977) w i t h the f o l l o w i n g m o d i f i c a t i o n s : 15 g d r i e d S e p h a r o s e 4-B ( d r i e d on a s c i n t e r e d g l a s s f u n n e l ) was made i n t o a s l u r r y w i t h d e i o n i z e d H^O and s t i r r e d s l o w l y on i c e . The pH o f the S e p h a r o s e s l u r r y was b r o u g h t t o 11-11.5 w i t h 2.0 M NaOH. 2.0mls o f a 250 mg/ml CNBr i n d i m e t h y l f o r m a m i d e s o l u t i o n was added d r o p w i s e , w h i l e m a i n t a i n i n g the pH a t 11 w i t h NaOH. T h i s was s t i r r e d 20-30 m i n u t e s , u n t i l the pH was s t a b l e a t a r o u n d 11 w i t h o u t a d d i t i o n o f NaOH. The a c t i v a t e d beads were washed on a s c i n t e r e d g l a s s f u n n e l w i t h 200 mis c o l d dH zO, t h e n 200 mis c o l d 0.1M sodium b o r a t e b u f f e r , pH 8.3 w i t h a z i d e . The S e p h a r o s e was washed i n t o a b e a k e r w i t h dH^O, a l l o w e d t o s e t t l e and e x c e s s H^O removed. A n t i b o d y c o n c e n t r a t e d t o 5 mg/ml was added to the S e p h a r o s e and s t i r r e d s l o w l y o v e r n i g h t a t 4°C. The c o u p l e d a n t i - p l 9 S e p h a r o s e beads were t h e n washed t h o r o u g h l y w i t h c o l d b o r a t e b u f f e r on a s c i n t e r e d g l a s s f u n n e l and wash s o l u t i o n s s a v e d and a s s a y e d f o r p r o t e i n c o n t e n t . Any 12 a c t i v a t e d g r o u p s on the S e p h a r o s e n o t c o u p l e d to a n t i b o d y were b l o c k e d by s t i r r i n g the S e p h a r o s e i n 0.15M m o n o e t h a n o l a m i n e pH 8.7 f o r 2 h o u r s a t 4 ° c . A f f i n i t y M a t r i x E l u t i o n . (1) . I n t e r n a l p r o be f o r e l u t i o n . In p r e - l a b e l e d e l u t i o n e x p e r i m e n t s , 10-20% o f the a f f i n i t y m a t r i x , a f t e r the i m m u n o p r e c i p i t a t i o n , was removed and p h o s p h o r y l a t e d a s d e s c r i b e d above under in_ v i t r o k i n a s e a s s a y . C o l d 10 mM T r i s - H C l pH 7.5 (100 ;ul) was added t o the r e a c t i o n , and the e n t i r e mix added back to the o r i g i n a l n o n - l a b e l e d a f f i n i t y m a t r i x . T h i s was mixed w e l l , and d i s t r i b u t e d t o t u b e s f o r e l u t i o n , a s m a l l p o r t i o n b e i n g r e s e r v e d as a n o n - e l u t e d c o n t r o l . (2) . B a t c h e l u t i o n o f a f f i n i t y m a t r i x . N o n - p h o s p h o r y l a t e d P140 c o m p l e x e d a f f i n i t y m a t r i x was d i s t r i b u t e d t o t u b e s f o r e l u t i o n a t 100 pi a f f i n i t y m a t r i x p e r t u b e . To e a c h tube was added 400 jul o f the e l u t i o n a g e n t . T h i s was i n c u b a t e d 3-4 m i n u t e s on i c e , t h e n p e l l e t e d i n a m i c r o f u g e , s u p e r n a t a n t removed and s a v e d . When a c i d o r base were us e d as e l u t i o n a g e n t s , the s u p e r n a t a n t s were n e u t r a l i z e d t o pH 7 i m m e d i a t e l y . The a f f i n i t y m a t r i x p e l l e t was washed w i t h T r i s and p e l l e t e d . P r o t e o l y s i s Of P140. S_. a u r e u s i m m u n o p r e c i p i t a t i o n o f P140 was done u s i n g 3 pi o f p o l y c l o n a l a n t i - p l 9 a n t i s e r u m and 60 u l S_. a u r e u s f o r a l l 1 3 p r o t e a s e e x p e r i m e n t s / as d e s c r i b e d above under the s e c t i o n " i m m u n o p r e c i p i t a t i o n s " , e x c e p t f o r the f o l l o w i n g : immune complex from 5 0 0 u l l y s a t e was us e d p e r r e a c t i o n and the s t a n d a r d i m m u n o p r e c i p i t a t i o n wash b u f f e r s were n o t u s e d i n t h e s e e x p e r i m e n t s to wash the immune complex p e l l e t . (1) . P r e - p h o s p h o r y l a t e d P140 p r o t e o l y s i s . 10-20% o f the immune complex p e l l e t was removed and p h o s p h o r y l a t e d i n the i n v i t r o k i n a s e r e a c t i o n / a s d e s c r i b e d . a b o v e . P e l l e t was t h e n washed w i t h c o l d 0.1 M T r i s - H C l pH 7.5 and added back t o the r e m a i n d e r o f the p e l l e t . The l a b e l e d p e l l e t was d i s t r i b u t e d t o m i c r o f u g e t u b e s f o r p r o t e o l y t i c d i g e s t i o n . A f t e r d i g e s t i o n / 70 pi o f l x SDS g e l sample b u f f e r was added t o p e l l e t and the s u p e r n a t a n t had added to i t e q u a l volume o f 2x SDS g e l sample b u f f e r i n p r e p a r a t i o n f o r SDS-PAGE. (2) . N o n - p h o s p h o r y l a t e d P140 p r o t e o l y s i s . The immune complex p e l l e t was washed w i t h b u f f e r a p p r o p r i a t e t o the e x p e r i m e n t / t h e n d i s t r i b u t e d t o m i c r o f u g e t u b e s f o r d i g e s t i o n . A f t e r d i g e s t i o n w i t h the p r o t e a s e ( s ) a s d e s c r i b e d p e r e x p e r i m e n t / the s u p e r n a t a n t s and p e l l e t s were i n c u b a t e d i n the i n v i t r o k i n a s e r e a c t i o n and p r e p a r e d f o r SDS-PAGE a s d e s c r i b e d a b o v e . ( i ) T r y p s i n p r o t e o l y s i s was done e s s e n t i a l l y a s d e s c r i b e d by Weinmaster e_t a_l (1983) e x c e p t f o r the f o l l o w i n g : the immune complex p e l l e t was washed 2 t i m e s w i t h the pH 9.0 c l e a v a g e b u f f e r t h e n r e s u s p e n d e d i n 30 pi o f the pH 9.0 b u f f e r . The a p p r o p r i a t e amount o f t o l y l s u l f o n y l p h e n l y a l a n y l c h l o r o m e t h y l k e t o n e t r y p s i n ( T P C K - t r y p s i n ) was i n c u b a t e d w i t h the p e l l e t 15 m i n u t e s a t 0 °c. The r e a c t i o n was t e r m i n a t e d by a d d i n g 2 mg o f trypsin i n h i b i t o r (aprotinin)/ then p e l l e t i n g the reaction in the microfuge and separating supernatant from p e l l e t . ( i i ) Chymotrypsin proteolysis was done by washing the immune complex p e l l e t 2 times with 0.1 M Tris-HCl pH 7.5 buffer, then resuspending the p e l l e t in 30 ul of the 0.1 M T r i s buffer. The appropriate amount of chymotrypsin, suspended in 50 mM NH^ HCO^  (as described per experiment), was incubated with the p e l l e t 15 minutes at 0o c. T n e reaction was terminated by adding 2 mg aprotinin, then p e l l e t i n g and separating supernatant from p e l l e t . / ( i i i ) Chymotrypsin-trypsin consecutive proteolysis was done by repeating the procedures for chymotrypsin digestion above, except that af t e r incubating the p e l l e t with chymotrypsin, 35 ul of the pH 9.0 buffer was added to the reaction, i t was pelleted and the supernatant s p l i t into 2 equal fr a c t i o n s . Aprotinin (2 pi) was added to one f r a c t i o n , reserved for l a t e r use. To the other f r a c t i o n was added 1.0 pg TPCK-trypsin, and incubated 15 minutes at 0 °c. This reaction was terminated by adding 2 jag trypsin i n h i b i t o r . Digestion supernatants from any one experiment were pooled to form a uniform source of proteol y t i c peptides for an experiment. These were stored in the gel f i l t r a t i o n column buffer at -80 °c u n t i l used. P u r i f i c a t i o n Of Proteolytic Peptides. (1). Gel f i l t r a t i o n . A Sephadex G-100 superfine (Pharmacia) column matrix was used in a 40 x 1.5 cm column. 15 T h i s sephadex f r a c t i o n a t e s p r o t e i n s i n the ra n g e 4/000-100,000 m.w.. The column was r u n by g r a v i t y f l o w w i t h a pH 9.0 T r i s b u f f e r (0.01 M T r i s - H C l pH 9.0, 1 mM EDTA, 0.25% DOC, 10% g l y c e r o l , 1 mg/100 mis a p r o t i n i n . To d e t e r m i n e the e x c l u s i o n and i n c l u s i o n volumes o f the column, 20 pi d e x t r a n b l u e and 20 j j l bromphenol b l u e were mixed w i t h 100 ^J1 o f b u f f e r and l o a d e d on the co l u m n . T h i s was e l u t e d f r o m the column, volumes o f b u f f e r r e q u i r e d f o r e l u t i o n o f e a c h dye d e t e r m i n i n g the e x c l u s i o n and i n c l u s i o n volumes r e s p e c t i v e l y . To f r a c t i o n a t e p e p t i d e s , d i g e s t i o n s u p e r n a t a n t was mixed w i t h 20 >il o f bromphenol b l u e , 70 pq human c a r b o n i c a n h y d r a s e , 30 )ig BSA and 100 pi column b u f f e r . T h i s was l o a d e d on the column and e l u t e d w i t h the b u f f e r , c o l l e c t i n g 0.5 ml f r a c t i o n s , u n t i l the bromphenol b l u e e x i t e d from the column. Column f r a c t i o n s were a s s a y e d f o r p r e s e n c e o f p r o t e o l y t i c p e p t i d e s by r e m o v i n g 35 pi sample, b r i n g i n g the sample up t o 10 mM MnCl^. and i n c u b a t i n g i n the i n v i t r o k i n a s e r e a c t i o n . A f t e r SDS-PAGE, the g e l was s t a i n e d to v i s u a l i s e the i n t e r n a l p r o t e i n m a r k e r s . ( 2 ) . Ion e x c h a n g e . A D E A E - S e p h a c e l m a t r i x was u s e d i n a 20 x 1.5 cm column. T h i s column was r u n by g r a v i t y f l o w w i t h a pH 7.5 T r i s b u f f e r (10 mM T r i s - H C l pH 7.5, 0.5% NP40, 0.5% DOC, 1 mg/100 mis a p r o t i n i n ) . P e p t i d e s were f r a c t i o n a t e d by l o a d i n g sample w i t h the i n t e r n a l p r o t e i n s t a n d a r d s o n t o the co l u m n . T h i s was washed t h r o u g h w i t h the pH 7.5 T r i s b u f f e r f o r 3-4 column volumes w i t h n o n - l a b e l e d sample, o r u n t i l the C e r e n k o v cpm were a t b a c k g r o u n d l e v e l s on p r e - l a b e l e d s a m ple. The column was e l u t e d w i t h a NaCl l i n e a r g r a d i e n t i n the pH 7.5 T r i s b u f f e r , 0.5 ml f r a c t i o n s b e i n g c o l l e c t e d . Column f r a c t i o n s were a s s a y e d by C e r e n k o v cpm f o r l a b e l e d s a mple, o r by i n c u b a t i n g 35 pi o f e a c h sample i n an i n v i t r o k i n a s e r e a c t i o n f o r 15 m i n u t e s , t h e n s u b j e c t i n g the f r a c t i o n s t o SDS-PAGE. Exogenous S u b s t r a t e P h o s p h o r y l a t i o n E n o l a s e s t o c k ( a t 10 mg/ml) was p r e p a r e d by a c i d t r e a t m e n t a s d e t a i l e d by Cooper e t a_l (1984) and u s e d as d e t a i l e d p e r e x p e r i m e n t . C H A P T E R I I M M U N O A F F I N I T Y P U R I F I C A T I O N OF P 1 4 0 I n t r o d u c t i o n In o r d e r t o c o n f i r m a n d / o r c l a r i f y p r e v i o u s work on FSV P140 ( e g . h a v i n g d e s c r i b e d the e n z y m a t i c a c t i v i t y a s s o c i a t e d w i t h P140, s u b s t r a t e a n d / o r s e l f p h o s p h o r y l a t i o n , the l o c a l i z a t i o n o f P140 i n FSV t r a n s f o r m e d c e l l s , r e l a t i n g the known p r i m a r y s s e q u e n c e o f P140 t o h i g h e r s t r u c t u r a l and f u n c t i o n a l o r d e r s ) i t w i l l be n e c e s s a r y t o o b t a i n i n t a c t P140 i n a p u r i f i e d s t a t e . I t was w i t h the aim o f p u r i f y i n g P140 t h a t I began t h i s work. T h i s c o i n c i d e d w i t h the i s o l a t i o n o f a m o n o c l o n a l a n t i b o d y d i r e c t e d t oward the gag gene e n c o d e d 19,000 m.w. p r o t e i n p l 9 , i n c l u d e d i n the N - t e r m i n a l end o f the g a g - f p s e n c o d e d P140 (Ingman-Baker e_t a_l 1 9 8 4 ) . A v a i l a b i l i t y o f an a n t i b o d y d i r e c t e d t oward the N - t e r m i n a l p o r t i o n o f P140 s u g g e s t e d an immunochemical a p p r o a c h t o p u r i f y i n g P140 i n r e l a t i v e l y p u r e , i n t a c t f o r m . A l o n g h i s t o r y o f u s i n g o n e - s t e p i m m u n o a f f i n i t y i n p u r i f y i n g a wide v a r i e t y o f a n t i g e n s , 18 a n t i b o d i e s and o t h e r p r o t e i n s i s known from the l i t e r a t u r e . F u r t h e r p r e c e d e n t f o r t h i s a p p r o a c h was the s u c c e s s f u l p u r i f i c a t i o n o f the RSV p p 6 0 s r c t r a n s f o r m i n g p r o t e i n by L e v i n s o n e t al (1980) and E r i k s o n e t a l ( 1 9 7 9 ) . R e s u l t s A n t i - p l 9 M o n o c l o n a l A n t i b o d y . The a n t i - p l 9 a n t i b o d y was made a v a i l a b l e a s mouse a s c i t e s f l u i d , a g i f t o f J . Ingman-Baker. B e f o r e u s i n g the a n t i b o d y i n i m m u n o a f f i n i t y p r o c e d u r e s , i t was n e c e s s a r y t o p u r i f y the a n t i b o d y from the a s c i t e s . Ammonium s u l f a t e p r e c i p i t a t i o n and i o n e x change column c h r o m a t o g r a p h y were us e d and p u r i f i c a t i o n f o l l o w e d by OD 280 p r o f i l e o f t o t a l p r o t e i n and the B i o - R a d p r o t e i n a s s a y ( s e e M a t e r i a l s and M e t h o d s ) . I n i t i a l a s c i t e . s f l u i d v a r i e d i n t o t a l p r o t e i n c o n t e n t from 16.5 mg/ml t o 30 mg/ml p r o t e i n . P r o t e i n r e m a i n i n g a f t e r the ammonium s u l f a t e p r e c i p i t a t i o n e l u t e d from i o n exchange w i t h a l i n e a r N a C l g r a d i e n t i n column b u f f e r i n 2 d i s t i n c t p e a k s , 0.09M-0.17M and 0.33M N a C l . The p r o t e i n peak e l u t i n g a t 0.09-0.17M NaCl c o n t a i n e d the a c t i v e a n t i - p l 9 a n t i b o d y . A n t i - p l 9 s p e c i f i c a n t i b o d y p r e s e n c e was d e t e r m i n e d w i t h the ELISA ( s e e M a t e r i a l s and M e t h o d s ) . A l l f r a c t i o n s c o n t a i n i n g the a c t i v e a n t i - p l 9 were p o o l e d and c o n c e n t r a t e d t o between 4.8 mg and 5.4 mg p e r ml t o t a l p r o t e i n a s a s s a y e d by the B i o - R a d p r o t e i n a s s a y . 19 A n t i b o d y c o n c e n t r a t e d t o 5 mg/ml was t e s t e d f o r a b i l i t y t o b i n d i n t a c t P140 i n FSV i n f e c t e d c e l l l y s a t e s as f u r t h e r c o n f i r m a t i o n o f the s p e c i f i c i t y o f the p u r i f i e d a n t i b o d y . B i n d i n g a b i l i t y was t e s t e d by p h o s p h o r y l a t i n g P140 i n the i n  v i t r o k i n a s e r e a c t i o n / t h e n i m m u n o p r e c i p i t a t i n g w i t h the p u r i f i e d m o n o c l o n a l a n t i b o d y and a s s a y i n g on 10% SDS-PAGE/ d e s c r i b e d i n M a t e r i a l s and Methods. R e l a t i v e t o a p o l y c l o n a l a n t i - p l 9 a n t i b o d y w h i c h p r e c i p i t a t e d a s i n g l e p r o t e i n a t 140K/ the m o n o c l o n a l a n t i - p l 9 p r e c i p i t a t e d 2 p r o t e i n s m i g r a t i n g t o g e t h e r , 143K and 140K/ i n r e l a t i v e e q u a l amounts on the SDS-PAGE, F i g u r e 1. 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. P r e p a r a t i o n o f m a t r i x . S e p h a r o s e 4-B i s a beaded d e r i v a t i v e o f a g a r o s e , and i s a w i d e l y u s e d s u p p o r t m a t r i x b e c a u s e of i t s h i g h p o r o s i t y and r e l a t i v e l y h i g h s u b s t i t u t i o n c a p a c i t y . A c t i v a t i o n o f the f u n c t i o n a l g r o u p s o f S e p h a r o s e 4-B was done w i t h CNBr, d e s c r i b e d i n M a t e r i a l s and Methods. M o n o c l o n a l a n t i b o d y was l i n k e d t o the a c t i v a t e d S e p h a r o s e m a t r i x a t 5 mg p r o t e i n p e r 1 gram S e p h a r o s e m a t r i x w i t h 80% c o u p l i n g e f f i c i e n c y , as d e t e r m i n e d by a s s a y i n g t o t a l mg p r o t e i n b e f o r e and a f t e r c o u p l i n g to the S e p h a r o s e . The a n t i - p l 9 i m m u n o a f f i n i t y m a t r i x was a s s a y e d f o r i t s a b i l i t y t o b i n d i n t a c t P140 i n FSV i n f e c t e d c e l l l y s a t e s , s i m i l a r t o the methods f o r the p u r i f i e d a n t i b o d y . The a n t i - p l 9 - S e p h a r o s e p r e c i p i t a t e d 2 p r o t e i n s i n the 140K r e g i o n , r e l a t i v e to a s i n g l e 140K p r o t e i n p r e c i p i t a t e d by t h e 20 Figure 1. A b i l i t y of the anti-pl9 a f f i n i t y matrix to bind intact P140. Sepharose 4-B anti-pl9 a f f i n i t y matrix was prepared as described in Materials and Methods. FSV infected c e l l lysate was phosphorylated and immunoprecipitated with the anti-pl9 a f f i n i t y matrix, polyclonal anti-pl9 antiserum and non-immune rabbit serum. The immunoprecipitation p e l l e t s were washed and prepared for SDS-PAGE by addition of SDS-gel sample buffer. Antibody was as follows: (lane 1) 0.8 ug anti-pl9 antibody per 100 pi Sepharose suspension, (lane 2) 3 pi non-immune serum, (lane 3) 3 pi polyclonal anti-pl9 antibody. P140 i s indicated by arrow in lane A. Molecular weights are indicated by bars in lane X. 205 P1 40 1 1 6 96 66 45 22 p o l y c l o n a l a n t i - p l 9 a n t i b o d y . The S e p h a r o s e m a t r i x bound a m a j o r i t y o f the p r o t e i n s p r e s e n t i n the SDS-PAGE, the 143-140K d o u b l e t b e i n g s p e c i f i c t o the a n t i - p l 9 - S e p h a r o s e , see F i g u r e 1. B. E l u t i o n c o n d i t i o n s o f the a f f i n i t y m a t r i x . Upon d e m o n s t r a t i n g the a b i l i t y o f the a f f i n i t y m a t r i x t o p r e c i p i t a t e P140 f rom FSV i n f e c t e d c e l l l y s a t e s , i t was n e c e s s a r y t o d e t e r m i n e the c o n d i t i o n s o f e l u t i o n o f the bound P140 from the a f f i n i t y m a t r i x , w i t h the d e s i r e d r e c o v e r y o f i n t a c t , e n z y m a t i c a l l y a c t i v e P140. The most common t r e a t m e n t s f o r d i s s o c i a t i n g a n t i g e n - a n t i b o d y c o m p l e x e s i n c l u d e low pH, h i g h pH ( i n some c a s e s ) , h i g h c o n c e n t r a t i o n s o f c h a o t r o p i c i o n s and u r e a ( L a n d o n , 1 9 8 1 ) . To d e f i n e the optimum c o n d i t i o n s f o r d i s s o c i a t i o n o f the a n t i - p l 9 - P l 4 0 complex a l o n g w i t h r e c o v e r y o f i n t a c t P140 r e t a i n i n g i t s a s s o c i a t e d e n z y m a t i c a c t i v i t y , I i n v e s t i g a t e d the p r o p e r t i e s o f d i s s o c i a t i o n and e l u t i o n o f s e v e r a l a p p r o a c h e s and r e a g e n t s . (1) KSCN e l u t i o n . The f i r s t a p p r o a c h t a k e n t o d i s s o c i a t e P140 from the a f f i n i t y m a t r i x i n v o l v e d the use o f c h a o t r o p i c a g e n t s . D a n d l i k e r e t a l (1967) and R u o s l a h t i (1976) have d e s c r i b e d the mechanism o f a c t i o n o f c h a o t r o p i c i o n s i n d i s s o c i a t i n g a n t i g e n - a n t i b o d y c o m p l e x e s . T h i o c y a n a t e , p e r c h l o r a t e and i o d i d e i o n s d i s s o c i a t e c o m p l e x e s i n the r e g i o n o f IM to 4M, a t n e u t r a l pH, f u n c t i o n a l l y a c t i v e a n t i b o d y h a v i n g been r e c o v e r e d i n e a c h c a s e . C h a o t r o p i c i o n s have been f o u n d e f f e c t i v e i n d i s s o c i a t i n g c o m p l e x e s i n the o r d e r SCN" > CICL"" > T I " > B r ~ > c l " ( R u o s l a h t i , 1 9 7 6 ) . P p 6 0 s r c h a v i n g been s u c c e s s f u l l y e l u t e d from an a f f i n i t y m a t r i x w i t h KSCN ( E r i k s o n 23 e t a l , 1979; P u r c h i o , 1982) o r NaSCN ( L e v i n s o n e t al, 1980) t r e a t m e n t / I t e s t e d the a b i l i t y o f KSCN i n d i s s o c i a t i n g P140 from the a n t i - p l 9 a f f i n i t y m a t r i x . KSCN was u s e d i n d i s s o c i a t i o n e x p e r i m e n t s a s d e s c r i b e d i n M a t e r i a l s and Methods and F i g u r e 2. The r e s u l t s a r e shown i n F i g u r e 2. Lane 1 r e p r e s e n t s the c o n t r o l S e p h a r o s e p e l l e t . L anes 2, 4, 6, 8 and 10 r e p r e s e n t the p r o t e i n s r e m a i n i n g bound to the a f f i n i t y m a t r i x p e l l e t a f t e r t r e a t m e n t w i t h 0.05M, 0.1M, 0.5M, 1.0M, 1.5M, and 2.0M KSCN r e s p e c t i v e l y . L anes 3, 5, 7, 9, 11 and 13 r e p r e s e n t the KSCN e l u a t e s o f the P140 bound a f f i n i t y m a t r i x . I t i s c l e a r t h a t P140 bound t o the a f f i n i t y m a t r i x , a s se e n i n the c o n t r o l l a n e . However, KSCN d i d n o t d i s s o c i a t e the bound P140 even a t the h i g h c o n c e n t r a t i o n o f 2.0M. I f any o f the bound P140 d i s s o c i a t e d from the S e p h a r o s e p e l l e t s , some d e c r e a s e i n the i n t e n s i t y o f the P140 band i n the p e l l e t l a n e would have been e x p e c t e d r e g a r d l e s s o f whether o r n o t t h a t P140 or some d e g r a d a t i o n p r o d u c t o f P140 showed up i n the e l u a t e l a n e . The i n t e n s i t y o f the P140 band r e m a i n e d c o n s t a n t a c r o s s the g e l , i n the p e l l e t f r a c t i o n s , and was o f h i g h e r i n t e n s i t y t h a n the c o n t r o l P140 band. KSCN t r e a t m e n t does n o t d i s s o c i a t e P140 f rom the a n t i - p l 9 a f f i n i t y m a t r i x . (2) H i g h and low pH e l u t i o n . F o l l o w i n g the f a i l u r e t o e l u t e P140 from the a f f i n i t y m a t r i x w i t h the c h a o t r o p i c a g e n t , e l u t i o n was a t t e m p t e d w i t h 2 i n c r e a s i n g l y h a r s h t r e a t m e n t s o f h i g h pH and low pH. D i e t h y l a m i n e , a s e c o n d a r y a l i p h a t i c amine, w i t h a s t a b l e pH a t 11 was us e d w i t h NaCl o v e r a r a n g e o f 0.IM to 1.0M (R. McMaster, p e r s o n a l c o m m u n i c a t i o n ) . S t o c k H C l , pH 2 4 F i g u r e 2. E l u t i o n o f P140 from A n t i - p l 9 A f f i n i t y M a t r i x . A 1400 u l volume a f f i n i t y m a t r i x - c e l l l y s a t e s u s p e n s i o n was i n c u b a t e d on i c e 30 m i n u t e s / spun and washed 3 t i m e s w i t h c e l l l y s i s b u f f e r , t w i c e w i t h k i n a s e a s s a y b u f f e r . The spun s e p h a r o s e p e l l e t was i n c u b a t e d i n the _in v i t r o k i n a s e r e a c t i o n t o p h o s p h o r y l a t e P140 a s i n t e r n a l p r o b e . A f t e r w a s h i n g w i t h k i n a s e b u f f e r , the s e p h a r o s e ( a s s u s p e n s i o n ) was d i s t r i b u t e d i n 7-200 u l f r a c t i o n s . KSCN was u s e d i n b a t c h e l u t i o n o f bound P140, 100 u l added p e r r e a c t i o n and i n c u b a t e d on i c e 3 m i n u t e s . Samples were spun o u t , s u p e r n a t a n t s s e p a r a t e d from s e p h a r o s e p e l l e t s and S D S - g e l sample b u f f e r added to a l l samples f o r SDS-PAGE. KSCN c o n c e n t r a t i o n s u s e d f o r e l u t i o n were a s f o l l o w s : ( l a n e 1) a f f i n i t y m a t r i x p e l l e t e l u t e d w i t h 0.1M T r i s - H C l (pH 7 . 5 ) as n e g a t i v e c o n t r o l , ( l a n e 2) a f f i n i t y m a t r i x p e l l e t e l u t e d w i t h 0.05M KSCN, ( l a n e 3) s u p e r n a t a n t from 0.05M KSCN e l u t i o n , ( l a n e 4) a f f i n i t y m a t r i x p e l l e t e l u t e d w i t h 0.1M KSCN, ( l a n e 5) s u p e r n a t a n t from 0. IM KSCN e l u t i o n , ( l a n e 6) a f f i n i t y m a t r i x p e l l e t e l u t e d w i t h 0.5M KSCN, ( l a n e 7) s u p e r n a t a n t from 0.5M e l u t i o n , ( l a n e 8) a f f i n i t y m a t r i x p e l l e t f r o m 1.0M KSCN e l u t i o n , ( l a n e 9) s u p e r n a t a n t from 1.0M KSCN e l u t i o n , ( l a n e 10) a f f i n i t y m a t r i x p e l l e t e l u t e d w i t h 1. 5M KSCN, ( l a n e 11) s u p e r n a t a n t from 1.5M KSCN e l u t i o n , ( l a n e 127 a f f i n i t y m a t r i x p e l l e t f r o m 2.0M KSCN e l u t i o n , ( l a n e 13) s u p e r n a t a n t from 2.0M KSCN e l u t i o n . P140 i s i n d i c a t e d by a r r o w , l a n e A. M o l e c u l a r w e i g h t s a r e i n d i c a t e d by b a r s i n l a n e X. 2 5 1.8/ was u s e d i n c o n c e n t r a t i o n s r a n g i n g from 0.05M t o 1.5M ( J . L e v y , p e r s o n a l c o m m u n i c a t i o n ) . In b o t h c a s e s , the e l u t i n g a g e n t was i n c u b a t e d w i t h a p o r t i o n o f the a f f i n i t y m a t r i x b i n d i n g p r e - l a b e l e d P140. S u p e r n a t a n t s and p e l l e t s were c o l l e c t e d , n e u t r a l i z e d i m m e d i a t e l y w i t h a c i d o r base and a n a l y z e d by 10% SDS-PAGE a s d e s c r i b e d i n M a t e r i a l s and Methods. A u n i f o r m b i n d i n g o f the l a b e l e d P140 o n t o the a f f i n i t y m a t r i x o c c u r r e d ( r e s u l t s n o t shown). No d e c r e a s e i n i n t e n s i t y o f the P140 f r o m a f f i n i t y m a t r i x samples i n c u b a t e d w i t h e l u t i o n a g e n t a s compared w i t h the n o n - e l u t e d c o n t r o l was a p p a r e n t and no p r o t e i n s i n the e l u a n t s u p e r n a t a n t s were o b s e r v e d . In f a c t , the r e s u l t s a r e u n i f o r m l y n e g a t i v e , even a t h i g h c o n c e n t r a t i o n s o f e i t h e r d i e t h y l a m i n e o r H C l . In n e i t h e r c a s e was P140 e l u t e d from the m a t r i x . D i s c u s s i o n C l e a r l y , d e v e l o p m e n t o f a m o n o c l o n a l a n t i b o d y s p e c i f i c t o the N - t e r m i n a l end o f P140 has a v a r i e t y o f a p p l i c a t i o n s i n d e t e r m i n i n g the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p o f P140 and a i d i n g i n d e f i n i n g the mechanisms o f o n c o g e n e s i s i n t h i s v i r a l s y s t e m . The a n t i b o d y , b e i n g m o n o c l o n a l l y s p e c i f i c t o the p l 9 p o r t i o n o f gag, s h o u l d be u s e f u l i n d e t e r m i n i n g the n a t u r e o f the e n z y m a t i c a c t i v i t y c u r r e n t l y a s s o c i a t e d w i t h P140, i n a d d i t i o n to i t s u t i l i t y a s an i m m u n o a f f i n i t y m a t r i x . By v i r t u e 27 o f b i n d i n g the a n t i g e n a t one p l a c e , as o p p o s e d t o s e v e r a l p l a c e s w i t h a p o l y c l o n a l a n t i - g a g a n t i b o d y , one s h o u l d be a b l e t o d i s t i n g u i s h between a P140 w h i c h has an i n t r i n s i c k i n a s e a c t i v i t y , and a P140 w h i c h i s t i g h t l y a s s o c i a t e d w i t h a n o t h e r p r o t e i n s p e c i f i c f o r the k i n a s e a c t i v i t y . W h i l e t h e r e a r e t e c h n i c a l p r o b l e m s i n u s i n g the S e p h a r o s e a f f i n i t y m a t r i x i n i m m u n o p r e c i p i t a t i o n s , i t may be a b e t t e r r e a g e n t than the c u r r e n t s t a p h A - a n t i b o d y p r o c e d u r e . The a n t i - p l 9 a f f i n i t y m a t r i x seemed as e f f i c i e n t a t p r e c i p i t a t i n g P140 i n my i n i t i a l t e s t s a s the s t a n d a r d p r o c e d u r e , e v e n though c o n d i t i o n s were n o t o p t i m i z e d f o r t h i s r e a c t i o n . W h i l e the a f f i n i t y m a t r i x seemed an i d e a l c a n d i d a t e a s an i m m u n o p r e c i p i t a t i o n r e a g e n t , i t was w i t h the g o a l o f p u r i f y i n g i n t a c t P140 t h a t i t was d e v e l o p e d . T h i s n e c e s s i t a t e d a method o f d i s s o c i a t i n g the a n t i - p l 9 - P 1 4 0 complex i n s u c h a way t h a t b o t h the P140 and the m a t r i x l i n k e d a n t i b o d y were p r e s e r v e d i n t a c t . To a c c o m p l i s h t h i s , s e v e r a l methods o f e l u t i n g P140 from the a f f i n i t y m a t r i x were i n v e s t i g a t e d . The most g e n t l e a p p r o a c h i n v e s t i g a t e d was the use o f the c h a o t r o p i c a g e n t KSCN t o e l u t e P140. The mechanism o f a c t i o n o f c h a o t r o p i c i o n s on a n t i g e n - a n t i b o d y c o m p l e x e s has been d i s c u s s e d by D a n d l i k e r e t a_l (1967) and R u o s l a h t i ( 1 9 7 6 ) . B e c a u s e o f i t s use a t n e u t r a l pH and d i s s o c i a t i o n o f a n t i g e n - a n t i b o d y c o m p l e x e s a t l e a s t p a r t i a l l y on the b a s i s o f i o n i c s t r e n g t h , t h i s method s h o u l d have been b e s t a b l e t o e l u t e an i n t a c t P140 p r o t e i n , b o t h s t r u c t u r a l l y and f u n c t i o n a l l y . E l u t i o n w i t h the h i g h pH o r low pH r e a g e n t s was n o t s u c c e s s f u l i n d i s s o c i a t i n g P140 f r o m the a f f i n i t y m a t r i x . T h i s 28 was s u r p r i s i n g c o n s i d e r i n g the s t r i n g e n c y o f s u c h methods and the f a c t t h a t h i g h pH c o n d i t i o n s do d i s s o c i a t e a n t i g e n - a n t i b o d y c o m p l e x e s and low pH c o n d i t i o n s n o t o n l y d i s s o c i a t e a n t i g e n - a n t i b o d y c o m p l e x e s b u t a r e us e d r o u t i n e l y f o r s t r i p p i n g an a f f i n i t y m a t r i x o f p r o t e i n s bound e i t h e r s p e c i f i c a l l y o r n o n - s p e c i f i c a l l y . A p p a r e n t l y the a n t i - p l 9 a f f i n i t y m a t r i x has bound the P140 a n t i g e n v e r y t i g h t l y . T h i s may be a r e s u l t o f the a v i d i t y o f the a n t i b o d y u s e d t o p r o d u c e the a f f i n i t y m a t r i x , r a t h e r t h a n a f a i l u r e t o use a s t r i n g e n t enough t e c h n i q u e . W h i l e t h e s e r e s u l t s a r e s u r p r i s i n g i t i s n o t n e c e s s a r i l y a u n i q u e s i t u a t i o n . T h e r e i s p r e c e d e n t , when a h i g h l y a v i d a n t i b o d y has been u s e d i n i m m u n o a f f i n i t y , where e l u t i o n o f bound a n t i g e n r e q u i r e d 8M u r e a - f o r m i c a c i d t r e a t m e n t ( R u o s l a h t i , 1 9 7 6 ) . However, f o r the p u r p o s e s o f f u r t h e r s t u d y o f P140, i t was n o t d e s i r a b l e t o a t t e m p t e l u t i o n o f P140 w i t h h a r s h e r t r e a t m e n t s t h a n t h o s e d e s c r i b e d . T r e a t m e n t w i t h SDS g e l sample b u f f e r o f the a f f i n i t y m a t r i x bound P140 p e l l e t was the one t r e a t m e n t t o s u c c e s s f u l l y d i s s o c i a t e P140 f r o m the a f f i n i t y m a t r i x . P r e s u m a b l y , the a c t i o n o f the SDS i n d e n a t u r i n g p r o t e i n s i s r e s p o n s i b l e f o r the d i s s o c i a t i o n o f t h i s immune complex, and s u b s e q u e n t e l u t i o n o f the P140 from the a f f i n i t y m a t r i x . T h i s a c t i o n a l l o w s a n a l y s i s o f the p r o t e i n s r e m a i n i n g bound t o the a f f i n i t y m a t r i x a f t e r t r e a t m e n t , b u t does n o t seem a s a t i s f a c t o r y a l t e r n a t i v e a s an e l u t i n g a g e n t f o r P140. The p r o b l e m s e n c o u n t e r e d i n d i s s o c i a t i n g P140 from the a f f i n i t y m a t r i x a p p e a r t o be i m m u n o l o g i c a l , r a t h e r t h a n t e c h n i c a l i n n a t u r e . I t i s u n l i k e l y t h a t P140 was b e i n g 2 9 d i s s o c i a t e d from the a n t i - p l 9 by one o f the t r e a t m e n t s b u t was t r a p p e d i n the i n s o l u b l e S e p h a r o s e m a t r i x . The S e p h a r o s e was p e l l e t e d a f t e r i n c u b a t i o n w i t h the e l u t i o n a g e n t , r a t h e r t h a n used i n column f o r m . Upon p e l l e t i n g , v i r t u a l l y a l l o f the e l u t i o n s u p e r n a t a n t i s removed from the a f f i n i t y m a t r i x p e l l e t , the p e l l e t b e i n g l e f t f a i r l y d r y . C e n t r i f u g a t i o n s h o u l d r e l e a s e a t l e a s t a p o r t i o n o f p r o t e i n r e l e a s e d from the a n t i b o d y , even i f t h i s p r o t e i n were b i n d i n g n o n - s p e c i f i c a l l y t o the S e p h a r o s e . In a d d i t i o n , i t i s h a r d t o i m a g i n e t h i s p r o t e i n s t i c k i n g n o n - s p e c i f i c a l l y t o S e p h a r o s e i f i t i s b e i n g r e l e a s e d from a s t r o n g e r a n t i g e n - a n t i b o d y b i n d i n g , g i v e n the p r e s e n c e o f the e l u t i o n a g e n t d u r i n g the p e l l e t i n g o f the S e p h a r o s e m a t r i x . No P140 was o b s e r v e d i n any o f the e l u a n t s u p e r n a t a n t s o f p e l l e t i n g the a f f i n i t y m a t r i x a f t e r t r e a t m e n t w i t h a s p e c i f i c e l u t i o n a g e n t . T h i s c o n t r a s t e d w i t h the p r e s e n c e o f an i n t e n s e P140 band i n the s u p e r n a t a n t a f t e r t r e a t m e n t w i t h SDS g e l sample b u f f e r . C l e a r l y , P140 was r e l e a s e d from the a f f i n i t y m a t r i x a t t h i s s t a g e , a s s i m i l a r p e l l e t i n g p r o c e d u r e s were c a r r i e d o u t to remove the g e l sample b u f f e r from the a f f i n i t y m a t r i x f o r SDS-PAGE. My r e s u l t s show t h a t t h e a n t i - p l 9 , w h i l e e f f i c i e n t l y p r e c i p i t a t i n g P140 from FSV i n f e c t e d c e l l l y s a t e s , i s u n s u i t a b l e f o r use i n a o n e - s t e p i m m u n o a f f i n i t y column t o p u r i f y P140. An a l t e r n a t i v e a p p r o a c h a t s t u d y i n g the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p o f i n t a c t P140, the u l t i m a t e g o a l i n p u r i f y i n g i n t a c t P140, was s u g g e s t e d by work p r e v i o u s l y done u s i n g p r o t e o l y t i c f r a g m e n t s o f P140. T h i s a p p r o a c h i s the b a s i s o f the work d i s c u s s e d i n the f o l l o w i n g c h a p t e r s . CHAPTER I I LIMITED PROTEOLYSIS OF P140 I n t r o d u c t i o n When i n v e s t i g a t i n g the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p o f a p r o t e i n , i t i s i m p o r t a n t t o u n d e r s t a n d a p r o t e i n a t the l e v e l o f i t s domain/ o r mod u l a r c o n s t r u c t . The o b s e r v e d p r o p e r t i e s o f many p r o t e i n s i n d i c a t e s t h a t the domain i s the b a s i c u n i t o f a p r o t e i n / r a t h e r t h a n the p r i m a r y p o l y p e p t i d e c h a i n . In p r o t e i n s o f unknown s t r u c t u r e / f u n c t i o n c an be mapped t o c e r t a i n r e g i o n s / o r domains. In many c a s e s , t h e s e f u n c t i o n a l domains have t u r n e d o u t t o be composed o f one o r more s p e c i f i c s t r u c t u r a l r e g i o n s / o r d o m a i n s . Thus the f u n c t i o n o f the p r o t e i n can be mapped t o s p e c i f i c s t r u c t u r a l d o m a i n s . In t h i s way the modular n a t u r e o f a p r o t e i n i s c o r r e l a t e d w i t h i t s f u n c t i o n ( s ) / and a mechanism o f a c t i o n o f the p r o t e i n e v e n t u a l l y e l u c i d a t e d . 31 L i m i t e d P r o t e o l y s i s L i m i t e d p r o t e o l y t i c d i g e s t i o n has been p a r t i c u l a r l y u s e f u l i n c h a r a c t e r i z i n g the domain s t r u c t u r e o f many g l o b u l a r p r o t e i n s . T h i s i s i l l u s t r a t e d i n the c h a r a c t e r i z a t i o n o f t h e a c t i o n o f the zymogens/ i n w h i c h l i m i t e d h y d r o l y s i s i s n o t o n l y the mechanism o f a c t i v a t i o n o f a s u b s t r a t e by the p r o t e a s e , b u t was a l s o the method by w h i c h the n a t u r e o f t h e p r o t e a s e i t s e l f was d e f i n e d . S t r o u d ' s g e n e r a l r e v i e w (1974) and N e u r a t h and Walsh (1976) d e s c r i b e d l i m i t e d h y d r o l y s i s a s b e i n g g e n e r a l l y d i r e c t e d t o w a r d s r e g i o n s on the s u r f a c e o f p r o t e i n s s u c h a s l o o p s o r e x p o s e d random s t r u c t u r e s / r a t h e r t h a n t o w a r d s s e q u e n c e s i n s i d e a g l o b u l a r r e g i o n o f the s u b s t r a t e p r o t e i n . Thus i t would be e x p e c t e d t h a t much i n f o r m a t i o n c o u l d be g a i n e d on the s t r u c t u r a l c o n f i g u r a t i o n o f p r o t e i n s by a s e l e c t i v e use o f p r o t e o l y t i c enzymes i n l i m i t e d h y d r o l y s i s o f the p e p t i d e s g e n e r a t e d from P140. R e c e n t work on p r o t e i n l o c a l i z a t i o n and s t r u c t u r e d e t e r m i n a t i o n has been done u t i l i z i n g the s p e c i f i c e n z y m a t i c a c t i v i t y o f the b e s t known s e r i n e p r o t e a s e , t r y p s i n , d e m o n s t r a t i n g i t s u s e f u l n e s s a s an a n a l y t i c a l t o o l . C o n t r o l l e d h y d r o l y s i s o f pl a s m a membrane p r o t e i n s i n e r y t h r o c y t e s ( S p e i c h e r e_t al_, 1980) and work on the RSV t r a n s f o r m i n g p r o t e i n p p 6 0 s r c i n membrane p r e p a r a t i o n s from t r a n s f o r m e d c e l l s ( L e v i n s o n e t a l / 1981; S c h e i d t m a n n e t a_T# 1982) a r e examples o f the u t i l i t y o f t r y p s i n i n work s u c h as the n a t u r e o f the a s s o c i a t i o n o f a p r o t e i n w i t h a membrane, and e l u c i d a t i n g s t r u c t u r a l and p e r h a p s f u n c t i o n a l domains o f a p r o t e i n . 32 L i m i t e d h y d r o l y s i s o f FSV P140 by t r y p s i n has been used t o p r o d u c e d i s c r e t e p e p t i d e f r a g m e n t s w h i c h r e t a i n e d an a s s o c i a t e d p r o t e i n k i n a s e a c t i v i t y i n an a t t e m p t t o l o c a l i z e t h e e n z y m a t i c a c t i v i t y a s s o c i a t e d w i t h t r a n s f o r m a t i o n by FSV t o a s p e c i f i c r e g i o n o f the P140 p r o t e i n ( Weinmaster e t a l , 1 9 8 3 ) . In t h i s p r e l i m i n a r y work, a c o n t r o l l e d p r o t e o l y s i s o f P140 i n an immune complex was done t o g e n e r a t e p r o t e o l y t i c f r a g m e n t s from the C - t e r m i n a l end o f P140. I t was d e m o n s t r a t e d t h a t 2 m a j o r s i t e s o f t y r o s i n e p h o s p h o r y l a t i o n o f P140 r e s i d e i n the C - t e r m i n a l end o f the p r o t e i n , t h a t t h e s e were i n v o l v e d i n the e n z y m a t i c a c t i v i t y o f t r a n s f o r m a t i o n by FSV and t h i s a r e a o f t h e p r o t e i n c o n t a i n e d s u c h e n z y m a t i c a c t i v i t y ( W einmaster e t a_l 1982; W einmaster e_t a_l 1 9 8 3 ) . D i s c r e t e p r o t e o l y t i c f r a g m e n t s f r o m the C - t e r m i n a l end o f P140 would a i d i n a v e r i f i c a t i o n o f t h e a c t u a l e n z y m a t i c a c t i v i t y o f P140, i t s r e l a t i o n s h i p t o the r e g i o n o f p h o s p h o r y l a t i o n on P140 a l o n g w i t h a p h y s i c a l l o c a l i z a t i o n o f s u c h e n z y m a t i c a c t i v i t y t o s p e c i f i c r e g i o n s o f P140. The o b j e c t i v e o f t h i s work was t o o b t a i n d i s c r e t e p r o t e o l y t i c P140 f r a g m e n t s , t o e v e n t u a l l y a i d i n d e f i n i n g the n a t u r e of the s t r u c t u r e - f u n c t i o n a l domain r e l a t i o n s h i p o f P140. R e s u l t s T r y p s i n P r o t e o l y s i s In my work, I a d a p t e d the a p p r o a c h o f Weinmaster e_t al_ (1983) t o c o n f i r m and e x t e n d t h e i r r e s u l t s on the f r a c t i o n a t i o n o f P140 w i t h t r y p s i n . The e x p e r i m e n t a l c o n d i t i o n s o f c l e a v a g e a r e d e s c r i b e d i n M a t e r i a l s and Methods and the r e l e v a n t f i g u r e s . I n i t i a l l y , t r y p s i n was u s e d a l o n e i n a t i m e - l i m i t e d h y d r o l y s i s o f P140. The r e s u l t s , F i g u r e 3 i n d i c a t e d P140 t o be e x t r e m e l y s e n s i t i v e t o d i g e s t i o n by t r y p s i n . In the p e l l e t s i n c u b a t e d w i t h t r y p s i n , l a n e s 3, 5, 7 and 9, t h e r e was an a b s e n c e o f the i n t a c t P140, r e l a t i v e t o the c o n t r o l p e l l e t , l a n e 1, n o t e x p o s e d to t r y p s i n . The p r i n c i p l e p r o t e o l y t i c f r a g m e n t s r e s u l t i n g from the l i m i t e d p r o t e o l y s i s by t r y p s i n were a 33,000 m.w. p e p t i d e , p r e s e n t i n the 1 ug t r y p s i n d i g e s t , l a n e 4, and a t r i p l e t o f p e p t i d e s r a n g i n g f r o m 22,000 t o 28,000 m.w.. The 33K p e p t i d e was d i g e s t e d a s h i g h e r t r y p s i n c o n c e n t r a t i o n s were u s e d , w h i l e the 3 p e p t i d e s i n the t r i p l e t were d i g e s t e d i n t o the l o w e r 2 p e p t i d e s , s e e m i n g l y s t a b l e t o i n c r e a s i n g t r y p s i n c o n c e n t r a t i o n s . A l l p r o t e o l y t i c f r a g m e n t s were d i g e s t e d a t the h i g h c o n c e n t r a t i o n o f 30 ug t r y p s i n . The t r y p s i n p r o t e o l y s i s r e s u l t p a r a l l e l s e a r l i e r work (Weinmaster e_t a_l, 1983) and d e m o n s t r a t e s the f r a c t i o n a t i o n o f P140 i n t o s m a l l p r o t e o l y t i c f r a g m e n t s by l i m i t e d h y d r o l y s i s w i t h t r y p s i n . The r e s u l t i n g t r y p t i c p e p t i d e s , w h i l e d i s c r e t e 34 Figure 3. Trypsin Proteolysis of P140. Immunocomplexed P140 was pelleted, the aureus p e l l e t incubated in the ijn v i t r o kinase reaction and washed as described in Materials and Methods. The p e l l e t was resuspended in 30 pi trypsin cleavage buffer (described by Weinmaster e_t a_l 1983) and incubated with trypsin, 1 ul per p e l l e t for 15 minutes on i c e . Proteolysis was terminated by addition of 2 pq aprotinin per sample, samples spun, supernatants removed and SDS-gel sample buffer added to both. Trypsin concentrations used were as follows: (lane 1) p e l l e t treated with 0.1M Tris-HCl (pH 7.5) as negative control, (lane 2) supernatant from 0.1M Tris-HCl (pH 7.5) treatment, (lane 3) p e l l e t treated with 1.0 pq trypsin, (lane 4) supernatant from 1.0 pq trypsin treatment, (lane 5) p e l l e t treated with 5.0 ug trypsin, (lane 6) supernatant from 5.0 pq trypsin treatment, (lane 7) p e l l e t treated with 15.0 pq trypsin, (lane 8) supernatant from 15.0 pq treatment, (lane 9) p e l l e t from 30.0 pq trypsin treatment, (lane 10) supernatant from 30.0 pq trypsin treatment. P140 and the p r i n c i p l e resulting fragments are indicated by arrows in lane A. Molecular weights are indicated by bars in lane X. 3 5 3 6 i n s i z e , were v e r y c l o s e i n t h e i r s i z e d i s t r i b u t i o n s . T h i s t r i p l e t o f r e l a t i v e l y s t a b l e p r o t e o l y t i c f r a g m e n t s was d e m o n s t r a t e d i n the p r e v i o u s work, tho u g h m i g r a t i n g a t a h i g h e r m o l e c u l a r w e i g h t r a n g e , and seemed to be a c o n s i s t e n t r e s u l t o f d i g e s t i o n o f P140 by t r y p s i n . To a i d i n the e v e n t u a l s e p a r a t i o n and p u r i f i c a t i o n o f t h e s e i n d i v i d u a l p r o t e o l y t i c f r a g m e n t s o f P140, i t was d e s i r a b l e t h a t the p r o t e o l y t i c f r a g m e n t s r e s u l t i n g from d i g e s t i o n o f P140 be f e w e r , o r a t l e a s t more d i s p a r a t e i n t h e i r m o l e c u l a r w e i g h t s . In t h e hope of a c h i e v i n g t h i s c h y m o t r y p s i n was p r e p a r e d and u s e d i n the f o l l o w i n g e x p e r i m e n t s under pH c o n d i t i o n s o p t i m a l f o r i t s e n z y m a t i c a c t i v i t y . C h y m o t r y p s i n P r o t e o l y s i s A. P r o t e o l y s i s o f p r e - p h o s p h o r y l a t e d P140. C h y m o t r y p s i n was u s e d i n s i n g l e d i g e s t i o n e x p e r i m e n t s on P140 i m m u n o p r e c i p i t a t e d from the u s u a l FSV i n f e c t e d c e l l l i n e l y s a t e , i n the e x p e r i m e n t r e p r e s e n t e d i n F i g u r e 4. The main p r o t e o l y t i c f r a g m e n t r e l e a s e d from the immunocomplexed P140 was a 45,000 m.w. p e p t i d e . T h i s 45K p e p t i d e was f a i r l y s t a b l e t o f u r t h e r d i g e s t i o n , p r e s e n t i n a l l s u p e r n a t a n t s , l a n e s 3, 5, 7 and 9. However, t h e r e was some d e c r e a s e i n i n t e n s i t y o f the 45k p e p t i d e w i t h c o n c o m i t a n t a p p e a r a n c e o f a 35,000 m.w. p e p t i d e a t h i g h e r c h y m o t r y p s i n c o n c e n t r a t i o n s , l a n e s 5, 7 and 9. C h y m o t r y p s i n t h u s r e s o l v e d P140 i n t o f ewer p r o t e o l y t i c f r a g m e n t s t h a n d i d t r y p s i n . 37 Figure 4. Chymotrypsin Proteolysis on Phosphorylated P140. The immunocomplexed P140 p e l l e t was incubated in the in  v i t r o kinase reaction as described in Materials and Methods. The labeled p e l l e t was washed with kinase buffer, then with 0.1M Tris-HCl ph 7.5 buffer thoroughly. The p e l l e t , resuspended in 150 u l of the Tris-HCl buffer, was aliquoted into 5 tubes, 2 pi volume of chymotrypsin added and incubated 30 minutes on i c e . Proteolysis was terminated by addition of 2ug aprotinin trypsin i n h i b i t o r , the reaction tubes spun and supernatants removed. SDS-gel sample buffer was added to both p e l l e t s and supernatants. Chymotrypsin was, used in the following concentrations: (lane 1) p e l l e t from 0.1M Tris-HCl treatment, as negative control, (lane 2) p e l l e t from 0.05 }iq chymotrypsin treatment, (lane 3) supernatant from 0.05 ug chymotrypsin treatment, (lane 4) p e l l e t from 0.1 ug chymotrypsin treatment, (lane 5) supernatant from 0.1 pq chymotrypsin treatment, (lane 6) p e l l e t from 0.4 ug chymotrypsin treatment, (lane 7) sup. from 0.4 ug chymo. treatment, (lane 8) p e l l e t from 0.7 pq chymo. treatment, (lane 9) sup. from 0.7 pq chymo. treatment. P140 and the 2 major peptides re s u l t i n g from this proteolysis are indicated with arrows in lane A. Molecular weight markers are indicated by bars in lane X. 38 39 B. P r o t e o l y s i s o f n o n - p h o s p h o r y l a t e d P140. These r e s u l t s were c o n f i r m e d and e x t e n d e d i n the r e v e r s e e x p e r i m e n t where c h y m o t r y p s i n was u s e d a l o n e t o d i g e s t u n l a b e l e d / immunocomplexed P140. In t h i s case, p e l l e t s were i n c u b a t e d w i t h 0.05 pq, 0.1 ^jg / 0.7 jjg and 1.0 ug c h y m o t r y p s i n f o r 15 m i n u t e s a t 0 ° c . T h i s r e a c t i o n was t e r m i n a t e d by a d d i t i o n o f t r y p s i n i n h i b i t o r . R e s u l t i n g p e l l e t s and s u p e r n a t a n t s were i n c u b a t e d i n the _in v i t r o k i n a s e r e a c t i o n / and the r e a c t i o n t e r m i n a t e d by a d d i t i o n o f SDS g e l sample b u f f e r . SDS-PAGE r e s u l t s o f t h i s p r o t e o l y s i s a r e shown i n F i g u r e 5/ l a n e s 2 t o 5. A number o f p e p t i d e f r a g m e n t s were l a b e l e d . A v e r y i n t e n s e l y l a b e l e d 45K d o u b l e t p e p t i d e was common t o a l l s u p e r n a t a n t s / and was r e l a t i v e l y s t a b l e t o f u r t h e r d i g e s t i o n . As the c h y m o t r y p s i n c o n c e n t r a t i o n i n c r e a s e d f r o m the low amount o f 0.05 pq i n l a n e 5, the 66/000 m.w. p e p t i d e d i s a p p e a r e d / the 58/000 m.w. p e p t i d e and 49/000 m.w. p e p t i d e s b o t h d i m i n i s h e d i n r e l a t i v e i n t e n s i t y u n t i l t h e y were a b s e n t from the 1.0 pq c h y m o t r y p s i n d i g e s t i o n s u p e r n a t a n t / l a n e 2. D i s a p p e a r a n c e o f the h i g h e r m o l e c u l a r w e i g h t p e p t i d e s was c o n c o m i t a n t w i t h the a p p e a r a n c e o f the 39/000 m.w. p e p t i d e a t the h i g h e r c h y m o t r y p s i n c o n c e n t r a t i o n s i n l a n e s 2, 3 and 4. None o f t h e s e p e p t i d e s were p r e s e n t i n the p e l l e t s o f the d i g e s t i o n r e a c t i o n s / a t l e a s t none t h a t p h o s p h o r y l a t e d i n the i j i v i t r o k i n a s e r e a c t i o n / l a n e 10. C h y m o t r y p s i n u s e d i n a l i m i t e d h y d r o l y s i s o f P140 p r o d u c e d p e p t i d e f r a g m e n t s w h i c h were more d i s p a r a t e i n m o l e c u l a r w e i g h t r a n g e t h a n t h o s e f r a g m e n t s p r o d u c e d by a t r y p s i n s i n g l e 40 Figure 5. Chymotrypsin and Chymotrypsin-Trypsin Consecutive Proteolysis of Non-Phosphorylated P140. (1) . Immunocomplexed P140 p e l l e t s were distributed in 4 aliquots. P e l l e t s were resuspended in 30 pi 0. IM Tris-HCl pH 7.5 and incubated with 1 ul chymotrypsin 15 minutes on ice, then 30 u l pH 9.0 trypsin cleavage buffer added to each sample. Pellets were spun and supernatants removed and s p l i t into 2 aliquots of 15 pi volume. Proteolysis was terminated in set 1 by addition of 2 pq aprotinin per sample (lanes 2-5). (2) . Trypsin proteolysis was continued in supernatant set 2 by addition of 1 pq trypsin per sample and incubating on ice 15 minutes. Proteolysis was terminated by addition of 2 pq aprotinin per sample (lanes 6-9). A l l supernatants were phosphorylated by the in. v i t r o kinase reaction and prepared for SDS-PAGE by addition of equal volume of SDS-gel sample buffer. Enzyme concentrations were as follows: (lanes 5, 4, 3, 2) supernatants from 0.05 pq, 0.1 jig, 0.7 pq and 1.0 pq chymotrypsin treatment respectively; (lanes 9, 8, 7, 6) supernatants from 0.05 pq, 0.1 pq, 0.7 pq and 1.0 pq chymotrypsin treatment respecively, treated with 1.0 pq trypsin each; (lane 1) supernatant from 1.0 jag trypsin treatment; (lane 10) supernatant from 0.1M Tris-HCl pH 7.5 treatment, as negative control. P r i n c i p l e peptides resulting are indicated by arrows in lane A. Molecular weight markers are indicated by bars in lane X. 4 1 42 d i g e s t i o n o f P140. In a d d i t i o n , the major 45K p e p t i d e was r e s i s t a n t to f u r t h e r d e g r a d a t i o n by the c h y m o t r y p s i n . C h y m o t r y p s i n - T r y p s i n C o n s e c u t i v e P r o t e o l y s i s A. P r o t e o l y s i s o f n o n - p h o s p h o r y l a t e d P140. As r e l a t i v e l y s t a b l e p e p t i d e f r a g m e n t s were p r o d u c e d i n a s i n g l e d i g e s t i o n o f P140 by e i t h e r t r y p s i n o r c h y m o t r y p s i n , a c o n s e c u t i v e d i g e s t i o n e x p e r i m e n t u s i n g the 2 p r o t e a s e s c o n c u r r e n t l y seemed a means t o d i s t i n g u i s h p o s s i b l e common p e p t i d e p o r t i o n s i n t h e v a r i o u s p r o t e o l y t i c f r a g m e n t s p r o d u c e d on d i g e s t i o n o f P140 by e i t h e r p r o t e a s e a l o n e . To t e s t t h i s , p r o t e o l y t i c f r a g m e n t s were p r o d u c e d by c h y m o t r y p s i n d i g e s t i o n o f P140 a s d i s c u s s e d a b o v e , and t h e s e f r a g m e n t s were t h e n s u b j e c t e d t o d i g e s t i o n by a u n i f o r m amount o f t r y p s i n p r i o r t o b e i n g p h o s p h o r y l a t e d i n the i n v i t r o k i n a s e a s s a y . The SDS-PAGE r e s u l t s i n F i g u r e 5, l a n e s 6 t o 9, i n d i c a t e a 35,000 m.w. p e p t i d e was the majo r p r o t e o l y t i c f r a g m e n t r e s u l t i n g f r o m t h i s d i g e s t i o n . The 35K f r a g m e n t was r e s i s t a n t t o f u r t h e r d i g e s t i o n by the t r y p s i n , shown by d o u b l i n g the d i g e s t i o n time ( r e s u l t s n o t shown), and by t h e f a c t t h a t i t was the major d i g e s t i o n p r o d u c t from t h o s e low m o l e c u l a r w e i g h t f r a g m e n t s r e s u l t i n g from h i g h l e v e l c h y m o t r y p s i n d i g e s t i o n a s w e l l a s t h o s e f r a g m e n t s o f h i g h e r m o l e c u l a r w e i g h t f r o m c h y m o t r y p s i n s i n g l e d i g e s t i o n . In a l l c a s e s , the p r e d o m i n a n t p r o t e o l y t i c f r a g m e n t p r o d u c e d was the 35K f r a g m e n t , w i t h a 28,000 m.w. f r a g m e n t a p p e a r i n g i n the d o u b l e d i g e s t i o n o f f r a g m e n t s from h i g h c o n c e n t r a t i o n c h y m o t r y p s i n s i n g l e 43 digestion. This 35K peptide phosphorylated quite readily in the ir\ v i t r o kinase reaction, and suggests an autophosphorylating a c t i v i t y was associated with the fragment. B. Proteolysis of pre-phosphorylated P140. To rule out the p o s s i b i l i t y that these p r o t e o l y t i c fragments were not true P140 derived fragments, but were contaminating peptides which were being labeled in the in v i t r o kinase, reaction, immune complexed P140 was incubated in the in v i t r o kinase reaction. This labeled, bound P140 was incubated with chymotrypsin and the supernatants s p l i t in 2 sets. Trypsin i n h i b i t o r was added to one set, the other set was subjected to digestion by trypsin. The resulting SDS-PAGE i s shown in Figure 6. In the chymotrypsin single digestions, the major peptide present in both supernatants was a 45K peptide, lanes 3 and 5. A 39K peptide was f a i n t l y v i s i b l e in the 0.05 jug chymotrypsin digestion, and increased in intensity in the 0.7 pq digestion supernatant, lane 5. There was no substantial evidence of higher molecular weight peptides in either digestion supernatant. However, the presence of the 45K and 39K peptides corroborates their presence in the previous results as being true digestion products of P140. In the chymotrypsin-trypsin consecutive digest, a 35K peptide was v i s i b l e in both supernatants, lanes 4 and 6, along with a small amount of the 28K peptide in the supernatant from the 0.7 pq chymotrypsin digestion. The presence of these peptides also corroborates the theory that these peptides are derived from proteolytic digestion of P140. Figure 6. Chymotrypsin and Chymotrypsin-Trypsin Consecutive Proteolysis of Phosphorylated P140. (1) . Immunocomplexed P140 was pelleted, washed and incubated in the in v i tro kinase reaction. The p e l l e t was distributed in 3 aliquots and incubated with l u l chymotrypsin on ice 15 minutes. After incubation 30 pi of pH 9.0 trypsin cleavage buffer was added to p e l l e t s , they were spun, supernatants removed and s p l i t into 2 sets, 15 pi per sample. Proteolysis was terminated in set 1 by addition of 2 pq aprotinin per sample (lanes 4 and 6). (2) . Trypsin proteolysis was continued in set 2 by addition of 1 pi trypsin per sample, incubated on ice 15 minutes. Proteolysis was terminated by addition of 2 pq aprotinin per sample (lanes 3 and 5). A l l samples were prepared for SDS-PAGE by addition of appropriate SDS-gel sample buffer. Enzyme concentrations were as follows: (lane 1) p e l l e t from 0.1M Tris-HCl pH 7.5 treatment, (lane 2) supernatant from 0. IM Tris-HCl pH 7.5 treatment, (lanes 3, 5) supernatants from 0.05 pq, 0.7 pq chymotrypsin treatment respectively, (lanes 4, 6) supernatants from 0.05 jug, 0.7 jag chymotrypsin treatment respectively, treated with 1.0 jiq trypsin per supernatant. P140 and pri n c i p l e resulting peptides are indicated by arrows in lane A. Molecular weight markers are indicated by bars in lane X. 45 D i s c u s s i o n P r o t e o l y t i c p e p t i d e d i s t r i b u t i o n s r e s u l t i n g from the above d e s c r i b e d e x p e r i m e n t s a r e summarized i n T a b l e I . The 22K, 25K and 28K t r i p l e t o f p e p t i d e s r e s u l t i n g f r o m a c o n t r o l l e d p r o t e o l y s i s o f P140 by t r y p s i n c o n f i r m s e a r l i e r work by Weinmaster e_t a_l ( 1 9 8 3 ) . These p e p t i d e s were q u i t e s t a b l e to f u r t h e r d i g e s t i o n by t r y p s i n . W h i l e t h i s r e s u l t i n d i c a t e d the p r a c t i c a l i t y o f u s i n g c o n t r o l l e d p r o t e o l y s i s t o p r o d u c e v a r y i n g s i z e d P140 f r a g m e n t s , the t r y p s i n g e n e r a t e d f r a g m e n t s seemed i n a d e q u a t e f o r e v e n t u a l s e p a r a t i o n and p u r i f i c a t i o n . P140 does seem to be s u s c e p t i b l e t o p a r t i a l p r o t e o l y s i s , t o y i e l d d i s c r e t e p e p t i d e s f o r f u r t h e r s t u d y . P140 y i e l d e d more d i s p a r a t e s i z e d p e p t i d e s on d i g e s t i o n w i t h c h y m o t r y p s i n . The h i g h e r m o l e c u l a r w e i g h t p e p t i d e s were q u i t e s e n s i t i v e t o the p r o t e a s e , and were r e a d i l y d i g e s t e d i n t o l o w e r m o l e c u l a r w e i g h t p e p t i d e s w i t h even low amounts o f c h y m o t r y p s i n p r e s e n t . A 45K p e p t i d e i s the a p p a r e n t common p e p t i d e to w h i c h the l a r g e r 66K, 58K and 49K p e p t i d e s were d i g e s t e d . T h i s 45K p e p t i d e was r e l a t i v e l y s t a b l e a g a i n s t f u r t h e r d i g e s t i o n , b u t was s e e m i n g l y f u r t h e r d i g e s t e d down t o a 39K p e p t i d e i n the p r e s e n c e o f h i g h p r o t e a s e l e v e l s . B o th the s t a b i l i t y and p r e d o m i n a n c e o f the 45K p e p t i d e i n a l l s a m p l e s may i n d i c a t e the 45K p e p t i d e t o be an e s s e n t i a l s t r u c t u r a l r e g i o n / d o m a i n o f P140 d i v e s t e d o f e x t r a n e o u s s e q u e n c e s , a s 47 r e p r e s e n t e d i n the l a r g e r p e p t i d e s . I t i s a p p a r e n t t h o u g h , t h a t even the p a r e d down s t r u c t u r a l e l e m e n t , a s a 39K p e p t i d e , has i n h e r e n t ( o r a s s o c i a t e d ) e n z y m a t i c a c t i v i t y . Thus the 45K p e p t i d e may c o n t a i n a l a r g e s t r u c t u r a l r e g i o n / d o m a i n w h i c h can be f u r t h e r d i g e s t e d i n t o an e s s e n t i a l r e g i o n a s a 39K p e p t i d e . B o t h 45K and 39K p e p t i d e s c o n t a i n e d a s i t e o f p h o s p h o r y l a t i o n . The same p e p t i d e s p r o d u c e d from d i g e s t i o n o f a p r e - p h o s p h o r y l a t e d P140, when p r o d u c e d from n o n - l a b e l e d P140, p h o s p h o r y l a t e d i n the .in v i t r o k i n a s e r e a c t i o n . A b i l i t y t o be p h o s p h o r y l a t e d ijn v i t r o i s s u b s t a n t i a l e v i d e n c e t h a t b o t h p e p t i d e s i n c l u d e a r e g i o n o f P140 c o n t a i n i n g the major t y r o s i n e a t p o s i t i o n 1075, a major s i t e o f p h o s p h o r y l a t i o n i n FSV t r a n s f o r m a t i o n . T h i s e v i d e n c e would i n d i c a t e b o t h p e p t i d e s , 45K and 39K, to c o n t a i n o r r e p r e s e n t an e s s e n t i a l s t r u c t u r a l domain o f P140. F u r t h e r d i g e s t i o n o f the c h y m o t r y p t i c p e p t i d e s o f P140 a l s o y i e l d e d a s t a b l e u n i q u e p e p t i d e , 35K i n s i z e , and t h e n a 28K p e p t i d e . The 28K p e p t i d e most l i k e l y o r i g i n a t e d i n the 35K p e p t i d e . The 35K p e p t i d e can be r e a d i l y p h o s p h o r y l a t e d i n the i n v i t r o k i n a s e r e a c t i o n , d e m o n s t r a t e d a l s o i n t h a t an i d e n t i c a l p e p t i d e c o n t a i n e d a major p r o p o r t i o n o f t h e l a b e l f r o m i n t a c t P140. T h i s p e p t i d e most l i k e l y r e p r e s e n t s a p a r e d down p o r t i o n o f the 45K c h y m o t r y p t i c p e p t i d e , and may i n f a c t be even c l o s e r t o an e s s e n t i a l s t r u c t u r a l domain o f P140 t h a n the 45K p e p t i d e . I n t e r e s t i n g l y , t h i s 35K s t a b l e p e p t i d e i s o n l y p r o d u c e d on t r y p s i n d i g e s t i o n o f l a r g e r p e p t i d e s p r o d u c e d from P140 by i n i t i a l c h y m o t r y p s i n d i g e s t i o n , and n o t f r o m i n i t i a l d i g e s t i o n o f P140 w i t h t r y p s i n . T h i s s u g g e s t s t h a t 48 c h y m o t r y p s i n p r o t e o l y s i s , w h i l e l e a v i n g r e l a t i v e l y l a r g e p o r t i o n s o f P140 i n t a c t a s 45K f r a g m e n t s , e x p o s e s s i t e s on the l a r g e p e p t i d e s w h i c h y i e l d u n i f o r m s m a l l p e p t i d e s w i t h t r y p s i n p r o t e o l y s i s . T r y p s i n a l o n e d i g e s t s P140 i n t o s m a l l p e p t i d e s , p e r h a p s d e s t r o y i n g an e s s e n t i a l domain w h i c h c h y m o t r y p s i n l e a v e s i n t a c t . From the e x p e r i m e n t a l d a t a , i t has been c o n f i r m e d t h a t P140 can be d i g e s t e d by s p e c i f i c p r o t e a s e s t o y i e l d d i s c r e t e p e p t i d e f r a g m e n t s . These p e p t i d e s l i k e l y o r i g i n a t e on the C - t e r m i n u s o f P140, i n d i c a t e d by t h e i r h i g h p h o s p h o r y l a t i o n l e v e l s i n  v i t r o , w h i c h c o r r e s p o n d t o the r e s i d e n c e i n the C - t e r m i n a l end o f i n t a c t P140 o f t y r o s i n e 1075, a s i t e o f a c t i v e p h o s p h o r y l a t i o n on i n t a c t P140 (Weinmaster e_t a_l 1 9 8 3 ) . From t h i s d a t a s e v e r a l p o i n t s o f d i s c u s s i o n emerge. F i r s t , the C - t e r m i n a l o r i g i n o f t h e s e p e p t i d e s r e m a i n s t o be d e f i n i t i v e l y p r o v e n . I t i s o f c o n s i d e r a b l e i n t e r e s t t h a t a s e l e c t i v e use o f 2 s e r i n e p r o t e a s e s , e a c h w i t h d i f f e r i n g s p e c i f i c i t i e s , w i l l r e s u l t i n a few u n i q u e p e p t i d e s o r i g i n a t i n g from P140, w h i c h r e t a i n a m a j o r s i t e o f p h o s p h o r y l a t i o n , and may r e p r e s e n t s p e c i f i c s t r u c t u r a l a n d / o r f u n c t i o n a l d o m a i n s . S e c o n d , a C - t e r m i n a l P140 o r i g i n o f t h e s e p e p t i d e s i s i n f e r r e d f r o m the k i n a s e a c t i v i t y a s s o c i a t e d w i t h them i n the i n v i t r o a s s a y . The k i n a s e a c t i v i t y a s s o c i a t e d w i t h the 35K f r a g m e n t r e s u l t i n g from the d o u b l e d i g e s t i o n o f n o n - l a b e l e d P140 i n c r e a s e s i n the i r i v i t r o k i n a s e a s s a y r e l a t i v e t o i t s i n t e n s i t y on SDS-PAGE a f t e r b e i n g p r o d u c e d from p r e - k i n a s e d P140. T h i s i n d i c a t e s a p o s s i b l e i n c r e a s e i n e n z y m a t i c a c t i v i t y o f t h i s s t r u c t u r a l domain once i t i s s e p a r a t e d from the r e s t o f the p r o t e i n by t h i s d o u b l e p r o t e o l y s i s . The e v i d e n c e g a t h e r e d s u p p o r t s the t h e o r y o f the C - t e r m i n a l o r i g i n o f t h e s e p r o t e o l y t i c f r a g m e n t s o f P140. T h i s i n f o r m a t i o n may be c r u c i a l i n d e t e r m i n i n g the e x a c t e n z y m a t i c a c t i v i t y o f P140, and may a i d i n l o c a l i z a t i o n o f s u c h a c t i v i t y on the i n t a c t p r o t e i n . Such s t r u c t u r a l i n f o r m a t i o n may a l s o a i d i n d e t e r m i n i n g the n a t u r e o f the p h y s i c a l i n t e r a c t i o n o f the P140 p r o t e i n w i t h s u b s t r a t e p r o t e i n s i n i n i t i a t i n g t r a n s f o r m a t i o n . A d e f i n i t i v e c o n f i r m a t i o n o f the o r i g i n and n a t u r e o f the p r o t e o l y t i c f r a g m e n t s p r o d u c e d from P140 w i l l n e c e s s i t a t e p u r i f i c a t i o n o f t h e s e p e p t i d e s and f u r t h e r s t u d y o f t h e i r p r i m a r y amino a c i d s t r u c t u r e . T h i s i n f o r m a t i o n w i l l be e s s e n t i a l i n d e t e r m i n i n g the r e l a t i o n s h i p o f the p e p t i d e s t o P140. Table 1 P1U0 PROTEOLYTIC PEPTIDES TREATMENT Trypsin kinase ( f i g -cleave 2 ) FRAGMENT S I Z E b 33k 28k 25k 22k FRAGMENT REPRESENTATIONc Chymotrypsin kinase cleave U5k ( f i g . 3 ) 35k cleave kinase ( f i g .,4 ) 6 6 k $ 8 k U 9 k l ; 3 k 3 9 k Chymotrypsin + Trypsin kinase cleave ( f i g . 5 ) 3 5 k cleave kinase ( f i g . 4 ) 3 f B 28k a . s p e c i f i c p r o t e o l y t i c enzyme u s e d and o r d e r o f p r o t e o -l y s i s i n r e l a t i o n t o p h o s p h o r y l a t i o n , d e s c r i b e d p e r e x p e r i m e n t . b. s i z e o f p r o t e o l y t i c p e p t i d e s o b t a i n e d d e t e r m i n e d on SDS-PAGE r e l a t i v e t o m o l e c u l a r w e i g h t m a r k e r s . c . s i z e r e p r e s e n t a t i o n o f 1mm e q u a l to 1000d m.w. 5 1 CHAPTER I I I PURIFICATION OF PROTEOLYTIC PEPTIDES I n t r o d u c t i o n G i v e n the a v a i l a b i l i t y o f p r o t e o l y t i c p e p t i d e s from P140 a l o n g w i t h the f a i l u r e t o p u r i f y t h e s e p e p t i d e s by 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 , i t seemed most e x p e d i e n t t o f r a c t i o n a t e t h e s e p e p t i d e s i n t o d i s c r e t e components f o r f u r t h e r s t u d y . L e v i n s o n e_t a_l (1981) have s u c c e e d e d i n f r a c t i o n a t i n g the p r o t e o l y t i c f r a g m e n t s o b t a i n e d from a l i m i t e d d i g e s t i o n o f p p 6 0 s r c by o n e s t e p g e i f i l t r a t i o n . Based on t h i s s u c c e s s I u s e d one s t e p g e l f i l t r a t i o n t o f r a c t i o n a t e the p r o t e o l y t i c c l e a v a g e p e p t i d e s p r o d u c e d from P140. F r a c t i o n a t i o n and e v e n t u a l p u r i f i c a t i o n o f t h e s e p e p t i d e s w i l l be n e c e s s a r y i n o r d e r to c o n c l u s i v e l y i d e n t i f y t h e i r o r i g i n and b i o l o g i c a l a c t i v i t y . O b t a i n i n g P140 d e r i v e d p e p t i d e s w i l l a l s o be a p p l i c a b l e t o w a r d s the l a r g e r q u e s t i o n o f t h e s t r u c t u r e and f u n c t i o n o f P140. 52 G e l F i l t r a t i o n G e l f i l t r a t i o n / s e p a r a t i n g m a c r o m o l e c u l e s on the b a s i s o f s i z e , i s g e n t l e on p r o t e i n s , i s h i g h l y r e p r o d u c i b l e and has a h i g h p e r c e n t a g e o f s o l u t e r e c o v e r y from the m a t r i x ( C o o p e r 1 9 7 7 ) . The s u c c e s s o f g e l f i l t r a t i o n i n p u r i f y i n g p r o t e i n s i s b a s e d on the t y p e o f m a t r i x u s e d . By a p p r o p r i a t e c h o i c e o f m a t e r i a l , t h i s method can be v e r y e f f e c t i v e i n t o t a l l y s e p a r a t i n g v a r i o u s s i z e d p r o t e i n s . A. Column p r e p a r a t i o n . In the f o l l o w i n g e x p e r i m e n t s , I us e d a. p o l y d e x t r a n g e l , Sephadex. T h i s i s c o m m e r c i a l l y p r e p a r e d i n a wide v a r i e t y o f mesh s i z e s , and f o r my p u r p o s e s a s u p e r f i n e Sephadex G-100 g e l was c h o s e n , w h i c h f r a c t i o n a t e s p r o t e i n s i n the range 4,000 t o 100,000 m.w.. The Sephadex was h y d r a t e d and d e f i n e d p r i o r t o use i n column form a s p e r e x p e r i m e n t . P r i o r to u s e , the e x c l u s i o n o r v o i d volume, and the i n c l u s i o n volume o f a g i v e n column were d e t e r m i n e d . D e x t r a n b l u e , m.w. 2x106 and Bromphenol b l u e , m.w. 600, were l o a d e d on the column, and the e l u t i n g volume o f b u f f e r measured. A pH 9.0 T r i s - H C l b u f f e r c o n t a i n i n g DOC and g l y c e r o l was u s e d i n o r d e r to p r e v e n t i n c l u s i o n o f the D e x t r a n B l u e i n the column. The v o i d volume was 13.5 m i s , and the i n c l u s i o n volume was 41.0 ml s. 53 B. Sample p r e p a r a t i o n . I t was n e c e s s a r y t o b e g i n the p u r i f i c a t i o n p r o c e d u r e s w i t h a l a r g e amount o f t h e p e p t i d e s t o be s e p a r a t e d , as w e l l as a u n i f o r m s t o c k from w h i c h t o draw on i n the work. To p r e p a r e l a r g e s a m p l e s o f P140 p r o t e o l y t i c p e p t i d e s , the r e a c t i o n c o n d i t i o n s o p t i m a l f o r the p r o d u c t i o n o f the p e p t i d e s were us e d i n m u l t i p l e a l i q u o t s r a t h e r t h a n b e i n g s c a l e d up, t o m a i n t a i n i d e n t i c a l c o n d i t i o n s . Ten-500 u l FSV i n f e c t e d c e l l l y s a t e s a l i q u o t s were i m m u n o p r e c i p i t a t e d , p e l l e t s o f w h i c h were s u b j e c t e d t o p r o t e o l y s i s , a s i n M a t e r i a l s and Me t h o d s . P r o t e o l y s i s s u p e r n a t a n t s were t h e n p o o l e d f o r u s e . R e s u l t s C h y m o t r y p s i n P e p t i d e s A. G e l f i l t r a t i o n . The f i r s t p r e p a r a t i o n t o be f r a c t i o n a t e d by g e l f i l t r a t i o n was a c h y m o t r y p t i c s i n g l e d i g e s t o f P140. The sample was mixed w i t h bromphenol b l u e , p r o t e i n s t a n d a r d s BSA and c a r b o n i c a n h y d r a s e , and l a y e r e d on t o p o f t h e column bed. Once the e n t i r e sample was i n the g e l m a t r i x , b u f f e r was a p p l i e d c o n t i n u o u s l y and the column e l u t e d under g r a v i t y . F r a c t i o n s c o l l e c t e d were a s s a y e d f o r the p r e s e n c e o f p r o t e o l y t i c p e p t i d e s o f P140 by the _in v i t r o , k i n a s e r e a c t i o n , see M a t e r i a l s and Methods and F i g u r e 7. In F i g u r e 7, e v e r y t h i r d f r a c t i o n o f the column, between 54 the v o i d volume and the i n c l u s i o n volume was a s s a y e d a s d e s c r i b e d a b o v e , a s w e l l a s the p r e - c o l u m n sample. Lanes 16-3 c o r r e s p o n d t o column f r a c t i o n s 13-60 r e s p e c t i v e l y . Lane 2 i s the p r e - c o l u m n p e p t i d e p r e p a r a t i o n . The c o n d i t i o n s c h o s e n f o r t h i s p a r t i c u l a r c h y m o t r y p t i c d i g e s t i o n o f P140 p r o d u c e d an abundance o f the 45K p e p t i d e a l o n g w i t h 68K, 58K, 39K and 18K p e p t i d e s w h i c h have a l l been o b s e r v e d b e f o r e as p r i n c i p l e p e p t i d e p r o d u c t s . In the column e l u a t e f r a c t i o n s , the 45K and 18K p e p t i d e s , a l o n g w i t h f a i n t 88K and 58K p e p t i d e bands c o - e l u t e d , a f t e r t h e v o i d volumn; t h e s e p e p t i d e s were i n c l u d e d i n the g e l m a t r i x . They e l u t e d a t a d i s t i n c t p o i n t f r o m the column as w e l l , r a t h e r t h a n i n a b r o a d s p r e a d a c r o s s s e v e r a l f r a c t i o n s . A t the same t i m e , the marker p r o t e i n s were f r a c t i o n a t e d by the column i n t o the e l u a n t f r a c t i o n s e x p e c t e d . B i n d i n g o r c o m p l e x i n g o f the P140 p e p t i d e s i s i n d i c a t e d by the r e s u l t s , r e s u l t i n g i n t h e i r c o - e l u t i o n from t h i s column. C h y m o t r y p s i n - T r y p s i n P e p t i d e s A. G e l f i l t r a t i o n . To i n v e s t i g a t e whether t h i s c o - e l u t i o n r e s u l t e d f r o m t h e s e p e p t i d e s h a v i n g o r i g i n a t e d from a s i n g l e l i m i t e d d i g e s t i o n , I u s e d the h a l f o f the l a r g e s c a l e d i g e s t i o n s u p e r n a t a n t r e s u l t i n g from c h y m o t r y p s i n - t r y p s i n d o u b l e d i g e s t i o n , as d e s c r i b e d a b o v e . I d e n t i c a l column c o n d i t i o n s were employed a s p r e v i o u s l y ; p r o t e i n m a r k e r s and bromphenol b l u e were added to the p e p t i d e sample b e f o r e l o a d i n g on the c o l u m n . A f r a c t i o n o f t h i s sample was r e s e r v e d f o r a n a l y s i s , the r e m a i n d e r c h r o m a t o g r a p h e d as p r e v i o u s l y . 55 F i g u r e 7. G e l F i l t r a t i o n o f P140 C h y m o t r y p t i c P e p t i d e s . N o n - p h o s p h o r y l a t e d P140 c h y m o t r y p t i c p e p t i d e s were p r o d u c e d and p o o l e d . A 0.5 ml sample o f p o o l e d p e p t i d e p r e p , was mixed w i t h 10 u l o f bromphenol b l u e , 30 pq BSA, 70 pq c a r b o n i c a n h y d r a s e and l a y e r e d on top o f a 20x1.5 cm Sephadex G-100 s u p e r f i n e column e q u i l i b r a t e d w i t h g e l f i l t r a t i o n b u f f e r . The column was e l u t e d w i t h g e l f i l t r a t i o n b u f f e r under g r a v i t y u n t i l bromphenol b l u e e x i t e d from the column, e l u e n t c o l l e c t e d i n 0.5 ml f r a c t i o n s . 30 pi s a m p l e s o f f r a c t i o n s were a s s a y e d f o r p e p t i d e s by the _in v i t r o k i n a s e r e a c t i o n and were p r e p a r e d f o r SDS-PAGE by a d d i t i o n o f SDS g e l sample b u f f e r . The SDS g e l was s t a i n e d w i t h Coomassie b l u e t o v i s u a l i z e the p r o t e i n s t a n d a r d s . E v e r y t h i r d f r a c t i o n was a s s a y e d from 13 t o 52 ( l a n e s 16 t o 3 r e s p e c t i v e l y ) ; ( l a n e 2) n o n - f r a c t i o n a t e d c h y m o t r y p t i c p e p t i d e sample u s e d i n the f r a c t i o n a t i o n . P r i n c i p l e p e p t i d e s r e s u l t i n g a r e i n d i c a t e d by a r r o w s i n l a n e A. M o l e c u l a r w e i g h t m a r k e r s a r e i n d i c a t e d by b a r s i n l a n e X. 57 The r e s u l t s o f g e l f i l t r a t i o n o f the c o n s e c u t i v e d i g e s t p e p t i d e s a r e seen i n F i g u r e 8. T h i s d i g e s t i o n d i d n o t go t o c o m p l e t i o n , r e s u l t i n g i n a 45K p e p t i d e , u n d o u b t e d l y r e m a i n i n g from the i n i t i a l c h y m o t r y p s i n d i g e s t i o n , and a 33K p e p t i d e , the major p r o d u c t o f the c h y m o t r y p s i n - t r y p s i n c o n s e c u t i v e d i g e s t i o n . L anes 2-8 (8B) show t h e column e l u t i o n f r a c t i o n s f o l l o w i n g the v o i d volume. The e n t i r e column e l u t i o n was a s s a y e d , and the 45K and 33K p e p t i d e s , as w e l l a s 49K and 39K p e p t i d e s f r o m the i n i t i a l d i g e s t a l l c o - e l u t e d from the column. They were i n c l u d e d i n the column m a t r i x , b u t e x i t e d i m m e d i a t e l y a f t e r the v o i d volume. A l s o , the marker p r o t e i n s were f r a c t i o n a t e d by the column and were seen l a t e r i n the e l u t i o n v o lume. B. Ion e x c h a n g e . In o r d e r t o a s c e r t a i n w hether t h e s e p e p t i d e s were c o m p l e x i n g • i n g e l f i l t r a t i o n , an i o n exchange m a t r i x was employed t o f r a c t i o n a t e the p e p t i d e p r e p a r a t i o n s . D E A E - s e p h a c e l i s an a n i o n e x c h a n g e r and the C - t e r m i n a l end o f i n t a c t P140 i s dense i n b a s i c amino a c i d s ( S h i b u y a and H a n a f u s a , 1982) a s compared t o the r e s t o f the p r o t e i n . P e p t i d e s o r i g i n a t i n g i n the C - t e r m i n a l end o f P140 may g i v e d i f f e r e n t i a l b i n d i n g o f t h e s e f r a g m e n t s t o the DEAE m a t r i x b a s e d on d i f f e r e n c e s i n c h a r g e d i s t r i b u t i o n r e s u l t i n g from v a r y i n g l e n g t h s a n d / o r p o r t i o n s o f the C - t e r m i n a l h a l f o f P140 b e i n g r e p r e s e n t e d i n the p e p t i d e s . Ion exchange would l i k e l y b i n d p e p t i d e s from the N - t e r m i n a l p o r t i o n o f P140 a s w e l l , b u t the h i g h p r o p o r t i o n o f b a s i c , and t r y p s i n l a b i l e amino a c i d s i n the C - t e r m i n a l end would f u r t h e r enhance f r a c t i o n a t i o n o f C - t e r m i n a l p e p t i d e s . In a d d i t i o n , a complex i s l i k e l y t o be 58 Figure 8. Chymotrypsin-Trypsin Consecutive Proteolysis of P140. Non-phosphorylated chymotrypsin-trypsin peptides were produced and pooled. A 0.5 ml sample of the pooled peptide preparation was mixed with 10 pi of bromphenol blue, 30 ug BSA, 70 jjg carbonic anhydrase and layered on a 20x1.5 cm gel f i l t r a t i o n column equilibrated with gel f i l t r a t i o n buffer. The column was eluted with gel f i l t r a t i o n buffer under gravity, 0.37 ml fractions were c o l l e c t e d . Eluent fractions were assayed for peptides by the iri v i t r o kinase reaction and prepared for SDS-PAGE by addition of SDS gel sample buffer. Protein standards were vi s u a l i z e d by staining the gel with coomassie blue. Fractions assayed were as follows: (Fig. 7-A) non-fractionated peptide sample used in the experiment; (Fig. 7-B,lanes 1 to 8) column elution fractions 28 to 35 respectively. P r i n c i p l e peptides resulting are indicated by arrows in lanes A and X. Molecular weights are indicated by bars in lanes A and X. 59 A 60 b r o k e n up i n an i o n exchange column, e s p e c i a l l y one s u c h a s DEAE s e p h a c e l w h i c h a l s o has g e l s i e v i n g p r o p e r t i e s w h i c h may a i d i n a c c o m o d a t i n g the p e p t i d e c omplex. (1) P r e - p h o s p h o r y l a t e d c h y m o t r y p t i c p e p t i d e s . I n i t i a l l y , t o d e t e r m i n e whether i o n exchange would f r a c t i o n a t e the p e p t i d e s w h i c h have p r e v i o u s l y c o - e l u t e d , I p o o l e d the f r a c t i o n s from the g e l f i l t r a t i o n column c o n t a i n i n g the c o - e l u t e d c h y m o t r y p t i c p e p t i d e s , d i s c u s s e d i n the p r e v i o u s c h a p t e r . T h i s sample was a p p l i e d t o and e l u t e d from the i o n exchange column, e m p l o y i n g , a l i n e a r N aCl g r a d i e n t i n the i o n exchange b u f f e r f o r e l u t i o n , a s d e s c r i b e d i n M a t e r i a l s and Methods. F i g u r e 9 shows the SDS-PAGE r e s u l t s o f the a s s a y o f t h e s e column f r a c t i o n s . E v e r y s e c o n d e l u t i o n f r a c t i o n was a s s a y e d by a d d i n g e q u a l volumes o f the sample t o SDS g e l sample b u f f e r . The l a b e l e d p r e DEAE column sample i n l a n e 1 shows the 45K and 18K p e p t i d e s were p h o s p h o r y l a t e d i n the p o o l e d sample, and p r e s e n t i n a p p r o x i m a t e l y e q u a l amounts, b a s e d on the r e l a t i v e i n t e n s i t i e s o f the b a n d s . The 45K p e p t i d e e l u t e d f r o m t h e . i o n exchange i n NaCl c o n c e n t r a t i o n r a n g e 0.7 to 0.85M, l a n e s 8-11 r e s p e c t i v e l y . The 18K p e p t i d e d i d n o t c o - e l u t e , and i n f a c t was n o t v i s i b l e on the g e l . T h i s c o u l d be due t o t i g h t b i n d i n g o f the p e p t i d e t o the i o n e x c h a n g e r , o r t o f u r t h e r d i g e s t i o n by some p r o t e a s e i n the b u f f e r o r column. Use o f i o n exchange and s u i t a b l e i o n i c s t r e n g t h b r o k e up the complexed p e p t i d e s , a l l o w i n g a t l e a s t the 45K p e p t i d e t o be f r a c t i o n a t e d t o some d e g r e e o f p u r i t y . In p u r s u i n g the f a t e o f the 1.8K p e p t i d e i n t h i s s y s t e m , the 61 Figure 9. Ion Exchange of P140 Chymotryptic Peptides. P140 chymotryptic peptides that co-eluted in gel f i l t r a t i o n were pooled. An internal probe of elution was produced by incubating a 40ul sample (10% of the volume of the entire sample) with 4 u l 0. IM MnCl and 10 jiCi [V-,2-P]-ATP 15 minutes on ic e . This was added back to the entire sample, mixed well and 40 ul retained while the rest was loaded on ion exchange equilibrated to the ion exchange buffer. The column was washed with buffer u n t i l wash was at background cpm. A 40 ml linear gradient 0 to 1. 5M NaCl in ion exchange buffer was used to elute the column, and 1.0 ml fractions were co l l e c t e d . Elution fractions were assayed for chymotryptic peptides on SDS-PAGE, samples prepared for SDS-PAGE by addition of SDS gel sample buffer. Samples assayed were as follows: (lane 1) non-fractionated pooled chymotrypsin-trypsin peptide sample used in the column, (lanes 2 to 18) every second f r a c t i o n eluted from 0 to 1. 5M NaCl li n e a r gradient respectively. Principle peptides resulting are indicated by arrows in lane A. Molecular weights are indicated by bars in lane X. 6 2 96 49 45 66 45 28 18 1 2 3 4 5 6 7 8 9 10 11 12 13 "14 15 1*6 1*7 1*? X 63 e l u t i o n c o n d i t i o n s were e x t e n d e d t o 2. 0M s a l t . A f r e s h c h y m o t r y p t i c p e p t i d e p r e p a r a t i o n was made under i d e n t i c a l c o n d i t i o n s t o the p r e v i o u s e x p e r i m e n t s . T h i s sample was p r e - p h o s p h o r y l a t e d i n the jln v i t r o k i n a s e as d e s c r i b e d p r e v i o u s l y ; and a p p l i e d d i r e c t l y t o i o n e x c h a n g e . E i u a t e f r a c t i o n s were f o l l o w e d i n cpm p r o f i l e / and a s s a y e d by SDS-PAGE. No a p p r e c i a b l e e l u t i o n o f the 18K p e p t i d e o c c u r r e d a t the i n c r e a s e d s a l t c o n c e n t r a t i o n o f 2.0M. However/ the 45K p e p t i d e e l u t e d i n a s i m i l a r r a n g e o f i o n i c s t r e n g t h a s i n t h e p r e v i o u s e x p e r i m e n t . I c o u l d n o t m a i n t a i n a s a l t c o n c e n t r a t i o n h i g h e r t h a n a p p r o x i m a t e l y 2M i n t h i s b u f f e r s y s t e m . S i n c e t h i s seemed to be the b e s t b u f f e r s y s t e m t o use w i t h DEAE s e p h a c e l / no f u r t h e r i n v e s t i g a t i o n s were done t o e l u t e the 18K p e p t i d e . However/ o b t a i n i n g the 45K f r a g m e n t i n i s o l a t i o n f r o m the o t h e r p e p t i d e s does d e m o n s t r a t e the p o s s i b i l i t y o f p u r i f y i n g t h e s e p e p t i d e s i n d i s c r e t e f r a c t i o n s . (2) N o n - p h o s p h o r y l a t e d c h y m o t r y p t i c p e p t i d e s . To complement the p r e v i o u s r e s u l t s , and t o c o n f i r m the p u r i f i c a t i o n of the p r e - l a b e l e d p e p t i d e s a s t h o s e w h i c h a r e d e r i v a t i v e s o f P140, i d e n t i c a l c o n d i t i o n s were employed t o p r o d u c e a c h y m o t r y p t i c p e p t i d e sample a s p r e v i o u s l y , and t h e s e u n l a b e l e d p e p t i d e s were s u b j e c t e d t o i o n exchange c h r o m a t o g r a p h y as a b o v e . Column f r a c t i o n s were a s s a y e d by i n  v i t r o k i n a s e a s s a y , r e s u l t s shown i n F i g u r e 10 A and B. E v e r y l i m i t e d d i g e s t i o n o f P140 by c h y m o t r y p s i n y i e l d e d d i f f e r e n t r a t i o s o f the v a r i o u s p e p t i d e s , even under s e e m i n g l y i d e n t i c a l c o n d i t i o n s . In a l l c a s e s , the major p e p t i d e 64 r e s u l t i n g was the 45K p e p t i d e - A wide r a n g e o f p e p t i d e s were p r o d u c e d i n t h i s e x p e r i m e n t w i t h the 45K p e p t i d e p r e d o m i n a t i n g ( s e e n i n the sample o f p e p t i d e p r e p a r a t i o n n o t r u n on the i o n e x c h a n g e , l a n e 10 F i g . 10B). The 45K p e p t i d e was p h o s p h o r y l a t e d i n the i o n exchange e l u t i o n f r a c t i o n s , and e l u t e d a t a p p r o x i m a t e l y 0.7M s a l t , l a n e s 7, 8 and 9, F i g u r e 10A. T h e r e was a l s o an i n t e n s e l y l a b e l e d p r o t e i n o f a p p r o x i m a t e l y 30K m.w. n o t s e e n i n any p r e v i o u s p r o t e o l y t i c d i g e s t i o n s o f P140. The 30K p r o t e i n e l u t e d from i o n exchange i n a h i g h s a l t c o n c e n t r a t i o n , i n 2 d i s t i n c t p e a k s , c o r r e s p o n d i n g to l a n e s 4-6 and 8-9 i n F i g u r e 10B. The 30K p r o t e i n d i d n o t p h o s p h o r y l a t e i n the p e p t i d e p r e p a r a t i o n p r i o r t o b e i n g f r a c t i o n a t e d on i o n e x c h a n g e . B o t h 30K p r o t e i n p eaks a r e s e e m i n g l y bound t i g h t e r t o the i o n exchange r e s i n t h a n the 45K p e p t i d e . In a d d i t i o n , the "minor" 68K, 58K, 39K and 18K p e p t i d e s c o n s i s t e n t l y d i s a p p e a r when the p r e p a r a t i o n i s a p p l i e d t o the c olumn. Exogenous S u b s t r a t e P h o s p h o r y l a t i o n To d e t e r m i n e u n e q u i v o c a l l y the e x i s t e n c e o f an e n z y m a t i c a c t i v i t y on t h e s e p r o t e o l y t i c p e p t i d e s , s p e c i f i c a l l y a t y r o s i n e s p e c i f i c , k i n a s e a c t i v i t y , a d v a n t a g e was t a k e n o f the f a c t t h a t n a t i v e P140 w i l l p h o s p h o r y l a t e exogenous s u b s t r a t e in. v i t r o . In f a c t , the amount o f p h o s p h o r y l a t i o n o f an e xogenous s u b s t r a t e i s much h i g h e r than t h a t f o r P140 i t s e l f , i n the same s y s t e m . E n o l a s e i s a g l y c o l y t i c enzyme w h i c h has been f o u n d t o be an exogenous s u b s t r a t e o f the k i n a s e a c t i v i t y a s s o c i a t e d 65 Figure 10-A/ 10-B. Ion Exchange of P140 Chymotryptic Peptides. P140 chymotryptic peptides were produced and pooled. A 0.5 ml sample was loaded on the column (50 J J I retained) equilibrated with ion exchange buffer and washed through the column with 4 column volumes of buffer. Sample was eluted with a 40 ml volume 0 to 2.0M NaCl li n e a r gradient in the buffer, 0.8 ml fractions were co l l e c t e d . Fractions were assayed for peptides by the in v i t r o kinase reaction and samples were prepared for SDS-PAGE by addition of SDS gel sample buffer. Samples assayed were as follows: (Fig. 9-A, lanes 1 to 14; F i g . 9-B, lanes 1 to 9) every second column elution f r a c t i o n assayed in l i n e a r NaCl gradient 0 to 2. 0M respectively; (Fig. 9-B lane 10) non-fractionated P140 chymotryptic peptide sample used in the experiment. Principle resulting peptides are indicated with arrows in lanes A. Molecular weights are indicated by bars in lanes X. 66 68 w i t h FSV f o r p h o s p h o r y l a t i o n ( C o o p e r e_t a_l, 1 9 8 4 ) . The amino a c i d s t y r o s i n e and s e r i n e a r e the o n l y ones p h o s p h o r y l a t e d , s u g g e s t i n g t h i s i s a l s o a s u b s t r a t e f o r the t y r o s i n e s p e c i f i c p r o t e i n k i n a s e a s s o c i a t e d w i t h the v i r u s i n t r a n s f o r m e d c e l l s . S i n c e the p h o s p h o r y l a t i o n o f e n o l a s e i r i v i t r o by P140 i s t a k e n as a measure o f the t y r o s i n e s p e c i f i c p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h P140, I u s e d e n o l a s e i n a s s e s s i n g the p r e s e n c e o f any t y r o s i n e s p e c i f i c p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h the p e p t i d e s p r o d u c e d from the above c o n d i t i o n s . T h i s w ould a i d i n d e t e r m i n i n g the o r i g i n o f s u c h p e p t i d e s , a s w e l l a s t h e i r e n z y m a t i c n a t u r e . E n o l a s e , t o be u s e d i n an i_n v i t r o a s s a y f o r k i n a s e a c t i v i t y , must be a c i d d e n a t u r e d f o r b e s t r e s u l t s . To do t h i s , I f o l l o w e d the p r o c e d u r e s o f Cooper e_t a_l (1984) w i t h some m o d i f i c a t i o n s ( s e e F i g u r e 1 1 ) . In t h i s a s s a y , I u s e d p e p t i d e s p r o d u c e d from a l i m i t e d c h y m o t r y p s i n d i g e s t i o n and p u r i f i e d on i o n exchange as a b o v e . R e s u l t s a r e shown i n F i g u r e 11. The c o - l a b e l i n g o f the 45K p e p t i d e and e n o l a s e s u b s t r a t e s e r v e d t o i d e n t i f y e a c h component, as w e l l a s g i v e some e s t i m a t e o f the e f f i c i e n c y o f s u b s t r a t e l a b e l i n g by the p e p t i d e . E n o l a s e p h o s p h o r y l a t e d w e l l i n a r e a c t i o n w i t h i n t a c t i m m u n o p r e c i p i t a t e d P140, l a n e 1, d e m o n s t r a t i n g the a b i l i t y o f e n o l a s e to a c t as a s u b s t r a t e i n p h o s p h o r y l a t i o n by P140. As a n e g a t i v e c o n t r o l , e n o l a s e was i n c u b a t e d w i t h l a b e l mix a l o n e , and d i d n o t s e l f - p h o s p h o r y l a t e ( d a t a n o t shown). In DEAE column f r a c t i o n s c o n t a i n i n g the 45K p e p t i d e , l a n e s 2-7, the 45K p e p t i d e p h o s p h o r y l a t e d e n o l a s e w e a k l y compared w i t h i n t a c t P140. E n o l a s e p h o s p h o r y l a t i o n d e c r e a s e d when the p r e s e n c e o f 69 Figure 11. Enolase Phosphorylation by P140 Chymotryptic Peptides. P140 chymotryptic peptides were produced and fractionated on ion exchange as in F i g . 10. To a 10% volume sample of the column elution fractions assayed were added MnCl to 20 mM, 5 ug. of acid treated enolase (prepared as directed by Cooper et a l 1984) and 1 jaCi [tf-^P] ATP (in 2 pi 0.1M Hepes pH 7.0-20mM MnCl buffer). The reaction was incubated at 30OQ 15 minutes and terminated by adding equal volume of 2X SDS gel sample buffer to column samples, and by spinning, removing supernatants and adding the appropriate SDS gel sample buffer to jS. aureus p e l l e t s and their supernatants. Samples were assayed on SDS-PAGE as follows: (lane 1) supernatant from enolase incubated with immunocomplexed intact P140, (lanes 2 to 7) ion exchange fractions containing the 45k peptide incubated with enolase, (lanes 8 to 10) ion exchange fractions containing the 30k peptide incubated with enolase. P140, enolase and p r i n c i p l e peptides are indicated by arrows in lane A.' Molecular weights are indicated by bars in lane X. 70 ! 71 45K p e p t i d e d e c r e a s e d (measured by band i n t e n s i t i e s ) . F r a c t i o n s from the DEAE column r e p r e s e n t e d i n F i g u r e 10, c o n t a i n i n g the 30K p e p t i d e were a l s o a s s a y e d , l a n e s 8-10. The s t r o n g 30K p e p t i d e was i n t e n s e l y p h o s p h o r y l a t e d w h i l e e n o l a s e was f a i n t l y p h o s p h o r y l a t e d . D i s c u s s i o n . G e l f i l t r a t i o n , w h i l e a b l e t o f r a c t i o n a t e the p r o t e i n s t a n d a r d s , was u n a b l e to f r a c t i o n a t e the p r o t e o l y t i c p e p t i d e s p r o d u c e d from P140 f o r b o t h the s i n g l e d i g e s t c h y m o t r y p t i c p e p t i d e s and the c h y m o t r y p s i n - t r y p s i n p e p t i d e s . B u f f e r c o n d i t i o n s were c h o s e n t o e l i m i n a t e o r m i n i m i z e l a r g e m i c e l l e f o r m a t i o n . I t i s l i k e l y t h a t the P140 p e p t i d e s were c o m p l e x i n g i n s o l u t i o n and n o t b r o k e n up i n t o i n d i v i d u a l p e p t i d e s by the g e l f i l t r a t i o n c o n d i t i o n s . The t h e o r y t h a t t h e s e P140 d e r i v e d p e p t i d e s were c o m p l e x i n g i s s u p p o r t e d by t h e r e s u l t s where an i o n e x change m a t r i x d i d f r a c t i o n a t e the p e p t i d e s . S i n c e i o n exchange i s a b l e t o d i s r u p t a complex h e l d t o g e t h e r by n o n - c o v a l e n t f o r c e s , the p e p t i d e s may have been c o m p l e x i n g by s t r u c t u r a l l y common components, s u c h as an e s s e n t i a l domain r e g i o n , o r s u r f a c e r e g i o n s o f e a c h p e p t i d e . T h i s c a n n o t be d e t e r m i n e d from the p r e s e n t work. Ion exchange r e s u l t e d i n the f r a c t i o n a t i o n o f the major 45K p e p t i d e from o t h e r p e p t i d e s , t h i s i s a s i g n i f i c a n t s t e p t o w a r d s the d e s i r e d r e s u l t o f o b t a i n i n g i s o l a t e d p e p t i d e s o r i g i n a t i n g f r o m p a r t i a l 72 p r o t e o l y s i s o f P140. The 45K p e p t i d e a p p e a r s t o c o n t a i n the major s i t e ( s ) o f p h o s p h o r y l a t i o n seen on P140. P140 C - t e r m i n a l o r i g i n o f the 45K p e p t i d e , w h i l e n o t c o n c l u s i v e l y i d e n t i f i a b l e f r o m t h e s e r e s u l t s , i s i n d i c a t e d . C o r r e l a t i o n o f e n z y m a t i c a c t i v i t y w i t h o r i g i n o f the 45K p e p t i d e w i l l d e t e r m i n e the major r e g i o n o f P140 f u n c t i o n a l i n t r a n s f o r m a t i o n . F u r t h e r s u p p o r t f o r the t h e s i s t h a t the 45K p e p t i d e c o n t a i n s a major f u n c t i o n a l domain o f P140 c o n s i s t s i n the f a c t t h a t an " i d e n t i c a l " 45K p e p t i d e r e s u l t e d from b o t h c h y m o t r y p s i n s i n g l e d i g e s t i o n and c h y m o t r y p s i n - t r y p s i n c o n s e c u t i v e d i g e s t i o n o f P140. As w e l l , t h i s same 45K p e p t i d e y i e l d e d an i n t e n s e l y l a b e l e d p e p t i d e i n b o t h p r e - d i g e s t i o n and p o s t - d i g e s t i o n p h o s p h o r y l a t i o n i n the ^ r i v i t r o k i n a s e r e a c t i o n . The u n i q u e 30K p r o t e i n , l a b e l i n g o n l y a f t e r f r a c t i o n a t i o n on i o n e x c h a n g e , i n d i c a t e s a p e p t i d e c o n t a i n i n g no s i t e s o f a c t i v e p h o s p h o r y l a t i o n on i n t a c t P140. The p h o s p h o r y l a t i o n o f the 30K p r o t e i n a f t e r f r a c t i o n a t i o n on i o n exchange i n d i c a t e s a p o s s i b l e s e c o n d a r y p h o s p h o r y l a t i o n s i t e n o t a c c e s s i b l e on i n t a c t P140. P h o s p h o r y l a t i o n o f the 30K p r o t e i n would t a k e p l a c e on a p e p t i d e r e l e a s e d from a complex o f p e p t i d e s , o r i n t a c t P140. Thus, the 30K p r o t e i n may be a p r o d u c t o f f u r t h e r p r o t e o l y t i c d i g e s t i o n o f P140 p e p t i d e s , i n d i c a t i n g the s u c c e s s i v e d i g e s t i o n o f l a r g e r p e p t i d e s to a s m a l l p e p t i d e c o n s i s t i n g o f a s t r u c t u r a l (and p o s s i b l y f u n c t i o n a l ) domain o f P140. I t w i l l be n e c e s s a r y t o p u r i f y the 30K p r o t e i n t o d e t e r m i n e i t s r e l a t i o n s h i p t o P140. F u r t h e r i n d i c a t i o n t h a t the 45K p e p t i d e c o n t a i n s a majo r f u n c t i o n a l domain o f P140 comes from the r e s u l t o f the 73 f r a c t i o n a t e d 45K p e p t i d e r e t a i n i n g an a b i l i t y t o p h o s p h o r y l a t e the exogenous s u b s t r a t e e n o l a s e , t h o u g h much weaker t h a n i n t a c t P140. The a c t i v i t y by the 45K p e p t i d e t o p h o s p h o r y l a t e e n o l a s e was d e m o n s t r a b l y p o s i t i v e , i f f a i n t , and d i s t i n c t f r o m o t h e r p e p t i d e s i n the sample. Such s p e c i f i c l a b e l i n g i n d i c a t e s the 45K p e p t i d e t o c o n t a i n an i m p o r t a n t r e g i o n o f P140. One c o u l d p o s t u l a t e many r e a s o n s why s u c h p o o r l a b e l i n g was c a r r i e d o u t by t h i s p e p t i d e . These w i l l o n l y be answered on p u r i f i c a t i o n o f the p e p t i d e . The n e g l i g i b l e a b i l i t y o f the 30K p e p t i d e t o p h o s p h o r y l a t e e n o l a s e may i n d i c a t e i t to be a p e p t i d e n o t c o n t a i n i n g a s t r u c t u r a l o r f u n c t i o n a l domain o f P140,but to have a s e c o n d a r y p h o p h o r y l a t i o n s i t e , a s d i s c u s s e d a b o v e . C o n v e r s e l y , i t may i n d i c a t e the p r e f e r e n t i a l a c t i o n o f a l a r g e r 45K p e p t i d e o f P140 t o p h o s p h o r y l a t e o t h e r s u b s t r a t e s w h i l e the s m a l l e r 30K p e p t i d e c o n t a i n s an a c t i v e s t r u c t u r a l domain f o r p h o s p h o r y l a t i o n o f s e l f , b u t n o t l a r g e enough t o a c t on a n o t h e r p r o t e i n . These a l t e r n a t i v e s were n o t i n v e s t i g a t e d i n the p r e s e n t work. However, I b e l i e v e I have d e m o n s t r a t e d t h a t by a s e l e c t use o f p r o t e a s e s i n c o n j u n c t i o n w i t h i m m u n o p r e c i p i t a t i o n and p u r i f i c a t i o n t e c h n i q u e s , s e v e r a l d i s t i n c t p e p t i d e s r e s u l t i n g f r o m f r a g m e n t a t i o n o f i n t a c t P140 can be p r o d u c e d . T h i s w i l l e n a b l e f u r t h e r s t u d y on the l o c a l i z a t i o n o f s t r u c t u r a l and f u n c t i o n a l domains on P140. 74 BIBLIOGRAPHY B a l t i m o r e / D. 1975. Tumor v i r u s e s . C o l d S p r i n g H a r b o r  Symp.Quant. B i o l . 39:1187-1200 Beemon, K. 1981. T r a n s f o r m i n g p r o t e i n s o f some f e l i n e and a v i a n sarcoma v i r u s e s a r e r e l a t e d s t r u c t u r a l l y and f u n c t i o n a l l y . C e l l 24:145-153 B i s h a i , F.R. 1981. L i g a n d A s s a y pp.183-204. E d i t e d by Langan, J . and C l a p p , J . J . 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